Volume 24, Horticultural Reviews

HORTICULTURAL REVIEWS Volume 24

Horticultural Reviews is sponsored by: American Society for Horticultural Science

Editorial Board, VoluDle 24 Matthew A. Jenks John \T. Possingham Margaret Sedgley

HORTICULTURAL REVIEWS Volume 24

edited by

Jules Janick Purdue University

John Wiley 8' Sons, Inc. NEW YORK / CHICHESTER / WEINHEIM / BRISBANE / SINGAPORE / TORONTO

This book is printed on acid-free paper.

0

Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4744. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, (212) 850-6011, fax (212) 850-6008, E-Mail: PERMREQ @ WILEYCOM. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold with the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional person should be sought. Library of Congress Catalog Card Number: 79-642829 ISBN 0-471-33374-3 ISSN 0163-7851 Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1

Contents

Contributors

ix

Dedication: Haruyuki Kamemoto

xi

Richard A. Criley 1. Bioreactor Technology for Plant Micropropagation Meira Ziv

I. II. III. IV. V. VI.

Introduction Plant Developmental Pathways in Bioreactors Plant Cell and Tissue Growth in Bioreactors Physical and Chemical Factors in Liquid Cultures Cell and Aggregate Density, Foaming and Medium Rheology in Bioreactors Summary and Conclusions Literature Cited

2. Biogenesis of Floral Scents Natalia Dudareva, Birgit Piechulla, and Eran Pichersky I. II. III. IV. V.

Introduction Pathways and Site of Synthesis Molecular Genetic Control Variation in Biosynthesis and Emission Over Time Conclusions Literature Cited

1

1

6 12

14 22 23

24 31

32

34 38 42 50 51 v

CONTENTS

vi

3. Triazoles as Plant Growth Regulators and Stress

Protectants

55

R. Austin Fletcher, Angela Gilley, Narendra Sankhla, and Tim D. Davis I. II. III. IV. V. VI. VII. VIII.

Introduction Translocation and Efficacy of Application Methods General Plant Responses to Triazoles Mode of Action Stress Protection Potential and Current Applications A Novel Seed Treatment Technology Summary Literature Cited

4. Ecologically-based Practices for Vegetable Crops Production in the Tropics Hector R. Valenzuela I. II. III. IV. V.

Introduction Integrated Cultural Management Nutrient Management and Soil Conservation Ecologically-based Pest Management Conclusions and Future Prospects Literature Cited

5. Lettuce Seed Germination Daniel J. Cantliffe, Yu Sung, and Warley M. Nascimento

I. II. III. IV. V. VI. VII. VIII.

Introduction Seed Structure Germination Environmental Factors Affecting Germination Restriction of Lettuce Seed Germination at High Temperature Increasing Thermotolerance in Lettuce Seed Changes in the Embryo and Endosperm During Germination Summary and Conclusion Literature Cited

56 59 61 66 76 81 115 116 118

139 140 146 156 172 201 203

229 229 231 232 233 236 242 254 263 264

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CONTENTS

6. Viroid Dwarfing for High Density Citrus Plantings Ronald J. Hutton, Patricia Broadbent, and Kenneth B. Bevington

277

Introduction Causal Agent Use of Viroids for Tree Size Control Vegetative Growth Reproductive Growth Intensive Viroid-Dwarfed Plantings Economic Considerations Management Summary and Conclusions Literature Cited

278 280 284 291 295 297 303 306 311 312

7. Growth, Development, and Cultural Practices for Young Citrus Trees

319

I. II. III. IV. V. VI. VII. VIII. IX.

Frederick S. Davies and James J. Ferguson I. II. III. IV. V. VI. VII. VIII. IX.

Introduction Growth and Development Tree Selection and Planting Irrigation Fertilization Freeze Hardiness and Protection Pruning Biotic Factors Summary Literature Cited

8. Fruit Growth Measurement and Analysis Lin us U. Opara I. II. III. IV. V. VI.

Introduction Fruit Developmental Stages Indices of Fruit Growth Growth Measurement Techniques Approaches to Fruit Growth Analysis Applications of Fruit Growth Data

320 321 329 330 338 343 347 348 361 362

373 374 379 383 388 390 409

CONTENTS

viii

VII. VIII.

Some Problems in Fruit Growth Measurement and Analysis Summary and Prospects Literature Cited

413 414 419

Subject Index

433

Cumulative Index

434

Cumulative Contributor Index

456

Contributors Kenneth B. Bevington, NSW Agriculture, Agricultural Research and Advisory Station, Dareton, Australia Patricia Broadbent, NSW Agriculture, Elizabeth Macarther Agricultural Institute, Menangle, Australia Daniel J. Cantliffe, University of Florida, IFAS, Horticultural Sciences Department, Gainesville, FL 32611-0690 Richard A. CrUey, Department of Horticulture, University of Hawaii, Honolulu, HI 96822 Frederick S. Davies, Horticultural Sciences Department, University of Florida, Gainesville, FL 32611-0690 Tim D. Davis, Texas A & M University, Texas Agricultural Experiment Station, Dallas, TX 75252 Natalia Dudareva, Horticulture Department, Purdue University, West Lafayette, IN 47907 James J. Ferguson, Horticultural Sciences Department, University of Florida, Gainesville, FL 32611-0690 R. Austin Fletcher, Department of Environmental Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada Angela Gilley, Department of Environmental Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada Ronald J. Hutton, NSW Agriculture, Yanco Agricultural Institute, Yanco, Australia Warley M. Nascimento, University of Florida, IFAS, Horticultural Sciences Department, Gainesville, FL 32611-0690 Linus U. Opara, Bioproducts Quality Research, Center for Postharvest & Refrigeration Research, Institute of Technology and Engineering, Massey University, Palmerston North, New Zealand Eran Pichersky, Biology Department, University of Michigan, Ann Arbor, MI 48109 Birgit Piechulla, Department of Molecular Physiology of Plants and Microorganisms, Gertrudenstrasse 11a, University of Rostock, Rostock 18051, Germany Narendra Sankhla, Texas A & M University, Texas Agricultural Experiment Station, Dallas, TX 75252 Yu Sung, University of Florida, IFAS, Horticultural Sciences Department, Gainesville, FL 32611-0690 Hector R. Valenzuela, Department of Horticulture, University of Hawaii at Manoa, Honolulu, HI 96822-2279 Meira Ziv, Agricultural Botany & the Warburg Center for Biotechnology in Agriculture, The Hebrew University of Jerusalem, Rehovot, 76100, Israel ix

Haruyuki Kamemoto

Dedication: Haruyuki Kamemoto Haruyuki Kamemoto is a distinguished horticulturist who has made significant contributions to the floriculture industry of Hawaii and the tropical world. His life's work has concentrated on breeding and development of anthuriums and dendrobium orchids at the University of Hawaii, where he is affectionately known by all as Kammy. His "official" retirement in 1986 to Professor Emeritus status did not slow his pace, as he merely continued his efforts without the distractions of committee meetings and other academic duties on a contractual part-time basis (if every day is considered part-time!) until his "final" retirement on June 30,1999.

Born in Honolulu in 1922 and raised on a family farm not far from the present-day campus of the University of Hawaii, Kammy enrolled in the College of Tropical Agriculture and earned both his BS and MS degrees there. In 1947, he enrolled for Ph.D. studies at Cornell University, where he was a member of the three K's of floriculture: Harry Kohl, Tony Kofranek, and Harry Kamemoto. He took great pride in achieving his degree in floriculture-plant breeding under the tutelage of several faculty. Returning to Honolulu in 1950, he was hired by the University of Hawaii as an Assistant Professor in the Horticulture Department and launched far-sighted flower breeding programs. Four decades plus of research into the culture and breeding of anthuriurns and dendrobium orchids propelled these two flowers into the leading floricultural crops of Hawaii. On the anthurium side, an anthracnoseresistant pink cultivar, 'Marian Seefurth', was released from his breeding program in 1963, followed by more than 20 new cultivars, including many interspecific hybrids and the "tulip" type anthuriums. His interspecific anthurium breeding program stimulated others to follow, and a number of potted and cut varieties now grace florist shops and cut flower stands around the world. UH-developed anthuriums now constitute a major part of Hawaii's anthurium industry. More than two dozen cut flower and potted dendrobium cultivars have originated through his work, which produced the first seed-propagated dendrobium cultivar, 'Uniwai Blush' in 1972. Many of these cultivars have become standards of Hawaii's orchid industry because of their high yields, long vaselife, and array of colors from white through dark xi

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DEDICATION: HARUYUKI KAMEMOTO

purple-violet. His potted dendrobiums have become a mainstay of Hawaii's potted floricultural exports. Dr. Kamemoto's program has been productive in turning out fine graduate students, as well, and he credits them with doing much of the "nitty-gritty" that led to the many orchid and anthurium releases: counting chromosomes, making crosses, growing out and maintaining the plants, recording and crunching data, starting tissue cultures, and authoring publications. No one left his program without knowing basic research methods as well as how to repot orchids and anthuriums. He has advised twenty-four students in master's programs and eleven students in doctoral programs. They have come from many parts of the world and many of them have returned to academia. Their appreciation of his guidance and knowledge instigated many invitations to make presentations in other parts of the world, as well as referrals of potential graduate students to his program. For many years, he taught the floriculture production course at the University of Hawaii and an Orchidology course, one of the few available in any university setting. Kammy is deeply concerned about the floriculture industry and has made innumerable presentations to grower groups to inform, to educate, to cajole, to incite (on occasion), and to develop the dendrobium and anthurium industries of Hawaii and elsewhere. The Kamemoto Scholarship, established in 1986 to help outstanding students interested in careers in floriculture, has been well-supported by former students, colleagues, friends, and the Hawaii floriculture industry because of their admiration and respect for his contributions. Dr. Kamemoto's valued contributions of seed-produced dendrobium orchid hybrids that are free of disease and his novel interspecific anthurium hybrids have made Hawaii's orchid and anthurium industries successful and competitive worldwide. Demands for his expertise have led to consultantships in India, Thailand, Okinawa, and Taiwan, and he has contributed to conferences and other horticultural meetings throughout Asia, as well as in the Caribbean, South America, Australia, Israel, and Europe. His contributions have been recognized by many awards from industry, professional organizations of which he is a member, and the University of Hawaii. His honors include the Alex Laurie award (1984) and Society of American Florists Hall of Fame (1991), Fellow of the American Society of Horticultural Science (1979), ASHS Distinguished Graduate Teaching Award, Norman Jay Coleman Award of the American Association of Nurserymen (1984), University of Hawaii Board of Regents Medal for Excellence in Research (1978), and the College of Tropical Agriculture and Human Resources Outstanding Alumnus

DEDICATION: HARUYUKI KAMEMOTO

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Award (1995). Societies have been profuse in recognizing his accomplishments, and he is a Fellow of the Orchid Society of Southeast Asia, Gold Medalist of Achievement from the Malayan Orchid Society and the American Orchid Society. Life Memberships in the Japan Orchid Society, American Orchid Society, and Orchid Society of Thailand, and the American Anthurium Society have been bestowed upon him. Breeding a new cultivar has never been the end product in itself. Kammy believed that you had to learn how to grow it, write about it, promote it, and use it to advance to the next level. He has authored over 220 papers in books, journals, newsletters, and, with Dr. Heidi Kuehnle, his successor, has co-authored Breeding Anthuriums in Hawaii (University of Hawaii Press, 1996). They have recently completed a second book, Breeding Dendrobiums in Hawaii, which highlights his unique breeding technique using amphidiploids to create bi- and tri-genomic combinations that can be readily reproduced from seed and features the many colorful and unique varieties that have been developed at the University of Hawaii, including the beautiful Dendrobium Ethel Kamemoto 'Splendor', named in honor of Kammy's wife. Kammy's life has been one of devotion to his profession, his students, his university, and his community. We proudly dedicate this volume of Horticultural Reviews to Haruyuki Kamemoto to honor a lifetime of accomplishments. Richard A. Criley Department of Horticulture University of Hawaii

1 Bioreactor Technology for Plant Micropropagation Meira Ziv Agricultural Botany and the Warburg Center for Biotechnology in Agriculture The Hebrew University of Jerusalem Rehovot 76100, Israel

I. Introduction II. Plant Developmental Pathways in Bioreactors A. Somatic Embryogenesis B. Organogenic Pathway C. Bud or Meristem Clusters D. Anomalous Plant Morphogenesis III. Plant Cell and Tissue Growth in Bioreactors IV. Physical and Chemical Factors in Liquid Cultures A. The Gaseous Atmosphere 1. Oxygen Level 2. CO 2 Effects 3. Ethylene B. Mineral Nutrients Consumption C. Carbohydrate Supply and Utilization D. pH Effects E. Growth Regulator Effects F. Temperature Effects V. Cell and Aggregate Density, Foaming and Medium Rheology in Bioreactors VI. Summary and Conclusions Literature Cited

Horticultural Reviews, Volume 24, Edited by Jules Janick ISBN 0-471<13374-3 © 2000 John Wiley & Sons, Inc. 1

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I. INTRODUCTION In vitro propagation is based on enhanced axillarybud proliferation and on the ability of differentiated, often mature plant cells, to redifferentiate, and develop new meristematic centers that are capable of regenerating fully normal plants. Regeneration is potentiated through two morphogenic pathways: organogenesis-the formation of unipolar organs, and somatic embryogenesis-the production of bipolar structures, somatic embryos with a root and a shoot meristem (Steward et al. 1970; Ammirato 1985). All plant somatic cells once isolated and cultured in vitro are capable of expressing totipotency. The injured cells in the outer layers of the isolated explant evolve ethylene that induces the inner layers of cells to undergo dedifferentiation. After the loss of coordinated control, which is ensued by cell division, the formation of new gradients of endogenous phytohormones in the dedifferentiated cells enhance cell divisions in response to the various growth regulators added to the medium. Cell division can take place in an unorganized pattern with callus formation, or in an organized pattern with the formation of meristematic centers directly in the explant tissues. Redifferentiation in the callus tissue takes place when new physiological gradients are formed in the non-organized parenchymatous tissue along with the formation of meristemoids (in vitro meristems), which can further differentiate into organized structures. In several plant species, meristematic centers form directly on the explant with very little or no callus formation and can develop into either shoots or somatic embryos. Plant regeneration can follow after the initial stages of controlled redifferentiation through either the organogenic or embryogenic pathways (Ammirato 1983, 1985; Ziv 1999). Plant regeneration in vitro is dependent on the manipulation of the inorganic and organic constituents in the medium, as well as the type of explant and the species. In most plants, successful regeneration from the callus or directly from the explants takes place after a series of subcultures in various media, in a sequence which is often specific to the species, variety, or the newly introduced genotype. The determining factors are the combination of the concentration in relation to mediumvolume and the composition of growth promoting and retarding regulators in the medium, the physiological status and competence of the cells and their capability for morphogenetic expression (Christianson 1985, 1987).

Micropropagation (in vitro propagation of axillary and/or adventitious buds as well as somatic embryos) is presently used as an advanced biotechnological system for the production of identical pathogen-free

1. BIOREACTOR TECHNOLOGY FOR PLANT MICROPROPAGATION

3

plants for agriculture and forestry (George 1996). The technique, however, is still costly due to intensive hand manipulation of the various culture phases and is not used commercially for all plant species. In addition, in some plants, the initial stage of establishment and response is slow and the survival of the plants in the final stage ex vitro is often poor, which further reduces the micropropagation production potential (Ziv 1995a; Aitken-Christie et al. 1995). Efficient commercial micropropagation depends on rapid and extensive proliferation along with the use of large-scale cultures for the multiplication phase. Furthermore, normal plant development during the acclimatization and hardening stage is mandatory to ensure a high percent of survival after transplanting to the greenhouse (Preece and Sutter 1991; Ziv 1991a,b, 1995b). Mechanization and automation of the micropropagation process can greatly contribute to overcoming the limitation imposed by existing conventional labor-intensive methods. Considerable attention has been directed toward automation of the repeated cutting, separation, subculture, and transfer of buds, shoots, or plantlets during the multiplication and transplanting phases (Levin et al. 1988; Aitken-Christie 1991, Aitken-Christie et al. 1995; Vasil 1994). Progress in tissue culture automation will depend on the use of liquid cultures in bioreactors, which will allow fast proliferation, mechanized cutting, separation, and automated dispensing (Alper et al. 1993; Sakamoto et al. 1995). These techniques were reported for some plants and were shown to reduce hand manipulation and thus reduce in vitro plant production costs (Levin et al. 1988; Ziv 1990b, 1992a,b, 1995b; Ziv and Hadar 1991; Vasil 1994; Aitken-Christie et al. 1995). The various propagation aspects of several plant species in bioreactors and some of the problems associated with the operation of bioreactors were recently reviewed by Takayama and Akita (1998). One major problem addressed was microbial contamination as affected by both the introduced plant material and the operation procedures of large-scale bioreactors. Liquid media have been used for plant cells, somatic embryos, and organ cultures in both agitated flasks or various types of bioreactors (Smart and Fowler 1984; Ammirato and Styer 1985; Stuart et al. 1987; Chen et al. 1987; Preil1991; Paque et al. 1992; Scragg 1992; Attree et al. 1994; Ziv 1995b; Archambault et al. 1995; Tautorus and Dunstan 1995). Although the use of bioreactors has been directed mainly for cell suspension cultures and secondary metabolites production, research directed at improving bioreactors for somatic embryogenesis has been reported for several plant species (Styer 1985; Preil et al. 1988; Nadel et al. 1990; Scragg 1990, 1992; Hale et al. 1992; Archambault et al. 1994; Tautorus et al. 1994; Attree et al. 1994; Ziv et al. 1994; Takayama and Akita 1998;

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Hvoslef-Eide and Munster 1998). The cultivation in liquid media using a temporary immersion system with different frequencies of immersion was reported to improve plant quality and multiplication rates of banana, coffee, and rubber (Alvard et al. 1993; Teisson and Alvard 1995; Etienne et al. 1997). Bioreactors were used also for the cultivation of hairy roots mainly as a system for secondary metabolite production (Rodriguez-Mendiola et al. 1991; Flores 1995; Weathers et al. 1989). The use of a simplified acoustic window mist bioreactor for transformed roots and for carnation shoots was reported to improve biomass growth (Chatterjee et al. 1997). Information on the use of bioreactors as a system for plant propagation (Table 1.1) through the organogenic pathway, although limited to a small number of plant species, is presently being applied to several ornamental and some vegetable and fruit crop plants (Takayama et al. 1991; Takahashi et al. 1992; Ziv 1992a,b; Akita and Takayama 1994; Ilan et al. 1995; Ziv and Shemesh 1996; Ziv et al. 1998). However, liquid cultures confer several problems associated with abnormal plant development, a phenomenon described as hyperhydricity, previously termed "vitrification" (Debergh et al. 1992), which causes poor plant development in vitro and later in ex vitro. Attempts were carried out to overcome hyperhydricity in liquid cultures by the use of growth retardants, inhibitors of gibberellin biosynthesis, which decreased hyperhydricity, reduced shoot elongation, and induced bud or meristem cluster formation (Ziv 1990a,b; Ziv and Ariel 1991; Ziv 1992b; Ziv and Shemesh 1996; Ziv et al. 1998). The clusters were shown to be an alternative propagation system for bioreactor cultures, providing a biomass with limited leaf elongation. Recently micropropagation of pineapple was scaled up using clusters in a temporary immersion system (ebb and flow) in bioreactors (C. G. Borroto 1998, pers. commun.). Bioreactors are presently being used for commercial micropropagation in the USA, Japan, Taiwan, Korea, Cuba, Costa Rica, Holland, Spain, Belgium, and France for ornamental and bulbous plants, pineapple, potato, and forest trees. The cost per propagule unit of foliage plants was estimated to be reduced from 17 to 6-7 cents (US) (R. Levin, pers. commun.). A recent review by Takayama and Akita (1998) describes several bioreactor techniques using plant propagules for large-scale propagation of potato, gladiolus, lilies, strawberry, Hyacinth, Amaryllis, and several Araceae. The present paper reviews various aspects of large-scale liquid culture in bioreactors for plant propagation, emphasizing the physical and chemical growth factors that control development and proliferation in liquid media for efficient plant production.

1. BIOREACTOR TECHNOLOGY FOR PLANT MICROPROPAGATION

Table 1.1.

5

Plants propagated in bioreactor cultures.

Species

Response

Amaryllis hippeastrum Ananas comosus Apium graveolens Araceae species Brodiaea complex Coftea arabica

Buds, plants, bulblets Shoot clusters Somatic embryos Shoots, plants Bud clusters, corms Shoot clusters, plants

Cyclamen persicum Daucus carota

Callus, somatic embryos Callus, somatic embryo

Dianthus caryophyllus Eschcholtzia cahfornica Euphorbia pulcherrima Fragaria ananasa Gladiolus grandiflorum

Shoots, plants Somatic embryos Somatic embryos Shoots, plants Bud clusters, plants, corms Shoots, plants, corms Buds, plants

Hevea brasiliensis Hyacinthus orientalis Lilium spp. Medicago sativa

Bulblets, plants Plants, bulblets Bulblets, plants Callus, somatic embryos

Musa spp.

Buds, plants

Nephrolepis exaltata

Buds clusters, plants Buds, plants

Nerine sarniensis Ornithogalum dubium Populus tremula Picea glauca Picea glaucaengelmannii Picea marianna Solanum tuberosum

Proembryogenic clusters, somatic embryos, bulblets Shoot clusters, plants, bulblets Bud clusters, shoots, plants Somatic embryos Somatic embryos Somatic embryos Plants, tubers Bud clusters, plants, tubers Shoots, plants, tubers

References Takayama and Akita 1998 Escalona et al. 1999 Nadel et al. 1990 Takayama and Akita 1998 Ilan et al. 1995 Alvard et al. 1993; Teisson and Alvard 1995 Hvoslef-Eide and Munster 1998 Jay et al. 1994; Archambault et al. 1995 Chatterjee et al. 1997 Archambault et al. 1994 Preil1991; Luttman et al. 1994 Takayama and Akita 1998 Ziv 1990; Ziv et al. 1998 Takayama and Akita 1998 Alvard and Teisson 1993; Teisson and Alvard 1995 Takayama and Akita 1998 Takayama 1991 Takayama and Akita 1998 Stuart et al. 1985, 1987; Chen et al. 1987; Stuart et al. 1987; McDonald and Jackman 1989; Denchev et al. 1992 Alvard and Teisson 1993; Teisson and Alvard 1995 Ziv et al. 1998 Levin et al. 1988; Ziv and Hadar 1991; Ziv et al. 1998 Lilien-Kipnis et al. 1994 Ziv et al. 1994 Ziv and Lilien-Kipnis 1997 McCown et al. 1988; Carmi et al. 1997 Attree et al. 1994 Tautorus et al. 1994 Tautorus et al. 1994 Akita and Takayama 1994 Levin et al. 1997a; Ziv et al. 1998; Ziv and Shemesh 1996 Takayama and Akita 1998

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II. PLANT DEVELOPMENTAL PATHWAYS IN BIOREACTORS

A. Somatic Embryogenesis The use of liquid cultures for the cultivation of somatic cells in recapitulating embryogeny was reported by Steward et al. and Reinert as early as 1958 in carrot (Daucus caTTata L.). The underlying concept for cell totipotency and the ability to renew growth and express morphogenesis was based on the assumption that isolation and bathing of the cells in a specific medium will simulate the zygotic embryo conditions in the ovary. The bathing liquid medium that contains various nutrients and growth regulators was assumed to be a source for similar stimuli that were present in the zygotic embryos' immediate environment. In addition, the segregation of embryogenic from non-embryogenic cells amplified the expression of cell totipotency (Steward et al. 1970). Somatic embryogenesis was achieved by the initial use of an induction medium containing an auxin, usually 2,4-dichlorophenoxyacetic acid (2,4-D), and coconut water; the latter was mainly a source of cytokinins, inositol, and reduced nitrogen compounds. Once the pro-embryonic clusters formed, the removal or lowering of the auxin concentration initiated a sequence of events similar to zygotic embryogeny (Halperin 1967; Ammirato 1985). Somatic embryogenesis was observed at first mainly in members of the Umbelliferae cultured in liquid media (Steward et al. 1970) and has since been reported in gymnosperms and angiosperms, including several ornamentals, and vegetable and field crops, as well as perennial woody plants. The embryogenic pathway, as opposed to the organogenic pathway, can be a more efficient and productive system for large-scale clonal propagation. Somatic embryogenesis may result in less variation through chimerism. However, somaclonal variation will be less likely to take place in direct regeneration, but might be more pronounced in somatic embryos developing from continuously cultured callus tissue. Since the embryo contains both a root and an apical shoot meristem, the rooting stage required in conventional in vitro bud or shoot propagation technology is obviated. Somatic embryos are small and can be adequately handled in scaled-up procedures. They are amenable to sorting and separation by image analysis, dispensing by automated systems, and can be encapsulated and either stored or planted directly, with the aid ofmechanized systems (Ammirato and Styer 1985; Cazzulino et al. 1991; Cervelli and Senaratna 1995; Sakamoto et al. 1995). Somatic embryogenesis in liquid shake or bioreactor cultures was reported in carrot (Ammirato and Styer 1985; Jay et al. 1992, 1994; Archambault et al. 1995), in caraway (Ammirato 1983), poinsettia (Preil1991),

1. BIOREACTOR TECHNOLOGY FOR PLANT MICROPROPAGATION

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alfalfa (Stuart et al. 1985,1987; Denchev et al. 1992; Kuklin et al. 1994), celery (Nadel et al. 1990; Saranga and Janick 1991), in Eseheholtzia ealiforniea (Archambault et al. 1994), in Nerine (Ziv et al. 1994), in Detea eatharinensis (Moura Costa 1992), in sweet potato (Harrell et al. 1994), in rubber (Etienne et al. 1997), and in spruce (Tautorus and Dunstan 1995). However, the ultimate goal of production of synthetic seeds through successful encapsulation of somatic embryos from liquid shake or bioreactor cultures was reported only for carrot (Kitto and Janick 1985; Redenbaugh et al. 1991; Sakamoto et al. 1995), alfalfa (Senaratna 1992; Fujii et al. 1992), celery (Kim and Janick 1989; Onishi et al. 1992, 1994), and white spruce (Attree et al. 1994) and is currently under continuous investigation for several other species. B. Organogenic Pathway The propagation of most plants is presently carried out commercially through the organogenic pathway in agar-gelled cultures, even though the protocols are long and costly. The advantages for the mechanization of the process are mainly for achieving a reduction in hand manipulation and labor costs. The process has to be scaled up using liquid cultures in bioreactors to amend it to automation (Levin et al. 1988; Kurata 1995). The information on the use ofbioreactors for unipolar structures such as protocorms, buds, or shoots is limited, mainly because of the problems of hyperhydricity of the leaves and shoots (Debergh et al. 1992). One of the first attempts in using liquid cultures for micropropagation of buds was reported for orchids that formed protocorms with minimal shoot elongation during the liquid culture stage (Morel 1974). The protocorms could be separated and induced to form new plants after subculture to agar-gelled medium. Several other ornamental plants also have been propagated through the organogenic pathway in liquid shake cultures and bioreactors. By controlling shoot growth and providing culture conditions that reduced abnormal leaf growth and enhanced the formation of bud or meristematic clusters, as was shown for potato, gladiolus, and Drnithogalum dubium (Fig. 1.1A, B, C), a high proliferation rate was achieved without the phenomenon of hyperhydricity (Levin et al. 1988; Ziv 1990a, 1991a, 1992a; Ziv and Hadar 1991; Ziv and Ariel 1991; Takayama 1991; Takayama et al. 1991; Takayama and Akita 1994; Han et al. 1995; Ziv et al. 1998). C. Bud or Meristem Clusters The development of spherical meristematic or bud clusters in liquid cultures was shown to provide a highly proliferative and rapid growing

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Fig. 1.1. Liquid cultured bud clusters of potato (A), meristematic clusters of gladiolus (B), and bulblet formation in Ornithogalum dubium bud clusters (C).

1. BIOREACTOR TECHNOLOGY FOR PLANT MICROPROPAGATION

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system amenable to automated inoculation, control of the medium components' mechanical separation, and efficient delivery to the final stage for plant growth and development (Levin et al. 1988; Ziv 1991b; Ziv et al. 1998). Cluster formation appears to be associated in most species with the continuous submergence, circulation, and agitation of the plant biomass in the medium, as well as with a balanced ratio of growth-promoting and growth-retarding regulators. Clusters can form from axillary buds and/or adventitious buds as well as from meristemoids in pro-embryogenic callus that later differentiate to somatic embryos (Fig. lolA, lo2A, B).

Fig. 1.2. Nerine proembryogenic clusters in a 2,4-D medium (A) and after 2,4-D removal with the addition of ZiP showing somatic embryos (B).

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The formation of condensed organized structures in which the shoots are reduced to buds or meristematic tissue was reported in several plant species. The clusters were made up of densely packed meristematic cells, actively dividing and forming new meristematic centers on the outer surface. The meristemoids surrounded loosely packed cells in the center and exhibited some vascularization, as was shown in liquidcultured poplar clusters (McCown et al. 1988). In banana, on the other hand, the clusters were made up of condensed buds surrounding a central core with a cavity (Ziv et al. 1998). Halperin in 1967 described the formation of pro-embryogenic masses in carrot root explants which were induced by the addition of 2,4-D to the liquid medium, resembling orchids protocorm-like bodies that formed when isolated buds were cultured in liquid medium in shake cultures (Morel 1974). Protocorm-like clusters also were induced in liquid-cultured gladiolus buds (Ziv 1989, 1990a) and in several species of the complex Brodiaea (Han et al. 1995) by the addition of paclobutrazol or ancymidol (gibberellin biosynthesis inhibitors) to the medium. In chicory, nodules were induced on leaves by IBA and BA in the medium (Pieron et al. 1992). In potato and banana, bud clusters were induced by a balanced ratio between kinetin and ancymidol in the liquid medium (Ziv et al. 1998). In woody species, McCown et al. (1988) and Aitken-Christie et al. (1988) described nodules in poplar in liquid medium and in radiata-pine in agar-gelled medium respectively, induced by the use of a balanced ratio of growth regulators. Levin et al. (1988), working with several ornamental species, described a severalfold increase of an organogenic biomass of clusters which proliferated in bioreactors and were separated mechanically prior to dispensing to agar-gelled cultures for further growth. The production of clusters in Philodendron cultured in liquid medium required the presence of benzylaminopurine (BA) and an inductive treatment for 24-48 h with ancymidol. The inductive treatment prevented a carry-over dwarfing effect of ancymidol on leaf and shoot development after transplanting the clusters to agar-gelled medium for further plant growth (Ziv and Ariel 1991). Gladiolus clusters cultured in a disposable 2-L bioreactor proliferated and made up to 60% of the vessel volume after 4-5 weeks. The aggregates which reached 0.5-1.5 em in diameter tended to sink to the bottom of the bioreactor as the biomass increased and could not be resuspended unless the rate of aeration was increased. An increase from 0.5 to 1.5 vvm (volume air/volume medium per minute) provided the required aeration for recirculation of the cluster biomass (Ziv et al. 1998). Potato and banana bud clusters proliferated in bubble and dis-

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posable plastic bioreactors at a faster rate than on agar-solidified medium (Levin et al. 1997b) Growth regulators, when given in a specific balanced ratio of promoting and retarding substances, apparently act as morphogenic signals and control the development of the spherical meristematic or bud clusters. The spherical meristematic or bud clusters were described by various terms: pro-embryogenic mass (PEM) in carrot (Halperin 1967), protocorms in orchids (Morel 1974), nodules in poplar (McCown et al. 1988) and radiata-pine (Aitken-Christie et al. 1988; Carmi et al. 1997), nubbins in daylilly (Krikorian and Kann 1981), and meristematic or bud clusters in gladiolus, Brodiaea, Nerine sarniensis, fern, Philodendron, potato, banana, and Ornithogalum dubium (Ziv 1989; Ziv and Hadar 1991; Ziv and Ariel 1991; Ziv et al. 1994; Han et al. 1995; Ziv and Shemesh 1996; Ziv et al. 1998; Ziv and Lilien-Kipnis 1997). It is suggested that the term cluster should be used, as it is more appropriate for describing these proliferative, usually rounded structures, which develop from buds or other proliferative tissue in liquid culture, through either the organogenic or somatic embryogenesis pathways.

D. Anomalous Plant Morphogenesis Scaling-up in bioreactors requires the use of liquid instead of agar-gelled media during the proliferation and biomass production stages. The culture of plants in liquid medium is known to cause anomalous morphogenesis, resulting in plant hyperhydricity (Debergh et al. 1992). The plants that develop in liquid media are fragile, have a glassy appearance, with succulent leaves or shoots and a poor root system (Paque and Boxus 1987; Werker and Leshem 1987; Ziv 1991a,c, 1995a). The leaves are the organs affected most severely in liquid cultures. They develop an unorganized mesophyll tissue that is made up mainly of spongy parenchyma tissue with large intercellular spaces (Werker and Leshem 1987; Gaspar et al. 1987), a deformed vascular tissue, and an abnormal epidermis. The epidermal tissue in hyperhydric leaves lacks a welldeveloped cuticle and possesses malfunctioning guard cells which cannot respond to closure signals (Sutter 1985; Ziv et al. 1987; Ziv 1991a; Ziv 1995a). Hyperhydricity affects plant survival after transplanting and causes loss of the in vitro developed leaves, or even whole plants, which often wilt and die. The two major processes carried out by the leaves, photosynthesis and transpiration, are not fully functional in hyperhydric leaves and thus cause the poor performance of the transplanted plants ex vitro (Preece and Sutter 1991; Ziv 1991c, 1995a; Ziv and Ariel 1994).

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In many plants propagated in vitro, the anomalous morphology and anatomical aberration often observed in plants result from deviation from the normal course of morphogenic events that are manifested in plants in vivo (Ammirato 1985; Ziv 1999). Some of the deformation, such as hyperhydric, malformed leaves and shoots, abnormal embryos, recurrent embryogenesis, and several other disorders (Ziv 1999), are apparently the result of interruption or faulty timing of the signals involved in the normal sequence of organizational events known to exist in vivo. These are problems that are manifested more severely in liquid medium and await further studies in order to understand and to help control plant morphogenic events in bioreactor cultures.

III. PLANT CELL AND TISSUE GROWTH IN BIOREACTORS The advantages provided by aerated liquid cultures in bioreactors include better contact between the plant biomass and the medium, no restrictions of gas exchange, the control of the composition of both the medium and the gaseous atmosphere, and the ability to manipulate the plant biomass in relation to the medium volume (Cazzulino et al. 1991; Heyerdahl et al. 1995; Leathers et al. 1995). The need for efficient circulation and mixing of the plant biomass, especially for cluster and embryogenic tissue, is essential to prevent sedimentation and allow optimal growth (Scragg 1992; Doran 1993). Various types of bioreactors with mechanical or gas-sparged mixing were used for plant cell cultures, to provide stirring, circulation, and aeration (Margaritis and Wallace 1984; Takayama 1991; Scragg 1992; Doran 1993; Takayama and Akita 1994). Mechanically stirred bioreactors depend on impellers, including a helical ribbon impeller (Archambault et al. 1994), magnetic stirrers, or vibrating perforated plates (Styer 1985; Preil1991; Cazzulino et al. 1991). Aeration, mixing, and circulation in bubble column or airlift bioreactors is provided by air entering the vessel from a side or bottom opening through a sparger; as the air bubbles rise, they lift the plant biomass and provide the oxygen gas required (Merchuk 1990). Mechanically stirred bioreactors are used for effective mixing, aeration, dispersion of air bubbles, and prevention of large cell aggregates formation in cell suspension cultures (Denchev et al. 1992; Scragg 1992; Doran 1993). In recent years, large-scale cultivation of plant cells, embryos, or organs has made use of airlift or bubble column bioreactors and, to a lesser extent, of mechanically stirred tank bioreactors, due to the former

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lower shearing force properties. Plant cells are relatively large in size, have sensitive cell walls which are highly susceptible to shearing forces, and are easily damaged. Furthermore, since plant cells, unlike microbial cultures, do not require high 0z, the use of bubble column (Fig. 1.3A) or airlift bioreactors was found to be adequate and advantageous for plant tissue cultured in liquid media (Margaritis and Wallace 1984; Smart and Fowler 1984; Merchuk 1990). In addition, it was shown that mixing by gas sparging in bubble column or airlift bioreactors lacking impellers or blades is far less damaging for clusters than mechanical stirring, since they were shown to have a lower shearing stress (Ziv and Hadar 1991; Han et al. 1995; Ziv and Shemesh 1996). The main advantage of airlift bioreactors is their relatively simple construction, the lack of regions of high shear, reasonably high mass and heat transfer, and relatively high yields at low input rates (Kawase 1989; Denchev et al. 1992). A bubble free oxygen supply bioreactor with silicone tubing was found suitable for embryogenic cell suspensions and provided foam-free cultures (Luttman et al. 1994). For hairy root culture, an acoustic mist bioreactor was found to increase root biomass significantly (Chatterjee et al. 1997).

Fig. 1.3. Glass bubble column bioreactors with gladiolus clusters (A) and a plastic disposable bioreactor with £ilium clusters (B)

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For efficient large-scale cultures of both somatic embryos and organogenic plant tissue, the bioreactor configuration and volume must be determined according to the mixing and aeration requirements of the specific plant or tissue propagated, as well as for minimizing the intensity of the shear stress (Doran 1993; Hvoslef-Eide and Munster 1998). One major problem encountered in large-scale liquid cultures is contamination. Fungi, bacteria, yeast, and insects are a source of serious contaminations, causing heavy losses of plant material in commercial laboratories. In scaled-up liquid cultures, the losses are even greater, as the source of contamination due to manipulation of the bioreactor apparatus is dependent on the various stages of preparing and maintaining the equipment. In several laboratories, the operation area is kept sterile by a positive pressure airflow, which decreases contamination risks. In addition, constant screening of the plant tissue for contaminants and continuous indexing is another safeguard (Cassells 1991). IV. PHYSICAL AND CHEMICAL FACTORS IN LIQUID CULTURES In order to control plant morphogenesis and biomass growth in bioreactors, various culture conditions must be manipulated, i.e., the gaseous atmosphere, oxygen supply and CO z exchange, pH, minerals, carbohydrates, growth regulators, and the liquid medium rheology and cell density (Heyerdahl et al. 1995). A. The Gaseous Atmosphere The atmosphere of the culture vessel is made up mainly of nitrogen (78%), oxygen (21 %), and carbon dioxide (0.036%). The culture vessel gas composition is influenced by the volume of the vessel and the extent of ventilation. Plants evolve CO z and consume Oz during respiration, while during photosynthesis CO z is used and Oz is produced. During the dark period, CO z levels were found to increase in cultures, and if photoautotrophic conditions prevail, its level decreased in the light (Fujiwara et al. 1987). Ethylene, ethanol, acetaldehyde, and other hydrocarbons are additional components of the gaseous atmosphere in vitro. Most of the effects of CO z, 0z, and CZH 4 on plant growth in vitro were reported for agar-gelled or cell suspension cultures (Buddendorf-Joosten and Woltering 1994). In bioreactors, the control of the gaseous phase depends on the gas flow and can be easily manipulated to provide the required levels of 0z, CO z, and CZH 4 • In airlift or bubble column

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bioreactors, the air supplied is used for both mixing and aeration (Scragg 1992; Doran 1993). The importance of aeration and the gaseous phase were shown in potato cultured in airlift bioreactors. Induction of tubers was inhibited under continuously submerged conditions. Microtubers developed only after the shoots elongated and reached the gaseous phase at the top of the bioreactor. Enrichment with O 2 and manipulation of the hormonal and osmotic conditions had no effect and could not explain the phenomenon. A two-phase culture substituting the growth medium with a tuber induction with 9% sucrose in the medium enhanced tuber formation from shoots that developed above the medium and were exposed to the gaseous phase (Akita and Takayama 1994).These results emphasize the importance of the gaseous phase in the bioreactors for specific developmental phases. 1. Oxygen Level. Oxygen levels in liquid cultures depend on the pres-

ence of O 2 in the gas phase above and in the air bubbles inside the medium, as well as in the dissolved O 2 in the medium. Air is released through a sparger located at the base of the bioreactor. The available oxygen for plant cells in liquid cultures determined by oxygen transfer coefficient (kLa values) is the part that dissolves in water. Its depletion as a function of the metabolic activity of the growing cell biomass can affect the culture yield. Plant cells have a lower metabolic rate than microbial cells and a slow doubling time and therefore require a lower O 2 supply. In general, high aeration rates appear to reduce the biomass growth (Cazzulino et al. 1991). The requirements for O 2 may vary from one species to another, and must be supplied continuously to provide adequate aeration, since it affects metabolic activity and energy supply as well as anaerobic conditions. The level of O 2 in liquid cultures in bioreactors can be regulated by agitation or stirring and through aeration, gas flow, and air bubble size. The use of a porous irrigation tube as a sparger generated fine bubbles, high kLa values, low mechanical stress and provided a high growth rate (Takayama and Akita 1998). Growth of poinsettia cell suspension in bioreactors was inhibited when the level of O 2 dropped below 10%. When the level of0 2 was elevated to 80%, cell number increased to 4.9 x 10 5 /ml as compared to 3.1 x 10 5 /ml at 40% O 2 (Preil et al. 1988). Somatic embryo development in alfalfa and poinsettia suspension cultures was enhanced at 78% and 60% O 2 levels, respectively (Stuart et al. 1987; Preil 1991). High aeration rates were found to inhibit cell growth in cell suspensions cultured in airlift bioreactors. This result was explained to be due to an effect of" stripping" of the volatiles produced by the plant cells, which are apparently necessary for cell growth (Smart

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and Fowler 1984). Increasing O 2 levels from 21 % to 80%, in bioreactor cultures of Boston fern clusters, enhanced growth values (final FW initial FW/initial FW) from 0.61 to 0.92. Reducing O 2 levels to 10% (v/v) affected cell differentiation in bioreactor cultures of carrot embryogenic tissue. Under these conditions embryo production was severely inhibited (Jay et al. 1992). The best production of somatic embryos from embryogenic cultures of Eschcholtzia californica and carrot in a helical ribbon impeller bioreactor was achieved under 20% oxygen concentration. When O 2 was low (5-10%), it inhibited biomass and somatic embryo production, while high O 2 (60%) favored undifferentiated biomass production (Archambault et al. 1994,1995). Additional studies are required to provide information on optimal dissolved O 2 requirements in large-scale liquid cultures. 2. CO 2 Effects. The effects of CO 2 were reported mainly for plants in agargelled cultures or for cell suspension cultures used for secondary metabolite production (Scragg 1992; Buddendorf-Joosten and Woltering 1994). The reports on the effects of CO 2 enrichment in sugar-free agargelled cultures suggest beneficial promotion of plant growth during plant acclimatization and transplanting ex vivo (Kozai et al. 1992). The contribution of CO 2 supply during the proliferation and multiplication stage in media supplied with sucrose in bioreactors is debatable. It is implicit that if photoautotrophic conditions do not prevail, CO 2 enrichment beyond the 0.36% in the air supply is unnecessary. There are reports that high aeration rates rather than excessive oxygen inhibit growth and that reduced growth could be due to depletion of CO 2 or to the removal of various culture volatiles including CO 2 (Hegarty et al. 1986; Kim et al. 1991). The requirement for CO 2 was not related to photosynthesis but to some other metabolic pathways involved in amino acid biosynthesis. In poinsettia, the growth of embryos in Erlenmeyer flasks was considerably higher than in bioreactors and it was suggested that the growth difference was related to the differences in the gaseous atmosphere in the headspace (Preil 1991). CO 2 enrichment in an illuminated bioreactor culture of Brodiaea clusters did not affect biomass growth. An increase from 0.3% to 1 % gave a similar growth value under the two CO 2 levels and supply of 135 l.lmols . m-2 • S-l photosynthetic photon-flux (Han et al. 1995). In Cyclamen persicum Mill, high CO 2 levels correlated with increased production of pro-embryogenic masses (Hvoslef-Eide and Munster 1998). 3. Ethylene. Ethylene level in the headspace in liquid cultures in flasks

differs from that in continuously aerated bioreactor cultures. Most of the

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reports on ethylene relate to agar-gelled or cell suspension cultures (Buddendorf-Joosten and Woltering 1994). In various plant species cultured in bioreactors, the rate of aeration will affect the level of ethylene. High rates of aeration, which are often required at high biomass densities, can cause "stripping" of volatiles that are apparently important for some plants grown in culture. In clusters of Brodiaea cultured in liquid medium, ethylene had no effect on growth, although its level was reduced in the presence of silver thiosulfate (an inhibitor of ethylene action). The level of ethylene was reduced from 0.38 to 0.12 mIlL in highly aerated bioreactor cultures without affecting biomass growth, which was similar under both levels (Han et al. 1995). B. Mineral Nutrients Consumption Revised MS medium (Murashige and Skoog 1962) or media with various partial modifications in the inorganic and organic constituents of MS are used for most plant species in agar-gelled or liquid cultures in vitro. The availability of mineral nutrients depends on the type of culture, whether agar-gelled or liquid, the type and size of the plant biomass, and the physical properties of the culture. Factors such as pH, temperature, light, aeration, the concentration of minerals, the medium volume, and the viscosity of the medium will determine the rate of absorption of the various nutritional constituents (Williams 1992; Debergh et al. 1994). Plant cells growing in liquid cultures are better exposed to the medium components and the uptake and consumption are faster. Agar-gelled and liquid shaken cultures often dehydrate by water evaporation to the headspace and out of the vessel and such a water loss concentrates the medium. The content of a certain component in the medium is a product of its concentration and the medium volume. The effects of the medium volume and the initial strength on potato were discussed by Kozai et al. (1995). In bioreactors, in which either humidified air or condensers are used to prevent dehydration, the level of the nutrients in the medium is affected mainly by the absorption rate and by cell lysis (Archambault et al. 1994). Differentiation and proliferation of micropropagated fern, gladiolus, and Nerine nodular clusters in bioreactors was better on half-strength than on full-strength MS minerals (M. Ziv, unpubl.). This was also true for Lilium bulblets differentiating on bulb scales that were cultured in bioreactors (Takayama 1991). A drop in pH to 4.5 and lower values and the subsequent increase to pH 5.5 was attributed to the initial utilization of ammonium and to the uptake at a later stage of nitrate. In several species the depletion of NH: is the first limiting factor of biomass growth and somatic embryos development.

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Increasing the concentration of NH: to 15 mM resulted in maximum somatic embryo production in carrot cultures in a helical ribbon impeller bioreactor (Archambault et al. 1995). A thorough and detailed study of nutrient uptake in Eschcholtzia californica embryonic cultures was achieved in a helical ribbon impeller bioreactor. Following a lag phase of about 140 h, first NH~ and then NO;, PO~, K+ and SO~ uptake was observed to coincide with biomass increase and somatic embryo production. After 300 h, PO~ was exhausted, while after 600 h NH~ was depleted and Mg++ and Ca++ composition continued to deplete after about 800 h. The K+ and Na+ ions were not depleted at that stage (Archambault et al. 1994). The composition of minerals, monitored during the culture of Begonia and Brodiaea in liquid media changed, with phosphate, ammonia, nitrate, and potassium depleting faster and prior to the depletion of Ca++ and Mg++ (Tormala et al. 1987; Han et al. 1995). In somatic embryos of spruce cultured in bioreactors, 80% of the ammonium was consumed by the growing biomass (Tautorus et al. 1994). In general, biomass growth is limited by the availability of phosphate, nitrogen, and carbohydrates and to a lesser extent by the availability of calcium, magnesium, and other ions. C. Carbohydrate Supply and Utilization Cultured plants require a constant supply of carbohydrates as their source of energy. Sucrose and to a lesser extent glucose, fructose, or sorbitol are the most commonly used carbohydrates in vitro. In general, sucrose is removed rather rapidly from the medium and after 10-15 days the sucrose can be completely depleted or reduced to 5-10 giL from an initial level of 30 giL in both agar-gelled and liquid cultures. At the same time, glucose and fructose that appear in the medium due to sucrose hydrolysis increase in the presence of invertase in the culture medium, and can reach levels of 5-10 giL. Cell suspensions of Catharanthus roseus cultured in a column airlift bioreactor showed a lag phase of 5 days, during which there was a total hydrolysis of the sucrose to glucose and fructose (Smart and Fowler 1984). In suspension cultures of alfalfa, sucrose also was hydrolyzed during the first 5 days. Most of the sugars uptake occurs after day 5 and glucose is taken up preferentially over fructose (McDonald and Jackman 1989). A higher yield of alfalfa embryos was obtained when 30 giL maltose combined with NH! was used instead of 30 giL sucrose that was used in combination with various nitrogen sources (Stuart et al. 1987). In embryonic suspension cultures of celery, the addition of mannitol reduced cell lysis and enhanced somatic embryogenesis. When 40 giL mannitol was added, a

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higher number of embryos was produced and the frequency of singulated normal embryos was increased (Nadel et al. 1989). In embryogenic cultures of Eschcholtzia californica grown in a helical ribbon impeller bioreactor, sucrose uptake started after 100 h and the sucrose was depleted after 600-800 h in culture (Archambault et al. 1994). In Picea species cultured in shaken flasks in a medium with 30 mM glucose, the carbohydrates depleted after 10-14 days (Lulsdorf et al. 1993). In mechanically stirred bioreactors, a level of 60 mM sucrose resulted in the highest cell biomass and somatic embryo number. The effects of various carbohydrates on the growth of spruce somatic embryos revealed that the response was species dependent (Tautorus et al. 1994). The biomass of fern meristematic clusters in a bubble column bioreactor was increased with the increase in sucrose concentrations from 7.5 giL to 30 giL, while higher levels caused a decrease in cluster growth. Elevated sucrose concentrations caused a decrease also in the chlorophyll content of the clusters and leaves (Ziv and Hadar 1991). Increasing sucrose concentrations from 30 giL to 60 giL in bioreactor cultured gladiolus clusters decreased the biomass FW by more than 50%. When 30 giL or 60 giL were combined with paclobutrazol, a further decrease in FW was observed. On the other hand, 60 giL sucrose induced a higher DW increment, which could be attributed to an osmotic effect and the subsequent water status of the clusters. Gladiolus clusters cultured in the presence of growth retardants had a higher level of starch, 845 as compared to 585 mglg DW in the control (Ziv 1992b). A higher number of bulblets were produced in bioreactor-cultured bulb scales of Lilium at 30 giL than at higher sucrose levels. However, larger size microbulbs were produced at 90 giL than at 30 giL sucrose in the medium (Takayama 1991). D. pH Effects The initial pH in most plant cell cultures ranges between 5.5-5.9. Since most media are not buffered, changes during autoclaving and during the biomass growth in culture occurs. A rapid drop in pH to 4.0-4.5 took place within 24-48 h in cell suspension, organ, and embryogenic cultures (Stuart et al. 1987; Preil1991; Ziv and Hadar 1991; Lulsdorf et al. 1993; Jay et al. 1994). These changes were related to an initial ammonium uptake and acidification due to cell lysis. However, the pH increased after a few days and reached a stable level around pH 5.0-5.5, which was related to the uptake of nitrates. In spruce species cultured in liquid medium, the pH levels were shown to increase to 6.5-6.8 after 14 days in culture (Tautorus and Dunstan 1995).

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In embryogenic cultures of poinsettia, various initial pH levels stabilized after 20 days in batch bioreactor cultures, between pH 5.3-5.7. Oxygen levels affected pH changes by 0.2 pH units (Preil1991). In fern cultured in an airlift bubble column bioreactor, the pH dropped to 4.2 after 24 hand subsequently increased to pH 5.4. Keeping a constant pH by titration with KOH did not affect the growth response (Ziv and Hadar 1991). In alfalfa, the development of somatic embryos was affected by the pH. A higher rate of embryo production was observed at a constant pH of 5.5 than at a nonbuffered medium or at lower pH levels (Stuart et al. 1987). Carrot cell differentiation in a controlled bioreactor was affected by the pH; the highest rate of embryo production was observed at a pH of 4.3. However, embryo development was arrested before the embryos reached the torpedo stage and continued only at pH 5.8. The changes in carrot embryo development were associated with sugar uptake and ammonium depletion and can be attributed to enzyme and metabolic activity at an optimal pH (Jay et al. 1994). It appears that pH requirements are species and developmental stage dependent.

E. Growth Regulator Effects The use of growth regulators in liquid cultures can be more effective in controlling the proliferation and regeneration potential than in agargelled medium due to the direct contact of plant cells and aggregates with the medium. The limited information available on regulators seems to indicate that similar levels of growth regulators were used in both agar-gelled and liquid cultures even though availability appears to be better in liquid medium. Somatic embryogenesis in many species was first induced in an auxincontaining medium that promoted rapid cell division. Expression of the embryogenic potential was achieved in auxin-free media, or with low levels of auxin in the medium (Halperin 1967; Steward et al. 1970). Further embryo conversion and maturation into plants was promoted by the addition of abscisic acid (ABA), which was found to control abnormal embryo and plant growth (Ammirato and Styer 1985). Bulblets development from scales in Lilium cultured in bioreactors was higher in the presence of BA than kinetin. However, high BA had an inhibiting effect on further bulblet growth (Takayama et al. 1991). High kinetin also stimulated bulblets differentiation, but inhibited further growth of the bulblets. However, the addition of 0.1 mg/L naphthalenacetic acid (NAA) was found to further enhance kinetin activity, which was, however, inhibited again in the presence of high (90 giL) sucrose levels (Takayama 1991; Takayama et al. 1991).

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In embryogenic cultures of Nerine, auxin and cytokinins were used to induce proembryogenic clusters. Embryogenic expression was achieved, however, only after a short exposure to 2-iso-pentenyladenine (2iP) and further subculture in a growth regulator-free medium (Lilien-Kipnis et al. 1992, 1994). Since one of the major problems in liquid-cultured plants is malformation of shoots, hyperhydricity, the induction of meristematic or bud clusters with arrested leaf growth (McCown et al. 1988; Ziv 1991b; Ziv and Shemesh 1996) was one of the solutions to hyperhydricity. The use of relatively high cytokinin levels and growth retardants which inhibit gibberellin biosynthesis was the most effective method to reduce shoot and leaf growth and promote the formation of meristematic clusters (Ziv 1990a,b). The information on abscisic acid is limited and was found effectual mainly in the later stages of somatic embryo development, promoting normal embryo growth and maturation in carrot and alfalfa embryogenic cultures (Ammirato and Styer 1985; Denchev et al. 1990). Growth of callus and leaf development in bulblets regenerated on bulb scales of Lilium in bioreactor cultures was inhibited by abscisic acid, thus providing singulated propagules for easier handling and storage (Takayama et al. 1991). Ethylene effects appear to be species dependent (see IVA3). Information on the effects of growth regulators in bioreactor cultures is inadequate and further research is needed to optimize culture conditions. F. Temperature Effects The control of the temperature in the liquid medium inside the bioreactor can be easily manipulated by a heating element in the vessel or by circulating water in an enveloping jacket outside the vessel. There is, however, limited information on the effects of temperature in bioreactor cultures, which is usually kept constant at 25°C, with small day and night fluctuations. Temperature effects on potato tuber formation in an airlift bioreactor were studied by Akita and Takayama (1994). A higher number and larger-size tubers developed at 25°C than at 17°C; the lower temperature caused a decrease in tuber size. Ziv and Shemesh (1996), working with potato internode explants in liquid cultures, found that tuber formation was best at a 16 h photoperiod and 18/15°C day and night temperature. When bulblets of Nerine were cultured in liquid medium, the FW increase was higher at 25°C than at 17°C. However, when subcultured from liquid to an agar-gelled bulb induction medium, rooting was better at 17°C (J. Vishinevetzky, M. Ziv unpublished).

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V. CELL AND AGGREGATE DENSITY, FOAMING AND MEDIUM RHEOLOGY IN BIOREACTORS Growth and proliferation of the biomass in bioreactors depends on airflow supply for the aeration and mixing, and for the prevention of the plant biomass sedimentation. In many plants cultivated in bioreactors, continuous aeration, mixing, and circulation cause shearing damage, cell wall breakdown, and accumulation of cell debris, which is made up mainly of polysaccharides. Cell debris accumulation results in foaming, adhesion of cells and aggregates to the culture vessel walls, and the development of a "crust" at the upper part of the bioreactor vessel. This layer prevents adequate circulation, causing additional cell debris formation and a demand for higher aeration rates that intensify the clogging problem (Scragg 1992). As the biomass increases and the cultures become viscous, higher rates of aeration are required to allow for oxygen supply and circulation. Medium viscosity and foaming were reduced by the use of half the concentration of MS minerals (Ziv 1992a) and by lowering the level of calcium in the medium (Takayama et al. 1991). In bioreactor cultures in which the plant biomass used was of an aggregate or cluster nature, the foaming and viscosity were not as severe as that observed in cell suspension cultures (Ziv 1991b). Surprisingly, in cluster cultures in bioreactors, even when the mixing was poor and the clusters sedimented, biomass growth was observed, suggesting that perhaps the continuous circulation accompanied by aeration was secondary in importance to oxygen supply (Ziv 1995b). Shearing stress and cell wall damage were greatly reduced in a Vibramix bubble-free oxygenated bioreactor, in which silicone tubing was used for air supply (Preil1991; Luttman et al. 1994). Embryogenic cells cultured in a helical ribbon impeller bioreactor produced poor-quality embryos when the mixing speed was increased from 60 to 100 rpm. This was attributed to shearing stress and cell damage (Archambault et al. 1994). The introduction of polyethylene glycol 6000 changed the rheology of the medium in alfalfa liquid cultures and improved somatic embryo development beyond the globular stage while it was arrested in a less viscous medium (Denchev et al. 1990). Disposable plastic presterilized bioreactors (Osmotek 'LifeReactor' TM, Fig. 1.3B), with a volume capacity of2 and 5 L, were used for the micropropagation of various plant species through bud or meristematic clusters. The plastic bioreactors were found to provide good circulation with reduced shearing damage and foaming (Ziv et al. 1998). The problems of cell damage, foaming, and culture viscosity can be better controlled by

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developing bioreactors with an optimal shape suitable for micropropagation through meristematic or bud clusters. Clusters that are compact and consist mainly of small meristematic cells are apparently less shear sensitive than highly vacuolated cells in parenchymatous cell suspension cultures. VI. SUMMARY AND CONCLUSIONS

The application of liquid cultures for micropropagation in bioreactors using the organogenic or the embryogenic pathway is becoming a more efficient alternative system for scale-up and automation in vitro (AitkenChristie et al. 1995). The successful exploitation ofbioreactors as a commercial micropropagation system will depend on careful studies of plant morphogenesis in liquid media and the understanding of the control mechanisms of organ and embryo development from meristematic or bud clusters. The chemical and physical environment, in relation to biomass growth and controlled regeneration, should be further investigated. The level of carbohydrates and specifically the levels and ratios of growth-promoting and -retarding regulators will need to be further studied in more detail. A major aspect which will have to be addressed is the problem of contamination in large-scale liquid cultures, which can cause severe losses (Leifert and Waites 1992). Attempts to control contamination in liquid cultures of foliage plants were reported by Levin et al. (1997a), who used continuous filtration systems to control bacterial growth in the medium. Harvest and quality assessment of bioreactor propagules is a major aspect in automation and was addressed by computerized image analysis (machine vision) to classify organogenic and embryogenic plant material (Harell et al. 1994; Kurata 1995). These are factors of major importance to the success of scaled-up automated in vitro propagation. In addition, the understanding of the effects of aeration, mixing, consumption, and depletion of the various components present in the medium will provide information for establishing semi-continuous or continuous culture systems and thus provide optimal conditions for biomass growth, differentiation, and eventual production of quality plants. Implementation of scale-up and mechanization are mandatory for the expansion of commercial micropropagation. New technologies in robotics and machine vision systems for cutting, sorting, and dispensing have been established (Aitkin-Christie et al. 1995). However, these technologies have actually increased the cost of plant micropropagation.

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Bioreactor cultures are being established in several commercial laboratories for ferns, spathiphylum, philodendron, banana, potato, lilies, poinsettia, sugar-cane, and some forest tree species such as eucalyptus, poplar, and early stages of conifer somatic embryos. The technique was estimated to render a 35% reduction in propagule unit costs when propagated through the organogenic pathway (R. Levin, pers. commun.). Using somatic embryogenesis as the propagation pathway in a semi-automated system (Cervelli and Senaratna 1995) reduced production costs by 24%. In conclusion, simple bioreactors combined with mechanized cutting, sorting, and delivery systems are the solution for low-cost micropropagation.

LITERATURE CITED Aitken-Christie, J. 1991. Automation. p. 363-388. In: P. C. Debergh and R. H. Zimmerman (eds.), Micropropagation: Technology and application. Kluwer Acad. Publ., Dordrecht, The Netherlands. Aitken-Christie, J., T. Kozai, and S. Takayama. 1995. Automation in plant tissue culture. General introduction and overview. p.1-18. In: J. Aitken-Christie, T. Kozai, and M. A. 1. Smith (eds.), Automation and environment control in plant tissue culture. Kluwer Acad. Publ., Dordrecht, The Netherlands. Aitken-Christie, J., A. P. Singh, and H. Davis. 1988. Multiplication ofmeristematic tissue: a new tissue culture system far radiata pine. p. 413-432. In: J. W. Hanover and D. E. Keathley (eds.), Genetic manipulation of woody plants. Plenum Press, New York. Akita, M., and S. Takayama. 1994. Induction and development of potato tubers in a jar fermentor. Plant Cell Tissue Organ Culture 36:177-182. Alper, Y., R. E. Young, J. W. Adelberg, and B. B. Rohdes. 1993. Mass handling of watermelon microcutting. ASAE meeting, Paper, 93-1019. Alvard, D., F. Cote, and C. Teisson. 1993. Comparison of methods of liquid medium culture for banana micropropagation. Plant Cell, Tissue Organ Culture 32:55-60. Ammirato, P. V. 1983. The regulation of somatic embryo development in plant cell cultures: suspension culture techniques and hormone requirements. Bio/Technology 1:68-74. Ammirato, P. V. 1985. Patterns of development in culture. p. 9-29. In: R. R. Henke, K. W. Hughes, M. J. Constantin, and A. Hollaender (eds.), Tissue culture in forestry and agriculture. Plenum Publ., New York. Ammirato, P. V., and D. J. Styer. 1985. Strategies for large scale manipulation of somatic embryos in suspension culture. p. 161-178. In: M. Zaitlin, P. Day, and A. Hollaender (eds.), Biotechnology in plant science: Relevance to agriculture in the eighties. Academic Press, New Yark. Archambault, J., 1. Lavoie, R. D. Williams, and C. Chavarie. 1995. Nutritional aspects of Daucus carota somatic embryo cultures performed in biareactors. p. 681-687. In: M. Terzi, R. Cella, and A. Flavinga (eds.), Current issues in plant molecular and cellular biology. Kluwer Acad. Publ., Dordrecht, The Netherlands. Archambault, J., R. D. Williams, 1. Lavoie, M-F. Pepin, and C. Chavarie. 1994. Production of somatic embryos in a helical ribbon impeller bioreactar. Biotech. Bioengin. 44:930-943.

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Kim, D. 1., H. Pederson, and C. K Chin. 1991. Cultivation of Thalictrum rugosum cell suspension in an improved airlift bioreactor: stimulatory effects of CO 2 and C2H 4 on alkaloid production. Biotech. Bioengin. 38:331-339. Kim, Y. H., and J. Janick. 1989. ABA and polyox-encapsulation or high humidity increases survival of desiccated embryos of celery. HortScience 74:674-676. Kitto, S. J., and J. Janick. 1985. Production of synthetic seeds by encapsulating asexual embryos of carrot. J. Am. Soc. Hort. Sci. 110:277-282. Kozai, T., K Fujiwara, M. Hayashi, and J. Aitken-Christie. 1992. The in vitro environment and its control in micropropagation. p. 247-282. In: K Kurata and T. Kozai, Transplant production systems. Kluwer Acad. Publ., Dordrecht, The Netherlands. Kozai, T., B. R. Jeong, c. Kubota, and Y. Murai. 1995. Effects of volume and initial strength of medium on the growth, photosynthesis and ion uptake of potato (Solanum tuberosum 1.) plantlet in vitro. J. Japan Soc. Hort. Sci. 64:63-71. Krikorian, A. D., and R. P. Kann 1981. Plantlet production from morphogenetically competent cell suspension of daylily. Ann. Bot. 47:679-686.

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Onishi, N., T. Mashiko, and A. Okamata. 1992. Cultural systems producing encapsulating units of synthetic seeds in celery. Acta Hort. 319:113-118. Onishi, N., Y. Sakamoto, and T. Hirosawa. 1994. Synthetic seeds as an application of mass production of somatic embryos. Plant Cell, Tissue Organ Culture 39:137-145. Paque, M., J. Bercetche, and K Dumas. 1992. Liquid media to improve and reduce the cost of in vitro conifer propagation. Acta Hort. 319:95-100. Paque, M., and P. Boxus. 1987. Vitrification: a phenomenon related to tissue water content. Acta Hort. 212:245-252. Pieron, S., M. Belaizo, and P. Boxus. 1992. Scheme for rapid clonal propagation of Chichorium intibus 1. through nodule culture. Acta Hort. 319:285-289. Preece, J. K, and K Sutter. 1991. Acclimatization of micropropagated plants to the greenhouse. p. 71-91. In: P. C. Debergh and R. H. Zimmerman (eds.), Micropropagation: Technology and application. Kluwer Acad. Publ., Dordrecht, The Netherlands. Preil, W. 1991. Application of bioreactors in plant propagation. p. 425-455. In: P. C. Debergh and R. H. Zimmerman (eds.), Micropropagation: Technology and application. Kluwer Acad. Publ., Dordrecht, The Netherlands. Preil, W., P. Florek, U. Wix, and A. Beck. 1988. Towards mass propagation by use ofbioreactors. Acta Hort. 226:99-106. Redenbaugh, K, J. A. Fuji, and D. Slade. 1991. Synthetic seed technology. p.35-74. In: 1. K Vasil (ed.), Scale-up and automation in plant propagation. Cell culture and somatic cell genetics of plants. Vol. 8. Academic Press, New York. Reinert, J. 1958. Morphogenese and ihre Kontrolle an Gewebekulturen aus Karotten. Naturwissenschaften 45:344-345. Rodriguez-Mendiola, M. A., A. Stafford, R. Cresswell, and C. Arias-Castro. 1991. Bioreactors for growth of plant roots. Enzyme Microbiol. Technol. 13:697-702. Sakamoto, Y., N. Onishi, and T. Hirosawa. 1995. Delivery systems for tissue culture by encapsulation. p. 215-244. In: J. Aitken-Christie, T. Kozai, and M. A. 1. Smith (eds.), Automation and environmental control in plant tissue culture. Kluwer Acad. Publ., Dordrecht, The Netherlands. Saranga, Y., and J. Janick. 1991. Celery somatic embryo production and regeneration improved protocols. HortScience 26:1335-1336. Scragg, A. H. 1990. Fermentation systems for plant cells. p. 243-263. In: B. V. Charlwood and M. J. C. Rhodes (eds.), Secondary products from plant tissue culture. Clarendon Press, London. Scragg, A. H. 1992. Large-scale plant cell culture: methods, applications and products. Current Opinion Biotech. 3:105-109. Senaratna, T. 1992. Artificial seeds. Biotechnology Adv. 10:379-392. Smart, N. J., and M. W. Fowler. 1984. An airlift column bioreactor suitable for large-scale cultivation of plant cell suspensions. J. Expt. Bot. 35:531-537. Steward, F. c., P. V. Ammirato, and M. O. Mapes. 1970. Growth and development oftotipotent cells; some problems, procedures and perspectives. Ann. Bot. 34:761-787. Steward, F. C., M. O. Mapes, and K Mears. 1958. Growth and organized development of cultured cells. II. Organization in cultures grown from freely suspended cells. Am. J. Bot. 45:705-708. Stuart, D. A., J. M. Nelson, S. G. Strickland, and J. W. Nichol. 1985. Factors affecting developmental processes in alfalfa cell cultures. p. 59-73. In: R. R. Henke, K W. Hugs, M. J. Constantin, and A. Hollaender (eds.), Tissue culture in forestry and agriculture. Plenum Publ. Corp., New York. Stuart, D. A., S. G. Strickland, and K. A. Walker. 1987. Bioreactor production of alfalfa somatic embryos. HortScience 22:800-803.

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Biogenesis of Floral Scents Natalia Dudareva Horticulture Department Purdue University West Lafayette, Indiana, 47907 USA Birgit Piechulla Department of Molecular Physiology of Plants and Microorganisms Gertrudenstrasse 11a University of Rostock 18051 Rostock Germany Eran Pichersky Biology Department University of Michigan Ann Arbor, Michigan, 48109 USA I. Introduction II. Pathways and Site of Synthesis A. Terpenes B. Phenylpropanoids, Benzenoids, and Their Esters C. Fatty Acids and Other Scent Compounds III. Molecular Genetic Control A. Terpenes B. Phenylpropanoids/Benzenoids IV. Variation in Biosynthesis and Emission Over Time A. Variation During Development B. Circadian Rhythm of Emission C. Differences in Rhythms of Emission of Specific Odor Components V. Conclusions Literature Cited

Horticultural Reviews, Volume 24, Edited by Jules Janick ISBN 0-471-33374-3 © 2000 John Wiley & Sons, Inc. 31

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I. INTRODUCTION Flowers of many plants emit scents, which are almost always a complex mixture of small (100-250 Da), volatile compounds (Fig. 2.1).The accumulated experience of research in the field and in controlled environment in the laboratory has shown that floral scents may function as both long- and short-distance attractants and nectar guides to a variety of animal pollinators (reviewed in Dobson 1993). Although little is known about how insects respond to individual components found in floral scents, it is clear that insects are able to distinguish between complex floral scent mixtures, and that discriminatory visitation based on floral scent has important implications for population structure and reproductive isolation (Galen 1985; Pellmyr 1986; Dodson et al. 1969).

Fig. 2.1.

Myrcene

Methylcinnamate

Linalool

Methylbenzoate

Methyljasmonate

Indole

Representative compounds of floral scent.

2. BIOGENESIS OF FLORAL SCENTS

33

Floral scent is also an important character in some crop plants, since the presence or absent of a scent appropriate to the locally available insect pollinators may have a substantial impact on the level of pollination and therefore on seed (and fruit) set (Traub et al. 1942; Sugden 1986; Henning et al. 1992). Plants imported into a new environment by humans may be especially disadvantaged in this regard, as they have not coevolved with the local pollinators (DeGrandi-Hoffman 1987). Even if a local pollinator is attracted to the flowers, it may not be physically suitable to be an effective pollinator. On the other hand, pollinators who may have the appropriate morphology (by chance) may not be successfully attracted to the plant. The specific neurological effects of scent on humans are also little understood. However, it has been assumed since antiquity that at least some floral scents have beneficial or otherwise exploitable effects on humans and human behavior. This has in fact been the impetus for most of the research on floral scent done at the level of the plant. Over the last few hundred years, the perfume industry has amassed a vast catalogue of floral scent compounds. The desire to identify and purify such compounds, for commercial purposes, has been the driving force behind the development and/or refinement of several chemical techniques now widely used in many fields of chemistry and biochemistry, gas chromatography and oil-based extraction, to name just two. Once a chemical from a floral scent has been identified, however, it could be synthetically made or obtained from sources other than flowers. Although some floral scent components are specific stereoisomers that cannot be synthetically made in pure form (others can be, by using chiral precursors), today the vast majority of perfumes (and scented food additives) are synthetically made. Perfumery has become an art that strives by chemical means to imitate life. Plant scents are dominated by fatty acid derivatives, terpenoid, phenylpropanoid, and benzenoid compounds, with a smattering of other chemicals (see the excellent reviews by Knudsen et al. 1993; Knudsen and Tollsten 1993), all of which are regularly defined as "secondary metabolites." In contrast to the chemical emphasis in floral scent research, there have been few studies concerning the biosynthesis of floral scents. Most of the biochemical pathways and the enzymes involved in the synthesis of these compounds have not yet been elucidated. However, many of the chemicals found in scent are also occasionally found in vegetative tissue, e.g., limonene, farnesol, where more extensive biochemical investigations have been carried out. Only recently has attention been given to scent biogenesis. In this review, we describe what is known to date about the control of scent emission, the biosynthetic

34

N. DUDAREVA, B. PIECHULLA, AND E. PICHERSKY

pathways of floral scent compounds, the properties and regulation of the enzymes catalyzing these reactions, and the genes encoding these enzymes. It should be noted that most of the recent data concerning genes and enzymes involved in scent production come from one model organism, Clarkia breweri, which we have studied. It is hoped that similar investigations with other scented species will be forthcoming.

II. PATHWAYS AND SITE OF SYNTHESIS

While some plants emit scent from all, or a subset of, their floral parts, e.g. Clarkia breweri (Raguso and Pichersky 1995), other plants have been found to possess specialized "scent glands," e.g., orchids (Stern et al. 1986; Curry 1987). It is not yet clear how prevalent scent glands are among all scented flowers. Investigations of scent glands have so far been conducted mostly on the anatomical levels, and the question of whether such glands represents sites of emission only or also of synthesis of scent volatiles has not yet been addressed. The determination of the location of synthesis of scent compounds has by necessity been tied to the elucidation of the pathways themselves. The first difficulty was trapping and identifying the actual volatile chemicals that are emitted under natural conditions, i.e., not by cut and/or dehydrated flowers. This was solved by the development of the "headspace" collection technique, in which flowers that are still attached to the rest of the plant are encased in a container from which the air is continuously purged. The volatiles in this "headspace" are collected onto a trapping sorbent, from which they are later eluted and analyzed (Kaiser 1991; Raguso and Pellmyr 1998). While this approach has given us accurate descriptions of the emitted scent components, the specific pathways that operate in floral tissues or elsewhere to generate them have, in most cases, remained obscure. Historically, secondary metabolites in general were investigated by chemists who looked for intermediates (by pulse-chase experiments, for example) to piece together the pathways that lead to the final products. More recently, the emphasis has been on the discovery of enzymes catalyzing specific reactions as a proof that a postulated reaction indeed occurs in the cell (and is not an artifact of the experimental system). However, biochemical investigations of secondary metabolism have commonly been hampered by the lack of knowledge of the pathways, and, even when a substrate-product relationship is known or suspected, by the lack of pure substrates, especially radiolabelled ones, to perform the enzymatic assays.

2. BIOGENESIS OF FLORAL SCENTS

35

Thus, the existence of scent biosynthetic enzymes in flower tissue has been demonstrated for the first time only recently. Because it had proven difficult to find such biosynthetic enzymes, many investigations have focused instead on the possibility that some floral scent compounds are synthesized elsewhere in the plant and are then transported to the flowers. Several observations prompted such a hypothesis. First, many floral scent components are often found in glycosylated forms (as well as in free form) in fruits, and some of these may have been transported from the vegetative part of the plant (Gunata et al. 1985; Tang et al. 1990). Second, glycosylated compounds are also often found in buds (which are usually not scented), and later in flowers (Ackermann et al. 1989; Loughrin et al. 1992; Watanabe et al. 1993). And lastly, a report which appeared in Russian with an English abstract (Pogorel'skaya et al. 1980) claimed that such transport into buds occurs in roses (although this has not yet been repeated by other groups). As is often the case in science, hypotheses that are relatively easy to test are tackled first. Thus, an important impetus for this line of investigation was simply the fact that it is relatively easy to measure the activity of the glycosidases, which were hypothesized to be the key enzymes releasing the aroma in the flower. In some cases it was reported that the levels of glycosidases increased during the development of the flower (Loughrin et al. 1992). It is important to note that the demonstration of glycosidase activity in flowers by itself says nothing about the site of synthesis of either the free scent component or its glycosylated form (both of which might of course occur in the flower itself). Moreover, when levels of glycosides present in the flowers at different stages are correlated with actual levels of emission, there is little to support the conclusion that glycosidically bound scent compounds are obligatory intermediates in scent biogenesis (Ackermann et al. 1989). This is because (1) the glycosides tend to accumulate more as the flower ages (i.e., they usually do not peak at or before the peak of scent emission), and (2) the pool of glycosides in the flower before or at peak emission time is only a small fraction of what the flower actually emits. In those studies neither the synthesis of the scent compounds themselves and their glycosylated forms in the vegetative tissues, nor the process of transporting them (or their glycosylated forms) into the flowers, have been examined. Thus, although it is possible that scent precursors are initially synthesized in vegetative tissue in some plant species, there is little empirical data to substantiate this scenario. This section will therefore review the growing evidence for flower-specific biogenesis of floral scent compounds. The published work deals with our model system Clarkia breweri, an annual flower from California. Recently, however,

36

N. DUDAREVA, B. PIECHULLA, AND E. PICHERSKY

similar results have been obtained with the cultivated ornamental species Antirrhinum majus (snapdragon) and Narcissus pseudonarcissus (daffodil) (N. Dudareva, J. D'Auria, and E. Pichersky, unpubl.). A. Terpenes Terpenes, especially monoterpenes but also some sesquiterpenes, are very common constituents of floral scent. Mettal and coworkers (Mettal et al. 1988) have shown that chromoplasts isolated from the petals of Narcissus pseudonarcissus flowers were capable of catalyzing the production of several monoterpenes, including limonene, myrcene, ocimene, and linalool. Interestingly, while the first three of these monoterpenes are regular constituents of the daffodil scent, linalool is not. The authors speculated that linalool is produced as an intermediate in the synthesis of other monoterpenes. Nevertheless, this work showed both that scent compounds could be synthesized in the flowers, and that the specific site of monoterpene synthesis is in the plastidic compartment. This is consistent with work on synthesis ofmonoterpenes in vegetative tissue, where it also occurs exclusively in plastids (McGarvey and Croteau 1995). It is now believed that in plants, monoterpenes (and diterpenes) are synthesized in the plastids via the Rohmer pathway, while sesquiterpenes are synthesized in the cytosol via the mevalonic acid pathway (Lichtenthaler et al. 1997). We have looked in some detail at the synthesis of linalool, an acyclic monoterpene alcohol, in flowers of Clarkia breweri. Its flowers emit copious amounts of this monoterpene (~20J.lg/flowerin 24 h), as well as a similar amount of two linalool oxide derivatives (Raguso and Pichersky 1995). Linalool synthase (LIS), the enzyme that catalyzes the formation oflinalool from geranyl pyrophosphate (GPP) (Pichersky et al. 1995), is most abundant in the petals, stigma, and style, and is not found in the vegetative parts of the plant (Pichersky et al. 1994). The petals constitute the bulk of the flower, and most of the linalool emitted by the flower is synthesized in, and emitted from, the petals. In situ hybridization experiments have shown that the LIS gene is expressed in petals, mostly in the cells of the epidermis (from which linalool can easily escape into the atmosphere after being synthesized). In stigma and style, the transmitting tissue shows the highest concentration of LIS, but the linalool produced there is apparently mostly converted into linalool oxides by additional enzyme(s) not yet characterized. The purpose of the production of linalool and its oxides in the transmitting tissue is not clear, but may be related to defense, as linalool is relatively toxic to animals and micro-organisms (Bruneton 1995).

2. BIOGENESIS OF FLORAL SCENTS

37

The gene encoding LIS has been isolated and characterized from several plant species (Cseke et al. 1998). The sequence of the deduced amino acid of the LIS protein shows that it is related to other terpene synthases, and LIS is therefore a member of a family of proteins that must have evolved from a common ancestor, although in a somewhat complicated manner (Bohlmann et al. 1998; Cseke et al. 1998). B. Phenylpropanoids, Benzenoids, and Their Esters The phenylpropanoids, derived from phenylalanine, constitute a large class of secondary metabolites in plants. Many are intermediates in the synthesis of structural cell components (e.g., lignin), pigments (e.g., anthocyanins), and defense compounds. These are not usually volatile. However, several phenylpropanoids whose carboxy group at C9 is reduced (to either the aldehyde, alcohol, or alkane/alkene) and/or which contain alkane additions to the hydroxy groups of the benzyl ring or to the carboxy group (i.e., ethers and esters) are volatiles. Such compounds include eugenol, methyleugenol, and methylcinnamate. The synthesis of methyleugenol and isomethyleugenol has been examined in C. breweri (Wang et al. 1997). An enzyme, S-adenosyl-Lmethionine:(iso)eugenol methyltransferase (IEMT) has been isolated and characterized. The enzyme is most abundant in petal tissue, which, as in the case of linalool, accounts for the majority of the production and emission of (iso)methyleugenol. Again, in situ experiments show that the IEMT transcripts are most abundant in the epidermal cells of the petals, as well as in the epidermal layer of other floral tissues such as anthers (J. Wang, N. Dudareva, and E. Pichersky, unpubl.). IEMT activity is not found in vegetative tissue, nor in petals of a C. breweri variety that does not emit (iso)methyleugenol (Wang et al. 1997). The sequence of IEMT is very similar (84% identity) to the sequence of caffeic acid methyltransferase (COMT) from C. breweri (Wang and Pichersky 1997). COMT is an enzyme found universally in plants and it is involved in lignin biosynthesis. Thus, it appears that IEMT has evolved, perhaps recently, from a common enzyme by gene duplication and divergence. Benzenoids, such as benzylalcohol (a component of petunia scent) are derived from phenylpropanoids by the loss of the C8-C9 carbons. Although the exact mechanism of their removal is still unclear, this process is probably not analogous to ~-oxidation (Schnitzler et al. 1992). Many benzenoids are found in floral scent in an ester form (e.g., methylbenzoate, benzylbenzoate). The biosynthesis of two benzenoid esters, methylsalicylate and benzylacetate, have been examined in C. breweri. The formation of benzylacetate is catalyzed in flowers of C. breweri by

38

N. DUDAREVA, B. PIECHULLA, AND E. PICHERSKY

the enzyme acetyl-CoA:benzylalcohol acetyltransferase (BEAT). This enzyme is also most abundant in petal tissue, from where the bulk of benzylacetate emission occurs. The BEAT protein is a member of a newly defined family of acyltransferases. S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (SAMT) is the enzyme that catalyzes the formation of methylsalicylate in petals of C. breweri (Dudareva et al. 1998a). IEMT, BEAT and SAMT, whose substrates are all phenylpropanoidslbenzenoids, are most likely localized in the cytosol. Recently, we have been able to demonstrate the activity of BEAT in petals of daffodils (J. D'Auria and E. Pichersky, unpublished). In addition, the activity of SAM:benzoic acid methyltransferase (BAMT), the enzyme that catalyzes the formation of methylbenzoate, a major component of the floral scent of Antirrhinum majus, has been demonstrated in their petals (N. Dudareva, unpublished). C. Fatty Acids and Other Scent Compounds Several floral scent components are derivatives of fatty acids. For example, the acetyl ester of cis-3-hexen-1-o1 is common, as is methyljasmonate. Both cis-3-hexen-1-o1 and jasmonate are breakdown products of linolenic acid (Creelman and Mullet 1997). Whereas it is well established that fatty acids themselves are synthesized in the plastids, the location of synthesis of their derivatives is still essentially uninvestigated. The jasmonates are important signaling compounds in vegetative tissues as well, and there it appears that at least the initial steps leading to their synthesis occur in the plastids. However, there are no reports that examine their synthesis in floral tissues. In this category are mostly scent compounds that contain nitrogen, such as indole. Again, little is known about the biosynthesis of alkaloids in general (Kutchan 1995), and even less is known about volatile ones. In fact no information is available concerning the synthesis of any volatile alkaloids in floral tissues. III. MOLECULAR GENETIC CONTROL A. Terpenes Although monoterpenes and sesquiterpenes are predominant in many floral fragrances, most of the work on terpene biosynthesis has been carried out on vegetative tissues, where these compounds also serve important functions such as defense (McGarvey and Groteau 1995). In the last few years, genes encoding the enzymes responsible for the synthesis of

2. BIOGENESIS OF FLORAL SCENTS

39

many of these compounds have been identified and characterized (Bohlmann et al. 1998). However, to date only the gene encoding linalool synthase has been characterized specifically in respect to its role in floral scent biosynthesis (Dudareva et al. 1996). Linalool synthase was purified to homogeneity from stigmata of Clarkia breweri flowers and a protein-based cloning strategy was employed to obtain a cDNA encoding this protein. This enzyme produces exclusively S-linalool, a component of floral scent of many plant species (Knudsen et al. 1993; Gerlach and Schill 1991). It is a monomer with apparent molecular weight of 76 kDa (determined by gel permeation chromatography and polyacrylamide gel electrophoresis), and with a strict requirement for a divalent metal cofactor, preferentially Mn z+ (Pichersky et al. 1995). The LIS gene is unique in C. breweri genome and its coding region is interrupted by 11 introns (Dudareva et al. 1996; Cseke et al. 1998). The complete amino acid sequence of LIS, derived from the cDNA clone, showed it to be related to several enzymes involved in terpene synthesis (Dudareva et al. 1996). LIS was also isolated from a linalool-scented relative of C. breweri, Oenothera arizonica, and from non-scented C. concinna (Cseke et al. 1998), the proposed progenitor of C. breweri. Both sequence comparisons and comparisons of the location of introns in the genes suggest that LIS is actually a composite gene. It includes a portion of a copalyl pyrophosphate synthase-like sequence at its N-terminus coding region and a portion of another terpene synthase (a limonene synthase-like) in most of its second half (Cseke et al. 1998). Expression of the LIS gene is temporally and spatially regulated during flower development. In scented C. breweri, LIS mRNA transcripts begin to accumulate in flower buds several days before opening the flower. One day before anthesis, LIS mRNA levels become approximately three and five times higher in petals than in pistil and stamens, respectively. There is a lag time of about a day between the peak levels of mRNA and the peak levels of LIS protein in petal and stigma tissues (Fig. 2.2), but not in style and anthers (Dudareva et al. 1996). During the lifespan of the flower, the protein levels in floral tissues show strong positive correlation with LIS activity (Fig. 2.2), indicating that the differences in LIS activity in different tissues and at different stages of flower development are due to changes in the amount of LIS protein and not to post-translational modifications. Clarkia breweri has arisen from the non-scented species C. concinna (Raguso and Pichersky 1995), which nonetheless has been shown to express its LIS gene at a low level only in the stigma and to emit 1000fold less linalool than C. breweri flowers (Pichersky et al. 1994; Dudareva et al. 1996). The most significant observation concerning the expression

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2. BIOGENESIS OF FLORAL SCENTS

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of LIS in C. breweri is that in addition to being upregulated significantly in the stigma compared with its non-scented ancestor, C. cancinna, the LIS gene is also highly expressed in other types of floral tissues such as styles, anthers, and petals. Up to 70% of the total LIS activity in the C. breweri flower was found in the petals, which are the major source of emitted S-linalool (Pichersky et al. 1994; Ragusa and Pichersky 1995). In addition to high expression of the LIS gene in petals, a strict pattern of spatial regulation was also observed: LIS transcripts were found mainly in epidermal cells at the surface of the petals (Dudareva et al. 1996). Overall, linalool production in C. breweri flowers involves a change in regulation of an existing gene. The expression of LIS gene was both upregulated and its range was expanded to include cells not expressing this gene in C. concinna, without the concomitant development of specialized "scent glands" as has been found in flowers of orchids (Stern et al. 1986). B. PhenylpropanoidslBenzenoids Two volatile phenylpropanoids, methyleugenol and isomethyleugenol, are important components of the floral scent of various species, including C. breweri (Ragusa and Pichersky 1995; Wang et al. 1997). It has been shown that they are produced in C. breweri flowers by the action of a single enzyme, IEMT, which catalyzes the transfer of a methyl group to the 4-0H group of the benzyl ring of both eugenol and isoeugenol, using S-adenosyl-L-methionine as a donor of methyl group (Wang et al. 1997). IEMT has been purified to homogeneity from C. breweri petals. The corresponding cDNA has been isolated from C. breweri flower cDNA library, sequenced, and the catalytically active enzyme has been expressed in Escherichia coli. IEMT is active as a homodimer with a subunit molecular mass of 40 kDa and does not require any cofactors for enzymatic activity (Wang and Pichersky 1998). IEMT is a single copy gene in the C. breweri genome and exhibits flower-specific and temporal expression patterns in (iso)methyleugenol emitters. The levels of IEMT activity and mRNA in different floral tissues strongly correlate with the production and emission of these two compounds by the same tissues, being highest in petals and absent in sepals and in leaf and stem tissue. Similar to LIS, the levels of IEMT mRNA in petals increased as the flower bud matured and peaked just before anthesis. Moreover, nonemitting plants did not have IEMT activity or IEMT mRNA in any floral tissues, but they do have the IEMT gene in their genome (Wang et al. 1997; J. Wang, unpubl.). These results suggest that similar to LIS, changes have occurred in the regulation ofIEMT gene expression.

42

N. DUDAREVA, B. PIECHULLA, AND E. PICHERSKY

In many plant species, volatile esters contribute significantly to the total floral scent output and are important in attracting insect pollinators. Benzylacetate in particular is one of the most commonly found esters in moth-pollinated flowers (Knudsen and Tollsten 1993) and is often found in the aromas of other flowers as well (Knudsen et al. 1993; Van Dort et al. 1993; Watanabe et al. 1993). The enzyme BEAT, which catalyzes the formation of benzylacetate, has been purified from C. breweri petals, a cDNA encoding this enzyme has been isolated and characterized, and enzymatically active protein has been expressed in Escherichia coli. (Dudareva et al. 1998b). The sequence of the protein encoded by BEAT cDNA does not show extensive similarity to any other known protein sequences, but a short segment within it has significant similarity to short segments in other proteins known or hypothesized to use an acyl-CoA substrate. There is a single copy of BEAT gene in the C. breweri genome. Its expression is developmentally and differentially regulated. Of the different parts of the C. breweri flower, petals contained the majority of BEAT transcripts, and no BEAT mRNA was detected in leaves. BEAT mRNA was first detected in petal cells just before the flower opened, and its level increased until it peaked on the day of anthesis (Dudareva et al. 1998b). These results are similar to those for LIS and IEMT, whose mRNA levels also peak at or around anthesis, suggesting a common regulatory mechanism. Taken together, these results clearly indicate that, at least in C. breweri flowers, scent compounds are produced de novo in the tissues from which they are emitted and the levels of activity of enzymes involved in scent production are regulated mainly at the mRNA levels at the site of emission. Expression of genes encoding scent biosynthetic enzymes is relatively uniform, being highest in petals just before anthesis, and restricted to surface of the floral tissue (epidermal cells), without development of specialized "scent glands" as found in orchid flowers (Stern et al. 1986) and in vegetative tissue of some terpene-producing plants (Lewinsohn et al. 1991, 1998; McGarvey and Croteau, 1995).

IV. VARIATION IN BIOSYNTHESIS AND EMISSION OVER TIME A. Variation During Development The composition of the odor emitted determines the attractiveness of the flowers to specific insects, and is therefore responsible for pollinator

2. BIOGENESIS OF FLORAL SCENTS

43

selectivity. For example, the intra- and inter-individual variation of odor composition of Ophrys sphegodes flowers and of flowers of other plants may influence the behavior of the male pollinator (Smith and Ayasse 1987). It is known that male bees can remember the floral bouquet of a visited flower and subsequently modify their response to it (Smith and Ayasse 1987; Ayasse et al. 1996, summarized in Schiestl et al. 1997). A decrease and/or an alteration of floral bouquets after pollination might lead to a lower attractiveness of these flowers. The advantage of these postpollination changes in scent emission is most likely that pollinators are directed to the unpollinated flowers of the plant. Scent production and maintenance by flowers is expensive in terms of energy resources, therefore many flowers have evolved various mechanisms for energy conservation, such as cessation of scent production and fast wilting immediately after pollination (Arditti 1979) or decrease in scent production of unpollinated plants as a result of aging. The changes in scent during floral development involve not only a decrease of total amount of scent produced, but also an alteration of the odor bouquet. The effect of pollination on floral scent composition and production was studied in the moth-pollinated orchid Platanthera bifolia. A significant decrease (about 200 times) in scent production was detected five days after pollination, although some decrease (about 3 times) was already found two days after pollination. All scent compounds were affected by pollination, even though some compounds had a larger impact on the overall scent reduction. There was also a drop in scent production during the lifespan of the flower in unpollinated plants: the average amount of total volatiles collected per flower decreased about two times, from 6911g at anthesis to 3711g at day five (Tollsten 1993). In addition to quantitative changes, qualitative alterations of odor emission were found in the Mediterranean orchid genus Ophrys after pollination. Pollinated flowers produced significantly different odor bouquets due to the change in the relative amount of each constituent volatile, and the total amount of scent emitted two to four days after pollination was significantly lower compared with unpollinated flowers (Schiestl et al. 1997). The comparative analysis of volatiles emitted at different stages of flower development in Clarkia breweri revealed variations in the quantitative contribution of the individual compounds to the floral fragrance (Pichersky et al. 1994; Wang et al. 1997; Dudareva et al. 1998a). Emission of volatiles began just before the flowers opened. Benzenoid esters (benzylacetate, benzylbenzoate, and methylsalicylate) were the first volatiles emitted from flower buds. The level of emission was 10-20% of the maximal level and remained relatively stable for the first 12 h after

N. DUDAREVA, B. PIECHULLA, AND E. PICHERSKY

44

anthesis. Unopened flowers (buds) emit no linalool, isoeugenol, and isomethyleugenol, but they do emit a small amount of linalool oxide, eugenol, and methyleugenoL Significant emission of volatile compounds begins at anthesis, peaks on day 1 or 2 (depending on compound) and declines thereafter. Developmentally, the activities of enzymes involved in the biosynthesis of these compounds of C. breweri scent follow two different patterns (Fig. 2.3). The activities of the first group of enzymes, such as LIS and SAMT (Fig. 2.3 A, D), increased in young flowers and declined in old flowers in parallel with emission of linalool and methylsalicylate, respectively. The activities of the second 2000

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2. BIOGENESIS OF FLORAL SCENTS

45

group of enzymes, such as IEMT and BEAT (Fig. 2.3 B, C), show little or no decline at the end of the lifespan of the flower, although emission of methyleugenol, isomethyleugenol, and benzylacetate do decline. The difference in developmental profiles of the latter two enzymes was that IEMT levels peaked on day 1 of anthesis and stayed stable afterward (Wang et al. 1997), whereas BEAT activity did not peak until the 4th day after anthesis (Dudareva et al. 1998a). The causes and consequences of high levels of activity of biosynthetic enzymes in old flowers, without concomitant emission of the volatile products, are unknown. Although it is possible that the biosynthetic pathways in which these enzymes participate are blocked elsewhere, another possibility that remains to be investigated is that the products produced in the reactions catalyzed by these enzymes are required for additional processes in the flowers other than scent emission. A third possibility is that as the flower ages, substrates may be diverted to other compartments and are not accessible to the scent biosynthetic enzymes. The mRNAs for three isolated genes encoding scent biosynthetic enzymes in the C. breweri flower (LIS, IEMT and BEAT) were first detected in petal cells just before the flower opened and their levels increased until it peaked at or around anthesis, and afterwards began to decline (Dudareva et al. 1996, 1998b; Wang et al. 1997). For all three enzymes, the peak of mRNA was always 1-2 days ahead of enzyme activity and emission of corresponding component. In case of linalool, levels of emission, enzyme activity, and mRNA in the petals all rose and fell in parallel until the end of the lifespan of the flower (Fig. 2.2), whereas the situation with IEMT and BEAT was somewhat different. Isomethyleugenol and methyleugenol emission, IEMT activity, and mRNA levels in the petals all increased in parallel as the buds matured and the flowers opened. However, starting from the 3rd day post-anthesis (1 day after the stigma becomes receptive and most pollination occurs), emission began to decline but IEMT activity remained relatively stable. IEMT mRNA levels actually went up a little after declining 25% from their peak on the day before anthesis. In case of benzylacetate, the levels of BEAT mRNA in petals increased as the bud matured, and peaked at anthesis, paralleling changes in BEAT activity and emission. However, after the second day post-anthesis, mRNA levels declined sharply, whereas BEAT specific activity continued to increase and only began to decrease on day 5 after anthesis, suggesting that the BEAT protein is relatively stable. Overall, the data show that strong positive correlation exists among levels of enzyme activity, mRNA at the site of biosynthesis and emission of corresponding component in the C. breweri flower before and one day after anthesis, suggesting that the activity of scent

46

N. DUDAREVA, B. PIECHULLA, AND E. PICHERSKY

biosynthetic enzymes is regulated at a pretranslational level. These results also suggest a common regulatory mechanism for genes involved in scent production. Quantitative and/or qualitative changes of floral scent chemistry during flower development may be part of a mechanism to differentiate attraction cues between pollinated and unpollinated flowers.

B. Circadian Rhythm of Emission While many plants continuously emit odor during flowering at a constant level, other flowers emit scent with the level of emission increasing or decreasing periodically. Yet other plants emit scent only at specific times during the day (diurnal emitters) or during the night (nocturnal emitters). In addition, some plants emit one set of compounds during the day and others during the night. Plant species that belong to one or the other category are listed in Table 2.1. The diurnal and nocturnal emission of odor correlates well (1) with the type of insect that pollinates the flower, e.g., the day-active insect (e.g. bees) and the nightactive moths, and (2) with the time of flower opening either at a particular time of day and/or during a particular developmental phase. Odor is usually only emitted during a short period of flowering (a few days); thereafter no volatile compound can be detected, not even at constant Table 2.1. Type Diurnal

Nocturnal

ConstanF

Fragrance emission pattern. Reference

Species

Citrus medica Odontoglossum constrictum Ophrys sphegodes Platanthera chlorantha Rosa hybrida (Hybrid tea) Cestrum nocturnum Hoya carnosa Hyacynthus orientalis Nicotiana sylvestris Nicotiana suaveolens Ophrys sphegodes Platanthera chlorantha Stephanotis floribunda Malus x domestica Nicotiana otophora

Matile and Altenburger Matile and Altenburger Schiestl et al. 1997 Nilsson 1983 Kaiser 1991 Overland 1960 Matile and Altenburger Kaiser 1991 Loughrin et al. 1991 Loughrin et al. 1991 Schiestl et al. 1997 Nilsson 1983 Matile and Altenburger et al. 1990 Loughrin et al. 1990

1988 1988

1988

1988

LV'.l.p,H.LLH

ZAromatic and benzyl alcohol compounds are present at constant levels throughout the day.

47

2. BIOGENESIS OF FLORAL SCENTS

levels. For example, in Nicotiana sylvestris, Hoya carnosa, and Stephanotis floribunda the diurnal/nocturnal emission of flower volatiles was observed for four to seven days after anthesis (Matile and Altenburger 1988; Loughrin et al. 1991; Altenburger and Matile 1988). The appearance of flower odor at specific times during the day is a prerequisite for pollination by diurnal or nocturnal insects. The regulation of the cycling of fragrance emission can be induced by either illumination or darkness, or alternatively be controlled by an endogenous clock. To find out whether diurnal or nocturnal odor emission is regulated by an internal mechanism, it is necessary to exclude possible external stimuli from the experiment. This is done by analyzing scent emission from plants grown under constant conditions, e.g. constant temperature and continuous illumination (LL) or constant temperature and continuous darkness (DD). If oscillations in scent emission are still observed under such conditions and have a periodic length of approximately 24 h, this means that such oscillations are controlled by an endogenous circadian clock. Results from such experiments are summarized in Table 2.2. The available data indicate that a circadian clock is likely to be a control mechanism involved in diurnal alterations of odor emission of several plant species. In the plants that show circadian rhythms, a peak fragrance emission occurs under free-running conditions (continuous light or continuous darkness) approximately every 25-28 h in flowers of intact plants and cut flowers of N. suaveolens, N. sylvestris, H. carnosa, O. constrictum, usually with a reduced or continuously dampening amplitude (Overland 1960; Loughrin et al. 1991; Altenburger and Matile 1988).

Table 2.2.

Fragrance emission under constant conditions. Rhythmic appearance of fragrance

Plant species

Cestrum nocturnum

Continuous illumination

Continuous darkness

+

Hoya carnosa

+

+ n.d.Y

Nicotiana suaveolens Nicotiana sylvestris

+ +

n.d. n.d.

Period length Z(h)

Reference

25 28

Overland 1960

29 x

Altenburger and Matile 1988 Loughrin et al. 1991 Loughrin et al. 1991

26-27 X

26-27

ZTime between a maximum and next maximum of fragrance emission. Yn.d. = not detected. XEntrainment adjusted after 2 days.

48

N. DUDAREVA, B. PIECHULLA, AND E. PICHERSKY

The period lengths of emission oscillations under free-running conditions are larger than 24 h, indicating that the endogenous clock controlling odor emission in flowers runs slower and is usually synchronized by environmental cues. The determination of odor emission to a precise time point during the day or night phase is predominantly synchronized by the day/night or night/day transitions, while alterations of temperature between day and night are usually less strong Zeitgebers (= time givers). Circadian oscillations are usually maintained throughout a wide temperature range (temperature compensation), but at unphysiological conditions, either the rhythm completely disappears or a rhythm with an extremely long period length occurs. The latter is observed in Cestrum nocturnum where a period length three times as long usually appears at 4°C (Overland 1960). The observation of daily alterations of volatile appearance begs the question of the molecular mechanisms. Issues as yet to be addressed are: From what cellular compartment is the fragrance ultimately derived, and is its release controlled by a mechanism that is able to measure time? Does the synthesis of odor compounds depend on the time of day, and if so, how? Do the enzymes of the biosynthetic pathway differentially accumulate at different time points during the day or is their amount constant but their activity regulated by processes controlled by a circadian clock? C. Differences in Rhythms of Emission of Specific Odor Components As mentioned earlier, different volatiles emanating from the same flower may show independent patterns of emission oscillations. It is therefore of interest to distinguish between different volatiles of an odor bouquet and their characteristic emission pattern, e.g. rhythmic or constant appearance. This aspect is summarized in Table 2.3. In H. carnosa, five volatiles-linalool, cineole, ~-pinene, iso-pentanol, and methylsalicylate (listed in order of their relative quantity: 21:4:2:1.5:1)-were identified that are major components of the odor, while a wealth of minor components of unknown identity possibly influence the scent specification as well. The emission of these five volatiles occurs during night (with a peak at 3 A.M.) and is in marked synchrony in continuous light conditions and light/dark entrained conditions (Matile and Altenburger 1988; Altenburger and Matile 1988). The oscillation in emission of these volatiles, with dampening amplitudes, is detected for four days in attached flowers of H. carnosa. Similar results were obtained with O. constrictum with two unidentified volatiles, and in C. medica, a

Table 2.3. Diurnal/circadian appearance of different odor volatiles. Daylength Long

Short day

12 h day

Species

Citrus medica Malus x domestica Nicotiana othophora Odontoglossum constrictum Ophrys sphegodes Z Platanthera chlorants Rosa hybrida

linalool, nerolidol cis-3-hexenyl acetate, cis-3-hexen-l-ol sabinene two unknown compounds nonanal, decanal, hexadecane, 2-nonanone ~-caryophyllene

Ophrys sphegodesZ

cis-hexenyl acetate, n-hexyl acetate, trans-2-hexenyl acetate myrcene, ipsdienol l-nitro-2-phenylethane linalool, 1,8 cineole, a-pinene, ~-pinene, iso-pentanol sabinene, methylsalicylate, methylbenzoate benzyl acetate, benzyl alcohol, benzaldehyde, methylbenzoate limonene, a-pinene, heptanal, 1.8-cineole, 2-nonanol,

Platanthera bifolia

methylbenzoate, linalool

Platanthera chloranta Platanthera stricta Stephanotis floribunda Malus x domestica

methylbenzoate, lilac alcohols, monoterpenes linalool linalool, methylbenzoate linalool, benzyl acetate, benzyl alcohol, 2-phenethyl alcohol myrcene, trans-f3-ocimene, benzaldehyde, benzyl acetate, benzyl alcohol caryophyllene, linalool, a-terpineol, cis-jasmone a-pinene, myrcene, limonene

Stanhopea anfracta Stephanotis floribunda Hoya carnosa Nicotiana suaveolens Nicotiana sylvestris

Nicotiana othophora Nicotiana sylvestris Platanthera stricta Z ~

Floral scent compounds

Reference Matile and Altenburger 1988 Loughrin et al. 1990 Loughrin et al. 1990 MaHle and Altenburger 1988 Schiestl et al. 1997 Nilsson 1978, 1983 Kaiser 1991 Curry et al. 1987 Matile and Altenburger 1988 Matile and Altenburger 1988 Loughrin et al. 1991 Loughrin et al. 1990,1991 Schiestl et al. 1997 Nilsson 1983; Tollsten and Bergstrom 1989 Nilsson 1978,1983 Patt et al. 1988 Matile and Altenburger 1988 Loughrin et al. 1990 Loughrin et al. 1990 Loughrin et al. 1990,1991 Patt et al. 1988

ZOriginal paper lists more than 20 compounds, but only a few that occur at relatively high levels are listed here.

50

N. DUDAREVA, B. PIECHULLA, AND E. PICHERSKY

major component with rhythmic appearance was linalool, while oscillations with very small amplitude were measured for nerolidol (MatHe and Altenburger 1988). It is worth noting that the volatiles of the latter two plant species were measured from excised flowers. This aspect is important, because excision can result in a dramatic loss of fragrance, e.g. H. carnosa (Matile and Altenburger 1988) and/or in major differences in fragrance composition compared to emission by attached flowers, e.g. yellow tea rose (Mookherjee et al. 1989; Kaiser 1991). Synchronized rhythmic emanation of 2,6-dimethyl-3(E),5(E),7octatrien-2-ol and its 5(Z) isomer, with a maximum between 6 and 10 P.M. was observed in H. orientalis (Kaiser 1991). Flowers of S. floribunda exhibit remarkable rhythmicity of three volatiles, methylbenzoate, RT 10.72 (tentatively identified as 1-nitro-2-phenylethane), and linalool (listed in order of their relative quantity: 4-7:2:1). Interestingly, the emission peaks of methylbenzoate and linalool occur at midnight, while the RT 10.72 compound peaks at noon, indicating that the appearance of the volatiles in this plant species does not coincide. Differences in daily odor composition was also detected in the genus Ophrys, a group of well-studied Mediterranean orchids. Comparison of volatiles at night versus day in O. sphegodes revealed that nocturnal emission contained significantly lower amounts of most aldehydes and 6-methyl-5-hepten-2-one and significantly higher amounts of most hydrocarbons, a-pinene, limonene, 1,8-cineole and 2-nonanol when collected during night, although the total amount of scent emission increased during night (Schiestl et al. 1997). Nilsson (1983) also found great differences in scent production during day and night of Platanthera chlorantha. The data summarized in this section indicate that, in many plants, the odor composition and total emission output vary between day and night. Although it is clear that odor variability is important in influencing the behavior of the pollinators, and therefore plant fitness, the mechanisms responsible for the circadian rhythm in scent emission and for the temporal changes in scent composition presently remain unknown. V. CONCLUSIONS

Fruit set in many agricultural and horticultural crops, such as most fruit trees, berries, nuts, oilseeds, and vegetables, rely on insect pollinators that are attracted by floral scents. Floral scent is typically a complex mixture of low molecular weight compounds, which gives the flower its unique characteristic fragrance. Although several thousand com-

2. BIOGENESIS OF FLORAL SCENTS

51

pounds have been identified from various floral scents and the chemical structures of most are known today, only a few studies have focused upon the biosynthesis of these compounds in planta. Recent investigations of floral scent production in Clarkia are the first examples of the isolation of enzymes and genes responsible for the formation of scent volatiles. In these investigations, it has been shown that scent compounds are produced de novo in open flowers, and that their emission levels, corresponding enzyme activities, and mRNA levels are all spatially and temporally correlated. However, our understanding of floral scent biosynthesis and its regulation is limited and based on analysis of a single model system in Clarkia. Obviously more research is needed in this field. An understanding of the molecular, genetic, and biochemical basis of scent formation in plants will provide the knowledge for engineering plants with improved scent quality. Bioengineering of the metabolic pathways responsible for the production of volatile compounds in plants can involve either the modification of existing pathways and/or the introduction of new enzymes to produce novel products not normally found in the plant. The availability of an increasing number of cloned genes encoding scent biosynthetic enzymes is the first step toward engineering transgenic plants with modified volatile composition. Development of crops with modified composition of volatiles and new introduced aroma could benefit agriculture by increasing crop productivity, pest resistance, and the value of ornamentals. Modified floral scent composition can increase attraction of flowers to pollinators and thereby increase reproduction efficiency and the yield of important agricultural crops. In addition, floral scent modification could be manipulated as a means of attracting beneficial insects and predators, and perhaps deter harmful ones. The manipulation of floral scents would be of immediate value for the floricultural industry. A large number of commercial flower cultivars have lost their scent during the selection and breeding processes due to the initial focus on maximizing postharvest shelf life and shipping characteristics. The lack of scent has long been recognized as a major problem in floriculture and a transgenic plant approach may help to solve this problem.

LITERATURE CITED Ackermann, I. K, D. V. Banthorpe, W. D. Fordham, J. P. Kinder, and I. Poots. 1989. ~­ glucosides of aroma components from petals of Rosa species: assay, occurrence, and biosynthetic implications. J. Plant Physiol. 134:567-572.

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Altenburger, R, and P. Matile. 1988. Circadian rhythmicity of fragrance emission in flowers of Hoya earnosa RBr. Planta 174:248-252. Arditti, J. 1979. Aspects ofthe physiology of orchids. In H. W. Woolhouse (ed.), Advances in botanical research, Vol. 7. p. 422-697. Academic Press, London. Ayasse, M., F. P. Schiestl, H. F. Paulus, D. Erdmann, and W. Francke. 1996. Does an Ophrys plant cheat a pollinating male bee more than once? Proc., XX Int. Congr. on Entomology, Firenze, p. 225. Bohlmann, J., G. Meyer-Gauen, and R Croteau. 1998. Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc. Nat. Acad. Sci. (USA) 95:4126-4133. Bruneton, J. 1995. Parmacognosy, phytochemistry, medicinal plants. Elsevier Publ., New York. Creelman, R A., and J. A. Mullet. 1997. Biosynthesis and action of jasmonate in plants. Annu. Rev. Plant Physiol. Plant Mol. BioI. 48:355-381. Cseke, 1., N. Dudareva, and E. Pichersky. 1998. Structure and evolution ofthe linalool synthase. Mol. BioI. Evol. 15:1491-1498. Curry, K. J. 1987. Initiation of terpenoid synthesis in osmophores of Stanhopea anfraeta (Orichadaceae): a cytochemic study. Am. J. Bot. 74:1332-1338. DeGrandi-Hoffman, G. 1987. The honey bee pollination component of horticultural crop production system. Hort. Rev. 9:237-272. Dobson, H. E. M. 1993. Floral volatiles in insect biology. p. 47-81. In: E. Bernays (ed.), Insect-plant interactions, Vol. V. CRC Press, Boca Raton, F1. Dodson, c., R Dressler, H. Hills, R Adams, and N. Williams. 1969. Biologically active compounds in orchid fragrances. Science 164:1243-1249. Dudareva, N., 1. Cseke, V. M. Blanc, and E. Pichersky. 1996. Evolution of floral scent in Clarkia: Novel patterns of S-linalool synthase gene expression in the C. breweri flower. Plant Cell 8:1137-1148. Dudareva, N., J. C. D'Auria, K. H. Nam, R A. Raguso, and E. Pichersky. 1998b. Acetyl CoA:benzylalcohol acetyltransferase-an enzyme involved in floral scent production in Clarkia breweri. Plant J. 14:297-304. Dudareva, N., R A. Raguso, J. Wang, J. R Ross, and E. Pichersky. 1998a. Floral scent production in Clarkia breweri. III. Biosynthesis and emission of benzenoid esters. Plant Physiol. 116:599-604. Henning, J. A., Y-S. Peng, M. A. Montague, and 1. R Teuber. 1992. Honey bee (Hymenoptera: Apidae) behavioral response to primary alfalfa (Rosales: Fabaeeae) floral volatiles. J. Econ. Entomol. 85:233-239. Galen, C. 1985. Regulation of seed set in Polemonium viseosum: floral scents, pollination and resources. Ecology 66:792-797. Gerlach, G., and R Schill. 1991. Composition of orchid scents attracting euglossine bees. Bot. Acta 104:379-391. Gunata, Y. Z., C. 1. Bayonove, R 1. Baumes, and R E. Cordonnier. 1985. The aroma of grapes. 1. Extraction and determination of free and glycosidically bound fractions of some grape aroma components. J. Chromatography 331:83-90. Kaiser, R 1991. Trapping, investigation, and reconstitution of floral scent. p. 213-248. In: P. Muller and D. Lamparsky (eds.), Perfume: Art, science, and technology. Elsevier Publ., New York. Knudsen, J. T., and 1. Tollsten. 1993. Trends in floral scent chemistry in pollination syndromes: floral scent composition in moth-pollinated taxa. Bot. J. Linn. Soc. 113:263284. Knudsen, J. T., 1. Tollsten, and G. Bergstrom. 1993. Floral scents-a check-list of volatile compounds isolated by head-space techniques. Phytochemistry 33:253-280.

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Kutchan, T. M. 1995. Alkaloid biosynthesis-the basis for metabolic engineering ofmedicinal plants. Plant Cell 7:1059-1070. Lewinsohn, E., N. Dudai, Y. Tadmor, I. Katzir, U. Ravid, E. Putievsky, and D. M. Joel. 1998. Histochemical localization of citral accumulation in lemongrass leaves (Cymbopogon citratus (DC.) Stapf., Poaceae). Ann. Bot. 81:35-39. Lewinsohn, E., M. Gijzen, T. J. Savage, and R. Croteau. 1991. Defense mechanisms in conifers. Relationship of monoterpene cyclase activity to anatomical specialization and oleoresin monoterpene content. Plant Physiol. 96:38-43. Lichtenthaler, H. K., M. Rohmer, and J. Schwender. 1997. Two independent biochemical pathways for isopentenyldiphosphate and isoprenoid biosynthesis in higher plants. Physiol. Plant. 101:643-652. Loughrin, J. H., T. R. Hamilton-Kemp, R. A. Anderson, and D. F. Hildebrand. 1990. Volatiles from flowers of Nicotiana sylvestris, N. otophora and Malus domestica: Headspace components and day/night changes in their relative concentrations. Phytochemistry 29:2473-2477. Loughrin, J. H., T. R. Hamilton-Kemp, R. A. Anderson, D. F. Hildebrand. 1991. Circadian rhythm of volatile emission from flowers of Nicotiana sylvestris and N. suaveolens. Physiol. Plant. 83:492-496. Loughrin, J., T. Hamilton-Kemp, H. R. Burton, R. A. Anderson, and D. F. Hildebrand. 1992. Glycosidically bound volatile components of Nicotiana sylvestris and N. suaveolens flowers. Phytochemistry 31:1537-1540. Matile, P., and R. Altenburger. 1988. Rhythms of fragrance emission in flowers. Planta 174:242-247. McGarvey, D. J., and R. Croteau. 1995. Terpenoid metabolism. Plant Cell 7:1015-1026. Mettal, D., W. Boland, P. Beyer, and H. Kleinig. 1988. Biosynthesis of monoterpene hydrocarbons by isolated chromoplasts from daffodil flowers. Eur. J. Biochem. 170:613616. Mookherjee, B. D., R. W. Trenkle, and R. A. Wilson. 1989. Live vs. dead, Part II. A comparative analysis ofthe headspace volatiles of some important fragrance and flavor raw materials. J. Ess. Oil. Res. 2:85-90. Nilsson, 1. A. 1978. Pollination ecology and adaptation in Platanthera chlorantha (Orchidaceae). Botaniska Notiser 131:35-51. Nilsson, L. A. 1983. Processes of isolation and introgressive interplay between Platanthera bifolia (L) Rich and P. chlorantha (Custer) Reichb. (Orchidaceae). Bot. J. Linn. Soc. 87:325-350. Overland, 1. 1960. Endogenous rhythm in opening and odor of flowers of Cestrum nocturnum. Am. J. Bot. 47:378-382. Patt, J. M., D. F. Rhoades, and J. A. Corkill. 1988. Analysis of the floral fragrance of Platanthera stricta. Phytochemistry 27:91-95. Pellmyr, O. 1986. Three pollination morphs in Cimicifuga simplex: incipient speciation due to inferiority in competition. Oecologia 8:304-307. Pichersky, E., E. Lewinsohn, and R. Croteau. 1995. Purification and characterization of Slinalool synthase, an enzyme involved in the production of floral scent in Clarkia breweri. Arch. Biochem. Biophys. 316:803-807. Pichersky, E., R. A. Raguso, E. Lewinsohn, and R. Croteau. 1994. Floral scent production in Clarkia (Onagraceae). I. Localization and developmental modulation of monoterpene emission and linalool synthase activity. Plant Physiol. 106:1533-1540. Pogorel'skaya, A. N., A. A. Prokof'ev, and S. A. Reznikova. 1980. Transport of monoterpenol and 2-phenylethanol alcohol glucosides from rose leaves to petals during flower maturation. Fiziol. Rast. (Moscow) 27:530-535.

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Raguso, R. A., and O. Pellmyr. 1998. Dynamic headspace analysis of floral volatiles: a comparison of methods. OIKOS 81:238-254. Raguso, R. A., and E. Pichersky. 1995. Floral volatiles from Clarkia breweri and C. concinna (Onagraceae): recent evolution of floral scent and moth pollination. Plant Sys. Evol. 194:55-67. Schiestl, F. P., M. Ayasse, H. F. Paulus, D. Erdmann, and W. Francke. 1997. Variation of floral scent emission and post pollination changes in individual flowers of Ophrys sphegodes subsp. Sphegodes. J. Chern. Ecology 23:2881-2895. Schnitzler, J. P., J. Madlung, A. Rose, and H. U. Seitz. 1992. Biosynthesis of p-hydroxybenzoic acid in elicitor-treated carrot cell cultures. Planta 188:594-600. Smith, B. H., and M. Ayasse. 1987. Kin-based male mating preferences in two species of halictine bees. Behav. Ecol. Sociobiol. 20:313-318. Stern, W., K. Curry, and W. M. Whitten. 1986. Staining fragrance glands in orchid flowers. Bul. Torrey Bot. Club 113:288-297. Sugden, E. 1986. Anthecology and pollinator efficacy of Styrax officinale subsp. redivivum (Styracaceae). Am. J. Bot. 73:919-930. Tang, J., Y. Zhang, T. G. Hartman, R. T. Rosen, and C. T. Ho. 1990. Free and glycosidically bound volatile compounds in fresh celery (Apium graveolens 1.). J. Agr. Food Chern. 38:1937-1940. Tollsten,1. 1993. A multivariate approach to post-pollination changes in the floral scent of Platanthera bifolia (Orchidaceae). Nord J. Bot 13:495-499. Tollsten, 1., and J. Bergstrom. 1989. Variation and post-pollination changes in floral odors released by Platanthera bifolia (Orchdaceae). Nord. J. Bot. 9:359-362. Traub, H., T. Robinson, and H. Stevens. 1942. Papaya production in the United States. U.S. Dept. Agr. Cir. 633. Van Dort, H., P. Jaegers, R. Ter Heide, and A. Van der Weerdt. 1993. Narcissus trevithian and N. geranium: analysis and synthesis of compounds J. Agr. Food Chern. 41:20632075. Wang, J., N. Dudareva, S. Bhakta, R. Raguso, and E. Pichersky. 1997. Floral scent production in Clarkia brewed (Onagraceae). II. Localization and developmental modulation ofthe enzyme SAM:(Iso)Eugenol O-methyltransferase and phenylpropanoid emission. Plant Physiol. 114:213-221. Wang, J., and E. Pichersky. 1997. Nucleotide sequence of caffeic acid O-methyltransferase from Clarkia breweri (Accesssion No. AF006009) (PGR97-104). Plant Physiol. 114:1567. Wang, J., and E. Pichersky. 1998. Characterization of S-adenosyl methionine: (iso)eugenol O-methyltransferase involved in scent production in Clarkia breweri. Arch Biochem. Biophys.349:153-160. Watanabe, N., S. Watanabe, R. Nakajima, J. H. Moon, K. Shimokihira, J. Inagaki, H. Etoh, T. Asai, K. Sakata, and K. Ina. 1993. Formation of flower fragrance compounds from their precursors by enzymic action during flower opening. Biosci. Biotech. Biochem. 57:1101-1106.

3

Triazoles as Plant Growth Regulators and Stress Protectants R. Austin Fletcher and Angela Gilley Department of Environmental Biology University of Guelph Guelph, Ontario Canada, N1G 2Wl Narendra Sankhla and Tim D. Davis Texas A & M University Texas Agricultural Experiment Station Dallas, Texas 75252 USA

I. Introduction II. Translocation and Efficacy of Application Methods III. General Plant Responses to Triazoles A. Morphological Effects B. Physiological Effects C. In Vitro Responses IV. Mode of Action A. Primary Effects 1. Gibberellin 2. Sterols 3. Abscisic Acid B. Secondary Effects 1. Cytokinins 2. Ethylene 3. Polyamines V. Stress Protection A. Drought B. Low Temperature C. High Temperature

Horticultural Reviews, Volume 24, Edited by Jules Janick ISBN 0-471-33374-3 © 2000 John Wiley & Sons, Inc. 55

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R. FLETCHER, A. GILLEY, N. SANKHLA, AND T. DAVIS

D. Environmental Pollutants and Other Stresses VI. Potential and Current Applications A. Ornamentals 1. Bedding Plants 2. Potted Plants 3. Foliage Plants 4. Woody Ornamentals 5. Geophytes 6. Orchids B. Turf and Amenity Grasses C. Arboriculture and Vegetation Management D. Agronomic Crops E. Vegetable Crops F. Spice Crops G. Fruit and Nut Species 1. Pome Fruits 2. Stone Fruits 3. Nut Crops 4. Small Fruits 5. Viticulture 6. Tropical and Subtropical Fruits VII. A Novel Seed Treatment Technology VIII. Summary Literature Cited

I. INTRODUCTION

In the late 1960s, several compounds from the chemical class of 1substituted imidazoles and 1, 2, 4-triazoles were commercially developed and successfully used for the treatment of plant and human fungal infections. These azole fungicides and antimycotics include the most active compounds known today for controlling plant diseases and human mycoses (BucheI1986). The azole fungicides belong to the large group of ergosterol biosynthesis inhibitors that interfere with the biosynthesis of fungal steroids. Certain azole compounds interfere with the biosynthesis of gibberellins and influence the morphogenesis of plants, indicating their possible use as plant growth regulators. Hence, several triazole derivatives (Table 3.1; Fig. 3.1) were developed and recommended for use worldwide as either fungicides or plant growth regulators (PGRs). The triazoles are the largest and most important group of systemic compounds developed for the control of fungal diseases in plants and animals (Siegel 1981). Commercial products have both fungitoxic and plant growth regulating properties, irrespective of whether they were

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

57

Table 3.1. Representative examples of triazole compounds recommended for use as fungicides or plant growth regulators. Compound

Application

Developer

Dichlobutrazole Paclobutrazol Propiconazole Etaconazole Ketaconazole Diconazole Uniconazole Triadimefon Triadimenol Triapenthenol Epoxiconazole BAS 111

Fungicide Plant growth regulator Fungicide Fungicide Fungicide (human) Fungicide Plant growth regulator Fungicide Fungicide Plant growth regulator Fungicide Plant growth regulator

ICI ICI Ciba-Geigy Ciba-Geigy Ciba-Geigy Sumitomo Sumitomo Bayer Bayer Bayer BASF BASF

released for one use or the other. They tend to be much more effective than many other PGRs, generally requiring relatively low rates of application (Fletcher et al. 1986; Jung et al. 1986; Davis et al. 1988; Gilley and Fletcher 1997). Of the various triazoles, paclobutrazol (PBZ) and uniconazole (UN!) thus far have been found to be the most active in OH

N

NLJ

CI

OH

CI

Paclobutrazol OH

L) N

N

Triapenthenol Fig. 3.1.

NLJ N

Uniconazole OR

o

©J0~ N

NLJ

BAS 111 ..W

Chemical structures of some triazole growth regulators.

58

R. FLETCHER, A. GILLEY, N. SANKHLA, AND T. DAVIS

retarding growth in both mono- and dicots (Fletcher et al. 1986; Gilley and Fletcher 1997). In an in vitro study with five different fungi, diconazole followed by uniconazole were the most fungitoxic (Fletcher et al. 1986). Epoxiconazole, a recently developed fungicide, has also exhibited PGR and stress protective properties (Siefert and Grossmann 1996). All triazole compounds are characterized by a ring structure containing three nitrogen atoms, a chlorophenyl, and a carbon side chain (Fletcher et al. 1986). Effectiveness as a fungicide or PGR is determined by the stereochemical configuration of the substituents on the carbon chain (Fletcher and Hofstra 1988). PBZ and triadimenol, for example, have four enantiomers, which differ in their relative activity as fungicides or inhibitors of gibberellin and sterol biosynthesis (Burden et al. 1987). There are indications that an R configuration at the chiral carbon bearing the hydroxyl group is the prime determinant for fungitoxicity, whereas enantiomers having an S configuration at this carbon atom are inhibitors of gibberellin biosynthesis and more effective as PGRs. From a comparative study using various enantiomers, Roberts and Mathews (1995) concluded that the PBZ-induced resistance of chrysanthemum plants derived from stage IV of micropropagation to desiccation was linked to the activity of the 2S, 3S enantiomer, presumably due to the inhibition of gibberellin biosynthesis. Triazoles have been tested extensively in the plant industry, and voluminous literature on their activity in plants has accumulated during the last decade. The current review is aimed at providing a comprehensive overview of the basic and applied aspects of triazole growth regulators in regulation of plant growth and development. We begin with a brief summary of the morphological, physiological, and in vitro responses elicited by triazoles, followed by an updated synthesis of their mode of action. Because the unique role of triazoles as plant multi-protectants deserves special attention, an in-depth coverage has been attempted to highlight their role in protection of plants from a variety of unrelated environmental stresses. The latter part of the review deals with their current and potential uses on ornamentals, fruit trees, agronomic crops, vegetable crops, turf and amenity grasses, arboriculture and vegetation management. It is not our intent to catalog all the published reports on the subject. Rather, we have used what we believe to be representative examples of plant responses to triazoles. For previous work, and aspects not covered in detail in the current review, the reader is referred to previously published reviews on the subject (Fletcher 1985; Davis et al. 1986, 1988; Fletcher and Hofstra 1988; Davis and Curry 1991; Rademacher 1991; Grossmann 1988, 1990, 1992).

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

59

II. TRANSLOCATION AND EFFICACY OF APPLICATION METHODS Triazoles are primarily believed to be transported acropetally in the xylem (Davis et al. 1988). However, recent findings show that PBZ is not exclusively xylem mobile as previously believed. In some plants (e.g. Pistachio chinensis Bunge, Ricinus communis 1.), PBZ has been detected both in xylem and phloem sap (Witchard 1997a,b), indicating that xylem is not the only route for translocation of triazoles (Browning et al. 1992; Hamid and Williams 1997). Results obtained with Ricinus, where PBZ was introduced through the hollow petioles, raise the possibility of introducing PBZ into the plant in a different way so that it is transported by both xylem and phloem, thus optimizing its effectiveness (Witchard 1997b). The metabolic fate of applied triazoles has not been studied thoroughly, though most have a high chemical stability (Jung et al. 1986) and thus tend to be catabolized by plants at a very slow rate (Davis and Curry 1991). It has been suggested that a strong correlation may exist between persistence of a triazole and its efficacy as a PGR (Reed et al. 1989). Two of the most active triazoles, PBZ and UNI, are comparatively resistant to degradation (Sterrett 1988) and this may limit their widespread use on food crops. A simple and efficient application method, capable of yielding consistent results, is a top priority in the commercial success of a growth regulator. Triazoles provide effective size control on many ornamental crops. However, when growers began to use these chemicals, many encountered difficulties in obtaining uniform marketable size. This was due partly to the high activity of the chemical and partly to the lack of an appropriate application procedure. In commercial practice, spray applications are often the method of choice, but can result in nonuniform plant size if suitable coverage is not obtained (Barrett and Nell 1990; Barrett et al. 1994a,b). It was observed that PBZ was more active when applied to the growing media and taken up through the roots (Davis et al. 1988) than when applied to leaves only. High spray volumes, which result in more thorough coverage of plant stems and a greater amount of solution entering the medium, increased the efficacy of PBZ (Barrett and Nell 1990). Barrett et al. (l994a) compared drench and spike application of paclobutrazol for height control of potted floriculture crops. PBZ drenches and spikes were effective for all crops tested, with a similar concentration response for all, except that drenches had greater efficacy than spikes on caladium that had the most rapid development rate. Both

60

R. FLETCHER, A. GILLEY, N. SANKHLA, AND T. DAVIS

drench and spike applications were also found suitable for controlling growth of tulips (Deneke and Keever 1992). Bailey (1989) demonstrated that UNI efficacy on chrysanthemum and poinsettia is not affected by spray carrier volume when concentration was varied to give the same active ingredient per plant. On the other hand, Gilbertz (1992) reported that PBZ and UNI had greater efficacy if applied soon after pinching, when there would be less leaf coverage on the medium. Barrett et al. (1994b) conducted experiments to determine effects of application site and spray volume on UNI efficacy. UNI applied only to mature leaves was less effective than were stem applications, whole-plant sprays, or medium drenches. The effect of UNI spray volume was also greater when the medium was not covered. These results indicate that the efficacy of UNI increased with increased stem coverage and with amount of chemical reaching the medium, which was achieved with high spray volumes. These findings are consistent with the view that UNI is also primarily transported via the xylem and does not move out of leaves via the phloem (Davis et al. 1988). Barrett (1982) reported reduced efficacy of soil-applied PBZ when used in conjunction with pine bark in the soil medium. The loss in activity was attributed to sequestering of the compound onto hydrophobic surfaces of the bark. For tree growth management under or near utility power lines, Profile 25SC(PBZ) can be applied around the base of a tree using either basal drench or soil injection technique in order to expose maximum root surface to the active ingredient (Davis et al. 1994). However, the magnitude of the response was found to be somewhat less than that observed with Cutless (flurprimidol) trunk implants. A peculiar situation occurs in mango when UNI is applied by conventional procedures. UNI was not efficacious on 'Tommy Atkins' or 'Keitt' mango trees unless the trees were pruned (Davenport 1994). Evidence indicates that the soil- or bark-applied UNI concentrates in the tree trunks and its effects are expressed only when buds residing in those areas of the trees are forced to sprout following severe pruning. All new growth from pruned scaffold limbs was severely stunted, resulting in permanent loss in productivity. Therefore, at least in mango, triazoles should be sprayed on developing leaves to promote direct uptake at the site of action and avoid concentration in trunks. It appears that in some fruit trees, especially plum and sour cherry species, the collar tissue (the vascular transition region), is a suitable location for influencing tree growth and fruiting (Grochowska and Hodun 1997). Relatively small amounts are applied to the tree and are separated in time and place from fruiting in subsequent years, and thus do not appear to pose any residual hazard to consumers or to the environment.

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

61

In some plants, efficacy of UNI is influenced by the stage of shoot development. For instance, in 'Prize' azalea, plants were taller and broader with later applications (Keever and Olive 1994), but these parameters decreased with increasing rate. Bypass shoot counts decreased quadratically with increased rate, but decreased when plants were treated at a later stage of shoot apex development. Application at a later stage increased time to flower and decreased flower count, while plants treated prior to shoot apex development flowered earlier with more blooms. In many plants, the growth regulatory effects oftriazoles are more pronounced when applied to seeds or young seedlings as a soil treatment. A novel seed treatment technology, which enables efficacious use of triazoles has been developed recently (Fletcher and Hofstra 1990). Compared to the conventional methods of application, the seed treatment procedure has several advantages. Because of its important implications in management of growth and yield, the salient features of this technology are described in detail in Section VII. III. GENERAL PLANT RESPONSES TO TRIAZOLES

A. Morphological Effects Triazoles are more potent than most other growth retardants and relatively low rates are required to inhibit shoot growth (Davis et al. 1988). However, even at high application rates they generally are not phytotoxic. The most pronounced effect of triazoles on plants is a reduction in height, with the treated plants being greener and more compact (Fletcher and Hofstra 1988). PBZ effectively suppresses growth in a wide range of species, including submerged aquatic species (e.g. Myriophyllum spicatum 1., Hydrilla verticillata (1. f.) Caspary) as well as noxious purple nutsedge (Netherland and Lembi 1992; Kawabata and DeFrank 1993). Reduction in shoot growth by triazoles occurs primarily as a consequence of reduced internode elongation and the effective dose varies within species and cultivars (Fletcher and Hofstra 1988; Davis and Curry 1991). PBZ reduced both cell number and length in safflower stems (Potter et al. 1993) and in chrysanthemums, cortical and pith cells were shorter and more tightly packed (Burrows et al. 1992). Initiation of secondary xylem in stem tissue of UNI-treated forsythia plants was suppressed and xylem length and width were rf:3duced. The treated plants also contained more phloem fibers with smaller cross sectional areas (Thetford et al. 1995). In peach shoots, PBZ reduced the proportion of xylem and increased that of phloem and cortex, and increased xylem density (Aguirre and Blanco 1992). Starch deposition

62

R. FLETCHER, A. GILLEY, N. SANKHLA, AND T. DAVIS

in the pith and xylem were also enhanced. In bean, gibberellin (GA)induced hollowing of the first internode was also significantly reduced by UNI (Takano et al. 1995). Similarly, in Rosa x hybrida 1., phyllody was reduced following PBZ application (Mor and Zieslin 1992). Triazoles have several morphological effects on leaves. They reduce leaf area, but increase epicuticular wax, width, and thickness (Gao et al. 1987) and hence leaf dry weight per unit area is increased (Davis et al. 1988). PBZ decreased length of wheat leaves by reduction of cell length rather than cell number (Tonkinson et al. 1995). In wheat and chrysanthemums, triazoles increased thickness of leaves by inducing additional layers of palisade mesophyll cells which had a smaller diameter (Gao et al. 1987; Burrows et al. 1992). Increased leaf width was correlated with additional vascular bundle formation in wheat (Gao et al. 1987). Treated leaves also tend to be darker green, possibly because of an increase in chlorophyll content per unit leaf area (Sankhla et al. 1985; Fletcher and Arnold 1986; Fletcher et al. 1988). In radish, triadimefon (TRIAD) induces the formation of sun-type chloroplasts, which allows a higher photosynthetic quanta conversion; this effect is probably related to increased cytokinin levels (Lichtenthaler 1979). In wheat leaves, both TRIAD and PBZ increased chloroplast size along both the long and short axes, being 34 and 30% longer than the control, respectively (Gao et al. 1987). In maize seedlings, PBZ increased the stromal lamella and number of thylakoids per granum stack, thereby increasing the cross sectional area, but decreasing the number of grana (Sopher et al. 1999). Triazoles have also been shown to influence root growth, although the effect may be either inhibitory or stimulatory, depending on the plant and the concentration of the triazole compound used (Fletcher and Hofstra 1988; Davis et al. 1988). PBZ caused thickening of maize root apices and increased their starch content. Immunofluorescence microscopy of cortical microtubules, coupled with a comparison of cell widths, lengths, and shapes indicated that the meristem and immediate post-mitotic zone were the targets of GA deficiency. Cortical cells in these regions were impaired in their ability to develop highly ordered transversal arrays of cortical microtubules. Consequently, the cells became wider and shorter (Baluska et al. 1993). PBZ treatment of primary pea roots inhibited root extension but promoted radial cell expansion (Wang and Lin 1992). Increased root diameter has been correlated with larger cortical parenchyma cells in soybean and maize (Barnes et al. 1989) and in chrysanthemum may be due to an increase in the number of rows and diameter of cortical cells (Burrows et al. 1992). Some triazole compounds have been found to be effective in promoting the formation of adventitious roots in plant cuttings (Davis and

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

63

Sankhla 1988). Both TRIAD and PBZ effectively stimulated rooting in bean hypocotyls (Davis et al. 1985; Fletcher et al. 1988). In mung bean cuttings, this effect was increased synergistically in the presence of indolebutyric acid (IBA) (Pan and Zhao 1994). Similar results were found with UNI and PBZ in English ivy (Geneve 1990). This stimulation in root growth may be related to the increased partitioning of assimilates towards the roots due to a decreased demand in the shoots (Symons et al. 1990; Wang et al. 1985). It has also been suggested, however, that this effect may be due to an increase in the activity of enzymes important for root formation (Upadhyaya et al. 1986; Bora et al. 1991). A mung bean rooting bioassay found PBZ to be particularly potent in promoting the rooting of cuttings relative to other triazole compounds studied (Porlingis and Koukourikou-Petridou 1996). Although the effects on root growth vary, a higher root-to-shoot ratio is usually a characteristic oftriazole-treated plants, primarily due to the drastic reduction in shoot growth (Fletcher and Hofstra 1988; Davis et al. 1988). An interesting observation relates to the growth of the roots of triazole-treated Ziziph us mauritiana Lam. seedlings during and after recovery from moisture stress (N. Sankhla 1998, unpublished results). The roots of PBZ- and UNI-treated seedlings not only continued to grow and produce new roots under moisture deprivation, but on rewatering a very rapid regeneration of new roots occurred. This may have a role in plant survival under moisture stress. B. Physiological Effects The foliage of triazole-treated plants typically exhibits intense dark green color compared to untreated controls. In most cases, this is due to enhanced chlorophyll content (Davis et al. 1988; Sankhla et al. 1996b, 1997) and more densely packed chloroplasts in a smaller leaf area (Khalil 1995). Just as they do with chlorophylls, the triazoles also greatly increase anthocyanin accumulation in carrot tissue cultures (Han and Dougall 1992). However, in general, triazoles have little direct effect on net photosynthesis rates on a leaf area basis, but indirectly, by inhibiting leaf expansion, they may decrease whole-plant photosynthesis (Davis et al. 1988). In several plants, the leaves on triazole-treated plants were retained longer than on controls and the onset of leaf senescence was also delayed considerably (Davis et al. 1988; Davis and Curry 1991). Although detailed studies on the effect of triazoles on respiration are lacking, available evidence indicates that respiratory activity is decreased following treatment with these chemicals (Davis et al. 1988; Wang and Lin 1992). In rat mitochondria, PBZ inhibited electron transport by 90%

64

R. FLETCHER, A. GILLEY, N. SANKHLA, AND T. DAVIS

at 3~M but was stimulatory at a low concentration (10-15 ~M). Thus, deficiency in energy supply, at high concentrations of PBZ, could be an additional mechanism for growth retardation by triazoles (Barr et al. 1996). Depending on the species, leaf conductance, transpiration rate, and water use may also be modulated following treatment with triazoles. Some species exhibit improvement of plant water status, while in others water use on a leaf area basis remains unaffected (Steinberg et al. 1991; Norcini 1991; Li and van Staden 1998a). Santakumari and Fletcher (1987) observed that triazoles partially close stomata in isolated epidermal strips of Commelina benghalensis 1. Since that initial study, partial stomatal closure following application of triazoles has been confirmed in several other plants, including pea, sesame, soybean, peanut, and chickpea (Bora et al. 1990a,b; Bora and Mathur 1990,1991, 1992). Triazoles are also known to shift assimilate partitioning from leaves to roots and could also alter mineral uptake and plant nutrition (Davis et al. 1988; Yelenosky et al. 1995). In canola leaves, TRIAD also promoted nitrate uptake and the activity of nitrate reductase, an enzyme that is also stimulated by cytokinins (Srivastava and Fletcher 1992). Although the triazoles affect the activities of several other enzymes, by far the most pronounced effect has been observed with the enzymes related to detoxification of active oxygen species and antioxidant metabolism (Sankhla et al. 1992b, 1996b, 1997; Kraus and Fletcher 1994; Kraus et al. 1995b). The role oftriazoles in enhancing antioxidant activity and thus protecting the plants from various environmental stresses is detailed in a later section.

C. In Vitro Responses At the time of publication of the earlier reviews on triazoles by Davis et al. (1988) and Fletcher and Hofstra (1988), the effect of triazoles on growth and differentiation in vitro had not been evaluated thoroughly. It has been subsequently observed that the triazole growth regulators, PBZ and UNI, reduced in vitro growth of moth bean callus and inhibited the differentiation of roots and shoots from the callus (Gehlot et al. 1989; Sankhla et al. 1991). Addition of GA 3 to the culture medium, in combination with the triazoles, restored callus growth to a level equivalent to that of the untreated control. PBZ also induced a number ofbiochemical changes in the callus cultures, including increased total sugars, free proline, soluble protein, and higher activities of peroxidase, protease, and RNase (Davis et al. 1988; Gehlot et al. 1989). In many plants,

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

65

these growth retardants promote in vitro formation of roots and shoot buds, and production of microshoots (Marino 1988; Ziv 199Z; Sankhla et al. 1994). In cultured melon cotyledons, PBZ mimicked the effects of auxin and induced roots at the basal cut portion. PBZ-induced temporal changes of reserve polypeptide PZO-Z5 also were similar to those induced by auxin (Leshem et al. 1994). In members of Araceae, the imidazole fungicides and PBZ, which share some structural features, strongly enhance the shoot-inducing effect of cytokinins (Werbrouck and Debergh 1996). In Lilium specisum Thunb., PBZ also reduced the dormancy of in vitro grown bulblets (Gerrits et al. 199Z). In Citrus sinensis Osbeck., where GA 3 suppresses somatic embryogenesis, PBZ stimulated the process (Spiegel-Roy and Saad 1986). The number of somatic embryos also increased following application of PBZ, but not UNI in Echinochloa frumentacea (Roxb.) Link (Sankhla et al. 199Za). Both compounds decreased embryo germination when added to germination medium. Likewise, Li and Wolyn (1995) observed that UNI and PBZ significantly enhanced the production of asparagus somatic embryos and their conversion to plantlets, although ancymidol and ABA were more effective than PBZ and UNI. Liquid cultures, used to scale-up micropropagation, tend to induce hyperhydric shoots with abnormal leaves. Treatment with triazoles inhibited leaf expansion and enhanced bud and/or protocorm proliferation in Aechmea faciata (Lindl.) Bak. (Ziv 1986) and gladiolus (Ziv 1990; Steinitz et al. 1991). In liquid shake or bioreactor cultures, philodendron proliferated profusely in the presence of PBZ (Ziv and Ariel 1991). PBZ inhibited leaf development and induced the formation of bud clusters. Regenerated plants resumed normal growth after transplanting. Rhizome multiplication, cormlet proliferation, and bulblet formation after triazole application have now been reported in several other plants (Table 3.Z). In Nerine x mansellii, Hort. ex Bak., as in other members of Amaryllidaceae, the natural propagation rate is low and conventional micropropagation techniques are labor intensive and expensive. However, using PBZ, proliferation and regeneration in liquid culture or in a bioreactor have become a distinct reality (Ziv et al. 1995). Meristematic tissues from differentiated peduncle explants, when subcultured in the presence of PBZ, proliferated into compact but friable meristematic clusters. Exclusion of PBZ from the medium resulted in the development of proembryogenic masses. Inclusion of 6y-y-dimethyl(allyl) amino purine (ZiP) in the media enhanced the differentiation of somatic embryos. These embryos could be induced to form plants in low growth regulator or growth regulator free media, or to differentiate into meristematic

66

R. FLETCHER, A. GILLEY, N. SANKHLA, AND T. DAVIS

clusters in the presence of PBZ. Bulblet formation was enhanced in the presence of auxin and high sucrose concentrations. Embryogenic tissue attached to the bulblets developed into secondary embryos. Thus, it appears that incorporation of PBZ in large-scale liquid culture for the production of embryogenic tissue is a potentially practical method for mass production of Nerine bulbs (Ziv et al. 1995). Microtubers have also become an important mode of rapid multiplication for pre-basic stock in seed tuber multiplication as well as germplasm exchange. Chandra et al. (1992) conducted experiments to evaluate the efficacy of two triazoles, TRIAD and UNI, compared to benzylaminopurine (BA) for in vitro microproduction in three leading Indian cultivars of potato (Kufri Jyoti, Kufri Badshah, and Kufri Sindhuri). At a low concentration, UNI was found to be more effective than BA in producing higher numbers and weight of microtubers. A similar response has been reported for other potato cultivars (Simko 1993; Li and Zhu 1994). Wattimena et al. (1991) used static liquid-liquid method to induce in vitro tuberization. Both PBZ and UNI showed significant effects in earliness oftuberization, number and size of microtubers, and their dormancy characteristics. Another consistent effect of triazoles in vitro relates to their ability to reduce wilting, improve desiccation resistance, and increase posttransplant survival in a number of plant species (Smith et al. 1990, 1991; Sankhla et al. 1992a; Eliasson et al. 1994). In carnation, triazoles also reduced the hyperhydricity caused by the presence of thidiazuron (Sankhla et al. 1996a), and this was accompanied with an increase in the antioxidant activity. IV. MODE OF ACTION The triazoles are characterized by a lone pair of electrons on the Sp2hybridized nitrogen atom in the heterocycle. This pair of electrons occurs on the periphery of the molecule, enabling it to interact with cytochrome P-450 dependent monooxygenases. As the 6th ligand, it bonds to the protoheme iron of cytochrome P-450, thereby displacing oxygen required for catalytic reactions (Grossmann 1990). In most fungi, ergosterol is an indispensable component of fungal membranes, and inhibition of this sterol leads to a loss of membrane integrity and ultimate cell death (Siegel 1981; Koller 1987). The demethylation of the ergosterol precursor at the C-14 position of 24-methylenedihydrolanosterol proceeds via several oxidation steps catalyzed by a cytochrome-P450 mixed function oxygenase (Koller 1987). The relative activity of enantiomeric

Table 3.2.

In vitro effects of triazole growth regulators. Plant

Aechmea faciata (Lindl.) Bak. Albizzia julibrissin Durazz. Allium trifoliatum Cyr. Alstroemeria 1. x Asparagus officinalis 1. Beta vulgaris 1. Citrus sinensis (1.) Osbeck Citrus reticulata Blanco Citrus sunki Hort. Ex Tan. Colchicum autumnale L. Dendranthema x grandiflora Ramat. Dianthus caryophyllus L. Echinochloa frumentacea (Roxb.) Link Gladiolus glandiflorus Hort. x G. tristis 1. Hyacinthus orientalis L. Lapageria rosea Ruiz & Pav. Lilium speciosum Thunb. Musa 1. sp. Nerine mansellii Hort. ex Bak. Pelargonium hortorum L. H. Bailey Philodendron hastatum C. Koch & H. Sello. OJ

'1

Response Protocorm proliferation Shoot bud formation from excised roots promoted In vitro propagation, germplasm storage Rhizome multiplication Production of somatic embryos, and their conversion to plantlets enhanced In vitro acclimatization Reduced winter injury Reduced winter injury Shoot proliferation Cormlet growth Reduction in wilting, improved desiccation resistance Number of microshoots increased, hyperhydricity reduced, antioxidant activity increased Number of somatic embryos increased, shoot elongation retarded, post-transplant survival increased Cormlet proliferation in liquid cultures Bulblet formation induced, leaf formation blocked Proliferation of rhizome buds Bulblet dormancy reduced In vitro hardening Induction of friable meristematic clusters Number of somatic embryos increased Prevention of abnormal leaf growth, bud proliferation

Reference Ziv 1986 Sankhla et al. 1993 Viterbo et al. 1994 Bond and Alderson 1993 Li and Wolyn 1995 Ritchie et al. 1991 Deng et al. 1991 Deng et al. 1991 Ramos et al. 1996 Ellington et al. 1997 Smith et al. 1990; Smith et al. 1991 Sankhla et al. 1994, 1996a Sankhla et al. 199Za Ziv 1990; Steinitz et al. 1991 Bach et al. 1992 McKinless and Alderson 1993 Gerrits et al. 1992 Murali and Duncan 1995 Lilien-Kipnis et al. 1992 Hutchinson et al. 1997 Ziv and Ariel 1991; Ziv 1992

Ol Q:)

Table 3.2.

(continued) Plant

Prunus cerasus 1. Prunus communis 1. Prunus serotina J. F. Ehrh. Rosa L. spp. Solanum tuberosum 1. Spathiphyllum floribundum Linden & Andre Vigna aconitifolia (Jacq.) Marecal Zea mays 1.

Response In vitro rooting enhanced, roots short, thick In vitro rooting enhanced, roots short, thick Enhanced acclimatization in vitro Wilting reduction Microtuber production enhanced, bud regeneration promoted, survival after transplantation increased Cytokinin-induced adventitious shoot proliferation enhanced Callus growth and differentiation suppressed, GA reversed the effect Embryo germination

Reference Marino 1988 Marino 1988 Eliasson et al. 1994 Roberts et al. 1992 Chandra et al. 1992; Simko 1993; Li and Zhu 1994; Opatrna et al. 1997 Werbrouck and Debergh 1996 Gehlot et al. 1989; Sankhla et al. 1991

White and Rivin 1993

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

69

forms of a triazole fungicide has been correlated to their affinity with this target enzyme (Carelli et al. 1992). In plants, triazole compounds interfere with the biosynthesis of gibberellin by inhibiting the oxidation of ent-kaurene to ent-kaurenoic acid (Graebe 1987; Izumi et al. 1985). The isoprenoid pathway (Fig. 3.2) contains polymers derived from a basic five-carbon isoprene unit. The pathway generates animal, fungal, insect, and plant hormones as well as other metabolites, including vitamins A and E, phytoalexins, steroids, allelopathic compounds, and insect anti-feedants (Fletcher 1985; Goodwin and Mercer 1990). Specific products of the isoprenoid pathway that are inhibited by the triazoles are ergosterol in fungi and gibberellins in plants (Fig. 3.2). It has been suggested (Fletcher and Hofstra 1985) that the PGR properties oftriazoles are mediated by interfering with the isoprenoid pathway and thus modulating the balance of important plant hormones, including GA, ABA, and cytokinins. The ultimate effect, therefore, would be dependent on

Acetate

~

Mevalonic acid

~

Antheridiol (f)

Isopentenyl-PP (C-5)

------i~.

Geranyl-PP ~ (C-10) lr------.~

Monoterpenoids

Farnesyl-PP (C-15)

Insect juvenile hormone (i)

I t

Squalene (C-30)

...

//

Isopentyl adenosine Cytokinins (p)

...

Sesquiterpenoids Abscisic acid (p)

Lanosterol Cholesterol

Tocopherols (Vitamin E)

If''-.

Testosterone (a) Estrogen (a) Carotenoid (C-40)

/\

Vitamin A

Geranylgeranyl-PP (C-20)

- -.... ~

I------!---..,)/¥o--....

... .......

Abscisic acid (p)

//

~

Phytol Diterpenoids Gibberellins (p)

Higher terpenoids

Fig. 3.2. The isoprenoid pathway is derived from a five carbon (C-5) isoprene unit and generates animal (a), fungal (f), plant (p), and insect (i) hormones. Triazoles inhibit (1/) ergosterol and gibberellins in fungi and plants, respectively.

70

R. FLETCHER, A. GILLEY, N. SANKHLA, AND T. DAVIS

the dynamic equilibrium of these hormones at a specific stage of plant growth and development. A. Primary Effects 1. Gibberellin. Regulation of the enzymes and products in the gibberellin biosynthetic pathway by triazoles has been reviewed by Graebe (1987) and Hedden and Kamiya (1997). Triazoles interfere with the first three steps in the pathway of ent-kaurene oxidation and thus the formation of ent-kaurenol, ent-kaurenal, and ent-kaurenoic acid are inhibited, whereas steps following ent-kaurenoic acid in the pathway are not affected (Izumi et al. 1985; Graebe 1987). These microsomal oxidation reactions are catalyzed by kaurene oxidase, a cytochrome P-450 hydroxylase (Izumi et al. 1985; Graebe 1987; Rademacher 1991). Interference with the different isoforms of this enzyme could lead to inhibition of GA biosynthesis and abscisic acid (ABA) catabolism (Rademacher 1997) as shown in Fig. 3.3. Inhibition of GA biosynthesis as the primary effect of the triazole PGRs is supported by the evidence that triazole-treated plants have lower concentrations of GA-like substances (Rademacher 1991; Graebe 1987) and that the PGR and stress protective properties of triazoles could be reversed with the application ofGA (Davis et al. 1988; Fletcher and Hofstra 1988; Guoping 1997; Gilley and Fletcher 1998). The effects of triazoles and GA are mutually antagonistic, as seen in examples of inhibition of triazole-induced physiological and biochemical processes. These studies, using different systems, indicate that the reversal of the triazole effects by GA is independent of the time of application, since similar results were obtained when GA was applied before (Davis et al. 1988), after (Davis et al. 1988; Lee et al. 1985) or simultaneously (Gilley and Fletcher 1998; Porlingis and Koukourikou-Peridou 1996; Fletcher et al. 1988). Based on the interactions of triazoles and GA, it is logical to conclude that the PGR and stress protective effects of the triazoles are a consequence of their primary action as inhibitors of GA biosynthesis.

2. Sterols. The fungicidal effects of triazoles can be attributed to their interference with the biosynthesis of ergosterol, resulting in depletion of the major sterol and an accumulation of 14-methyl sterols (Siegel 1981; Koller 1987). There is evidence that suggests that certain triazole isomers interfere with sterol synthesis in plants. In barley seedlings, treatment with triadimefon and triadimenol reduced levels of C-4, 4desmethylsterols and increased sterols with C-4 methyl groups (Buchenauer and Rohner 1981). Similar results were obtained with PBZ and triapenthenol, which reduced shoot growth of barley seedlings,

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

ABA-Catabolism

GA-Anabolism

t

t GGPP

i

Kaurene

71

Monooxygenases ...................

t t (Violaxanthin) t t C6~~

Kaurenol ~~~J

[6'-OHMe-ABA]

}_~:I_____~:~:",, // l-;~:~~~c Acid

Kau

Kaurenoic Acid

GA 12-aldehyde

Cyt. P-450-

dependent

Conjugates

~

j

Fig. 3.3. Proposed involvement of different isoforms of cytochrome P450-dependent monooxygenases in GA and ABA metabolism (after Rademacher 1997).

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induced the accumulation of abnormal sterols, and also changed the ratios of campesterol, stigmasterol, and sitosterol (Burden et al. 1987). This change in sterol balance may involve changes in cell membranes that may be reflected in increased acclimatization and frost hardiness. When white spruce seedlings were treated with a soil application of TRIAD, transpiration rates declined immediately after treatment. In roots, TRIAD significantly increased the ratios between free sterols and sterol esters and decreased the ratios between sterol esters and acylated sterol glycosides (Sailerova and Zwiazek 1997). Treatment of celery cell suspension cultures with PBZ and three other triazoles resulted in decreased growth and accumulation of 14 alpha-methyl sterols accompanied by a loss of campesterol, stigmasterol, and sitosterol, and growth could be restored by adding sterols to the culture (Yates et al. 1993). It was observed that to achieve substantial alterations in the sterol profiles of barley shoots, comparatively high doses of triazole were necessary (Grossmann 1992). A decline in stigmasterol/sitosterol ratio has been observed after treatment with PBZ and tetcyclacis and it has been suggested that inhibition of cell division in cell suspensions and sub apical meristems by high concentrations of these growth regulators is mediated by a change in sterol synthesis and thus modified membrane properties (Grossman 1992). Although the involvement of sterols in plant growth and development is not well established, it has been suggested that the PGR effect of triazole compounds may be related to their effects on sterol biosynthesis (Davis and Curry 1991). 3. Abscisic Acid. ABA, commonly considered a "stress hormone" (Zeevart and Creelman 1988), has been implicated in plant acclimation and protection against various environmental stresses such as heat, chilling, drought, and flooding (Mackay et al. 1990). ABA can be derived directly from farnesyl-P-P or indirectly from carotenoids, more specifically from xanthoxin (Zeevart and Creelman 1988). Although experimental evidence favors the indirect pathway, in either case mevalonic acid is the precursor (Fig. 3.2). In bean plants, the triazole fungicide, TRIAD, induced a transient rise in ABA levels, reduced transpiration, and protected the plants from drought (Asare-Boamah et al. 1986b). Similar transient increases in ABA levels after treatment with triazoles have been observed in cell suspensions, detached leaves and in young seedlings (Grossmann 1992). The ABA level in mandarin fruitlets treated with UNI was about four-fold higher than in controls (Kojima et al. 1996). In UNI-treated bean plants, the increase in ABA level in the primary leaves was biphasic, which correlated closely with stomatal resistance and the accumulation of UNI in the leaves (Mackay et al. 1990).

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Water uptake and transpiration rates were reduced by triapenthenol, while there was up to a twenty-fold increase in ABA (Lurssen 1987). Increases in the levels of ABA have also been associated with triazoleinduced cold hardiness (Tafazoli and Beyl 1993). There is evidence to suggest (Hauser et al. 1990) that the increase in ABA levels by triazoles is due to prevention of its catabolism to phaseic acid (Fig. 3.3), an enzymatic step that is catalyzed by a cytochrome P-450-dependent monooxygenase (Zeevaart et al. 1990). Although most reports have demonstrated an increase in levels of ABA in plants after triazole treatment, there are reports to the contrary. In wheat seedlings, levels of ABA were similar between control and UNI treatment (Buta and Spaulding 1991), whereas in rice seedlings PBZ reduced ABA levels (Izumi et al. 1988). In celery cell suspension cultures, ABA levels transiently increased shortly after treatment with triazoles, but later fell below those of the controls at the higher concentrations oftriazoles (Hauser et al. 1992). Similar reduction in ABA levels after triazole treatment has been reported in apple (Wang et al. 1985), rape (Hauser et al. 1990), and soybean (Grossmann 1990). These apparent discrepancies may be ascribed to the use of different growth conditions, application methods, plant species, developmental stage, and the concentration oftriazole used (Fletcher and Hofstra 1985; Buta and Spaulding 1991; Grossmann 1990). Because the hormonal balance of plants is in a dynamic state (Fletcher and Hofstra 1985), the estimated ABA levels could also depend on the time of analysis after triazole treatment. The transient rise in ABA levels after triazole treatment (Asare-Boamah et al. 1986b; Mackay et al. 1990; Grossman 1992) has also been observed during cold hardening of plants (Chen et al. 1983). Since increases in ABA levels have been associated with plant stress protection (Zeevaart and Creelman 1988), it is suggested that triazole-induced stress protection could be mediated at least partially, via its effects on ABA levels (Fletcher and Hofstra 1988). Recently, interactions of growth retardants with the metabolism of ABA have attracted special interest (Rademacher 1997). Results with enantiomers and their variable effects on ABA metabolism have confirmed that different isoforms of the monooxygenases exist which may have overlapping functions (Rademacher 1997). It is likely that regulation of such monooxygenases could be a part of the plant's response to cope with a stressful environment. It is envisaged (Fig. 3.3) that, under favorable conditions, most monooxygenases might be "switched on," resulting in high GA but low ABA levels. This may result in intensive shoot growth and photoassimilation. On the other hand, under unfavorable situations such as moisture stress, these enzymes could be "turned off," leading to low GA and high ABA levels (Rademacher 1997)

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and resulting in reduced shoot growth, photoassimilation, and transpiration. However, it should be borne in mind that the monooxygenases have several isozymes, and it is generally difficult to study and characterize them. Instead, characterization and cloning of kaurene synthase may offer an attractive possibility in mimicking triazole action. B. Secondary Effects 1. Cytokinins. Cytokinins stimulate cell division, and it is now widely

accepted that cytokinins produced in roots move in the xylem to the shoot where they regulate both development and senescence (Letham and Palni 1983; Binns 1994). Although previous studies had shown that cytokinins retard senescence in excised leaves, similar effects on intact plants were first reported by Fletcher in 1969. Later, cytokinins were shown to stimulate chloroplast differentiation and chlorophyll biosynthesis (Fletcher et al. 1982). TRIAD and triadimenol delayed leaf senescence and it has been suggested that they had cytokinin-like properties (Buchenauer and Rohner 1981). Using the cucumber cotyledon bioassay developed for cytokinins (Fletcher et al. 1982), it was found that TRIAD was not active as a cytokinin, but rather it induced the plant to produce more cytokinins, probably by stimulating root growth (Fletcher and Arnold 1986). BAS 111 (1-phenoxy-3-(lH-2,4-trizol-lyl) 4-hydroxy-5,5dimethylhexane), delayed senescence of pumpkin cotyledons and stimulated greening in oilseed rape cotyledons, these effects being associated with increased cytokinin levels (Grossmann 1992). It has been suggested that the enhanced levels of cytokinins found in the roots of soybean seedlings after treatment with various retardants, including BAS 111, could be due to either a stimulated synthesis of cytokinins that are transported to the shoot or prevention of cytokinin degradation (Grossman 1992). In rice (Izumi et al. 1988) and soybean seedlings (Grossmann 1992), the increased cytokinins were identified as trans-zeatin, dihydrozeatin, and its ribosides. Several reports indicate that triazoles delay senescence in various plants, including Kentucky bluegrass (Goatley and Schmidt 199Gb), grapevine (Hunter and Proctor 1992), blueberry (Basiouny and Sass 1993), pumpkin, oilseed rape (Grossmann 1992), and soybean (Sankhla et al. 1985; Kraus et al. 1993). Although UNI delayed senescence of intact soybean cotyledons, it had no effect on excised cotyledons. Furthermore, triazoles are ineffective in extending the shelf life of cut flowers (R. A. Fletcher, unpublished) and it is suggested that the triazoles are more effective in actively metabolizing tissues. Because previous studies have shown that cytokinins or chemicals like thidiazuron with

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cytokinin-like activity stimulate chlorophyll synthesis (Visser et al. 1992) and retard senescence (Fletcher 1969; Letham and Palni 1983; Binns 1994), it could be concluded that these and similar effects induced by triazoles are a consequence of increased cytokinin levels. 2. Ethylene. In several plant species, triazoles reduce ethylene formation

(Grossmann 1992; Grossmann et al. 1993). Following heat stress in wheat or after application oftriclopyr, an auxinic herbicide on soybeans, ACC levels increased by 40 and 400% respectively in both the control and UNI treated seedlings. This was accompanied by an increase in ethylene production from the control, but not from the UNI-treated seedlings (Kraus et al. 1991). These results suggest that UNI inhibits ethylene synthesis by interfering with the conversion of ACC to ethylene by ACC oxidase. Subsequent studies with ACC oxidase suggest that cytochrome P-450 monooxygenase reactions may be involved in the conversion of ACC to ethylene (Kraus et al. 1992). Similarly, BAS 111 inhibited ethylene production in the exponential growth phase of sunflower cell suspensions with a concomitant increase of ACC which was converted to N-malonyl-ACC, which led to the conclusion that the triazole inhibits ACC oxidase activity (Grossmann 1992). Through its inhibitory effects on ethylene biosynthesis, UNI and PBZ were found to delay senescence of intact soybean cotyledons (Kraus et al. 1993) and oilseed rape cotyledons (Grossmann et al. 1994). The proposal that the effects of triazoles are mediated by a change in hormonal balance (Fletcher and Hofstra 1985) is supported by the observation that retardation of senescence in pods of oil seed rape is accompanied by an increase in cytokinins and a decrease in abscisic acid and ethylene levels (Grossmann et al. 1994). 3. Polyamines. S-adenosylmethionine (SAM) is the shared common precursor for both ethylene and polyamine biosynthesis (Yang and Hoffman 1984). Inhibition of ethylene biosynthesis should therefore affect polyamine metabolism. Increases in the levels of spermidine and spermine have been reported after treatment with PBZ and UNI in apple and mung beans respectively and, in both these cases, higher polyamine levels were associated with increases in root number (Hofstra et al. 1989). In sunflower cell suspensions treated with BAS 111, the conversion of ACC to ethylene was blocked and it was proposed that SAM was diverted from ethylene biosynthesis and increasingly incorporated into spermidine and finally spermine (Grossmann et al. 1993). Consequently, free putrescine in the cells, the direct precursor of these biologically more active polyamines, simultaneously decreased. The increased polyamine levels observed after triazole treatment may in part account

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for some of the senescence and root-promoting effects observed after triazole treatment (Fletcher and Hofstra 1988). V. STRESS PROTECTION

Crop plants are often subjected to environmental stresses that interfere with their normal physiological processes, affecting growth, development and, ultimately, crop yield. In addition to their growth regulatory and fungicidal effects, triazoles have been found to be highly effective in protecting plants from various environmental stresses (Davis et al. 1988; Fletcher and Hofstra 1988). TRIAD is a fungicide which is highly active against several economically important fungal diseases, including powdery mildew, smut, bunt, and rust (Buchenauer and Rohner 1981). In addition to its fungicidal action, it was demonstrated that TRIAD protected plants from injury due to biotic and abiotic stresses, including powdery mildew, drought, chilling, ozone, heat, and air pollutants. Hence, the triazoles were referred to as "plant multi-protectants" (Fletcher and Hofstra 1985). A. Drought TRIAD treatment is known to improve the survival of plants during periods of drought (Fletcher et al. 1988). Previous studies with TRIAD showed that it reduced transpiration in soybean, radish, and pea, and increased yield under water stress conditions (Fletcher and Hofstra 1988). Reduction of transpiration and protection from drought in bean by triazoles was associated with a reduction in shoot weight and length, leaf area, and increased diffusive resistance, indicating partial closure of stomates and a transient rise in ABA levels (Asare-Boamah et al. 1986b). Triazole-treated plants characteristically use less water and may be able to withstand drought better than untreated plants (Davis et al. 1988). Water use by UNI-treated plants was reduced by 35% due to reduction in leaf area and lower stomatal conductance (Fuller and Zajicek 1995). In maize, treatment with UNI resulted in maintenance of a higher relative water content and diffusive resistance, and decreased the relative conductivity and transpiration rate in the seedlings of a drought-resistant cultivar (Li and van Staden 1998a). The increased tolerance to drought induced by UNI in the drought-resistant cultivar of maize was due to the maintenance of increased antioxidant activity (Li et al. 1998). Even the callus of the drought-resistant cultivar of maize indicated higher antioxidant activities under water stress, and exhibited

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less oxidative damage as indicated by the levels of hydrogen peroxide and malondialdehyde (Li and van Staden 1998b). In wheat grown under moisture stress conditions, TRIAD reduced transpiration, increased the relative water content, membrane stability and significantly increased yield (Sairam et al. 1995). There are also several reports of reduced transpiration rates and increased drought resistance by triazoles in tree seedlings, including white spruce (Sailerova and Zwiazek 1997), black locust (Shen and Zeng 1993), jack pine (Marshall et al. 1991), and elm seedlings (Ashokan et al. 1995). PBZ protected black spruce seedlings against predisposition to gray mold induced by a combination of drought and high temperature (Zhang et al. 1994). The use of PBZ as an antitranspirant in conifers is well established and it is now registered for use under the trade name "Confer" by Zeneca Corp., Ontario, Canada. Due to the beneficial effects of triazoles in water conservation, their use in plant micropropagation has increased. PBZ and UNI added to culture media reduced water loss and increased survival of in vitro plants of Prunus seratina J.F.Ehrh. (Eliasson et al. 1994), and Echinachlaa frumentacea (Roxb.) Link plantlets after transplantation (Sankhla et al. 1992a). PBZ treatment reduced wilting of micropropagated plants of grapevine (Smith et al. 1992), chrysanthemum, and sugar beet (Ritchie et al. 1991; Roberts and Mathews 1995). Regenerated rice plants cultured in the presence of PBZ survived longer than the control plants (Zhao et al. 1991). Banana plantlets conditioned with TRIAD obviated the need for hardening and the treated plants were turgid and healthy compared to the controls (Murali and Duncan 1995). B. Low Temperature In the field, winter survival of autumn-sown pea and cereal crops were increased (Davis et al. 1988) and early frost injury to tomato, maize, and canola was reduced, while yields were significantly increased by PBZ (Fletcher and Kraus 1995). PBZ delayed chilling injury in pepper fruits (Lurie et al. 1995) and cucumber seedlings (Whitaker and Wang 1987; Upadhyaya et al. 1989). Protection against cold injury is attributed to an inhibition of chilling-induced degradation of membrane lipids (Whitaker and Wang 1987). Further evidence for this was found in a study involving the exposure of tomato seedlings to low temperature, where UNI did not influence fatty acid saturation but, instead, prevented the loss of phospholipid and accumulation of free fatty acids that were characteristic of the control (Senaratna et al. 1988). UNI also increased the levels of the antioxidants, tocopherol, and ascorbic acid in tomato seedlings, and it has been suggested that membrane damage is the result of oxygen

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R. FLETCHER, A. GILLEY, N. SANKHLA, AND T. DAVIS

free radicals generated by low temperatures and that triazoles protect membranes by preventing or reducing oxidative injury (Senaratna et al. 1988). This suggestion is supported by the observations that increased antioxidant enzyme activity is associated with triazole-induced low temperature thermotolerance in cucumber (Upadhyaya et al. 1989) and rape seedlings (Yang et al. 1994). In a chilling sensitive maize cultivar, PBZ-induced chilling tolerance was associated with several changes in the antioxidant enzyme profiles and increased activities of superoxide dismutase and ascorbate peroxidase (Pinhero et al. 1997). Chilling and freezing tolerance have also been associated with increased ABA levels (Zeevaart and Creelman 1988). Similar increases in ABA levels have been observed with triazole-induced chilling tolerance in banana (Zhou et al. 1995). PBZ and UNI have also been reported to increase cold hardiness and alter carbohydrate metabolism and endogenous ABA concentrations in Actinidia spp. (Tafazoli and Beyl1992, 1993; Zhang and Beyl 1997). Therefore, the ability of triazole compounds to alter the ABA balance may playa role in protection from low temperature stress. C. High Temperature High temperatures can induce numerous physiological and biochemical effects in plants, including protein denaturation, enzyme inactivation, altered metabolic rates, membrane damage, and reduced chloroplast biochemical activity (Blum 1988). These effects of heat stress are often confounded with those of water stress, since high temperatures are accompanied by increases in transpiration rate and dehydration (Kramer 1980). The close relationship between these two stresses is supported by a study that found increased thermal tolerance in triazole-treated wheat plants that had been pre-exposed to water stress (Fletcher and Hofstra 1988). One of the earliest reports of triazole-induced protection from heat stress showed that TRIAD protected bean plants from high temperature stress and prevented electrolyte leakage, a decline in chlorophyll, and a decrease in weight (Asare-Boamah and Fletcher 1986a). Subsequently, PBZ was shown to protect both wheat (Kraus and Fletcher 1994) and corn (Pinhero and Fletcher 1994) seedlings from damage due to high temperatures. The coleoptile membranes of maize seedlings were examined and results indicated that control membranes begin to degrade in response to heat stress, while those treated with PBZ merely altered their membrane properties to facilitate the removal of damaged areas (Paliyath and Fletcher 1995). UNI-induced thermotolerance of wheat seedlings has been attributed, in part, to its ability to increase the transpiration

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rates of treated plants, thereby resulting in lower leaf temperatures (Booker et al. 1991). One of the major sites of heat stress damage is the chloroplast (Blum 1988) and, therefore, reduced leaf temperature may be important in maintaining a functional photosynthetic system (Booker et al. 1991). Triazoles were also effective in protecting plants from a combination of heat and drought in wheat (Fletcher and Hofstra 1988) and jackpine seedlings (Marshall et al. 1991). Protection from heat shock (42°C) in soybean seedlings by UNI was accompanied by the production of low molecular weight proteins, not found in the controls (Larsen et al. 1988). PBZ was effective in protecting light-grown wheat seedlings from exposure to an intense heat treatment that severely damaged the control plants. During this period, control seedlings synthesized several heat shock proteins (HSP) that were not found in the PBZ-treated seedlings. Thus, HSP synthesis during heat shock is perhaps a manifestation of stress perception by the seedlings and might not have a function in triazole-induced thermotolerance (Kraus et al. 1995c). Evidence also indicates that protection of wheat (Kraus and Fletcher 1994) and soybean (Upadhyaya et al. 1990) seedlings from high temperature damage by triazole compounds may be related to increases in the activities of important antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase, peroxidase, and catalase. Wheat seedlings treated with PBZ were found to have higher levels of antioxidant enzymes under optimal conditions and this activity was maintained at a higher level than in control seedlings under heat stress conditions (Kraus and Fletcher 1994). Thus, triazole-induced protection from heat stress may be related to a more efficient free radical scavenging system. D. Environmental Pollutants and Other Stresses Triazoles have been shown to decrease injury due to air pollutants. Symptoms of ozone injury in wheat leaves are chlorosis, necrotic lesions, solute leakage, reduced Hill activity, a decrease in phospholipids, and a concomitant increase in free fatty acids indicative of free radical damage. These phytotoxic symptoms were prevented in beans by TRIAD (Fletcher and Hofstra 1985) and in wheat plants by UNI, and it was suggested that this might be due in part to increases in lipid soluble antioxidants (Mackay et al. 1987). Injury from sulfur dioxide was suppressed by PBZ in snap bean (Lee et al. 1985), and by UNI in cucumber (Upadhyaya et al. 1991). Although it has been suggested that triazoles may cause ABA-induced stomatal closure resulting in sulfur dioxide exclusion (Fletcher and Hofstra 1988; Davis et al. 1988), the mechanism

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of protection from these atmospheric pollutants is not fully understood (Lee et al. 1985). In the absence of any stress, triazoles enhanced the activity of antioxidant enzymes such as ascorbate peroxidase, monodehydroascorbate reductase, and glutathione reductase, superoxide dismutase, and catalase (Sankhla et al. 1992b, 1996a,b, 1997). Similar results were observed in PBZ-treated wheat seedlings, and it was further demonstrated that the higher activities of antioxidant enzymes were conserved after stress imposed by directly exposing wheat leaves to paraquat, a bipyridinium herbicide that generates oxygen free radicals in the chloroplast (Kraus and Fletcher 1994). Application of paraquat induced photoinhibition and a loss of photosynthetic pigments, protein, fresh mass and membrane integrity in wheat leaves. These symptoms of damage were reduced by PBZ, which stimulated an overall increase in antioxidant enzyme activity and elevated the plants to a higher level of oxidative stress tolerance (Kraus et al. 1995b). PBZ also antagonized the inhibitory effects of simazine, another herbicide, on the early growth of strawberry plants (Atkinson and Harrison 1984). PBZ protected wheat seedlings from injury due to waterlogging, where all symptoms of damage were reduced by the PBZ treatment at three different growth stages (Webb and Fletcher 1996). This was attributed to the development of an extensive root system, improving aeration and nutrient acquisition. PBZ-treated citrus seedlings, however, did not show increased tolerance to waterlogged conditions (Yelenosky et al. 1995). Pre-treatment of moth bean seedlings with PBZ increased tolerance to salt stress, prevented growth inhibition by NaCl, and decreased root sulpholipid content (Trivedi et al. 1988), while TRIAD ameliorated salt stress in peanut, radish, and soybean seedlings (Muthukumarasamy and Paneerselvam 1997; Paneerselvam et al. 1997, 1998). PBZ also modulated salt-induced alterations in pigment content, proline level, lipid peroxidation, and antioxidant activity in leaves of zuzuba seedlings (Sankhla et al. 1996b). Similarly, propiconazole was shown to alleviate the symptoms of salinity stress in Kentucky bluegrass (Nabati et al. 1994). In wheat seedlings, cadmium-induced inhibition of chlorophyll and carotenoid accumulation was prevented by UNI (Thomas and Singh 1996). PBZ increased the tolerance of soybean seedlings to elevated levels of ultraviolet-B radiation, and it has been suggested that the simultaneous effects of triazole treatment on increased leaf thickness, epicuticular wax deposition, and free radical scavenging activity may be some of the factors contributing to the observed tolerance (Kraus et al. 1995a). In chickpea, TRIAD protected the seedlings against UV-B dam-

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age by maintaining the level of glycoproteins and lipoproteins (Abbas and Zaidi 1997). Insecticidal phytotoxicity is a problem in ornamental production due to the variety of species and the lack of specific phytotoxicity tests. In Salvia splendens F. Sellow, application of PBZ prior to bendiocarb, a carbamate insecticide, treatment not only reduced damage to the plant, but also improved recovery from phytotoxicity (Latimer and Oetting 1994). An interesting interaction between PBZ and waterhyacinth weevil in controlling the eradication of waterhyacinth [Eichhornia crassipes (Mart.) Solms.] has recently been reported (Van and Center 1994). In combination, PBZ and weevil synergistically accelerated leaf mortality rates of waterhyacinth leading to early plant death. Thus, PBZ has great potential for increasing the effectiveness of waterhyacinth weevil. It has also been reported that TRIAD reduced root colonization and sporulation of vesicular arbuscular mycorrhizae fungus Glomus etunicatum Becker & Gerdemann on Eucalyptus grandis W. Hill ex Maiden (De-Paula and Zambolim 1994). However, this effect may be more related to the fungitoxic property of this chemical rather than any changes in the ability of the plant to host the mycorrhizae. VI. POTENTIAL AND CURRENT APPLICATIONS

A. Ornamentals 1. Bedding Plants. For many bedding plants, application of PGRs is a standard practice to maintain quality and compactness prior to sale, and to extend the marketing period (Davis and Curry 1991; Keever and Foster 1991). Both PBZ and UNI have received considerable interest (Table 3.3) as a bedding plant growth retardant (Keever and Foster 1991; Song et al. 1991). These compounds are highly active in reducing shoot growth of a number of bedding plant species and increasing the tolerance to water stress. High root-shoot ratio, following treatment with triazoles, also increases post-transplant survival. However, optimum rates are species specific and caution must be exercised while optimizing dosage due to high potency and persistence of these compounds. As an example of plant-to-plant variability in response to triazoles, Runkova and Shakhova (1992) studied the effect of PBZ and UNIon numerous tropical plants belonging to Acanthaceae, Lythraceae, Rubiaceae, Euphorbiaceae, and Geraniaceae. While Hypoestes phyllostachys Bak, Graptophyllum pictum (L.) Griff., and Pachystachys coccinea M. J. Roem. did not respond to treatment, the best response was obtained in

OJ N

Table 3.3.

Important bedding plant species responding to triazoles. Species

Response

Antirrhinum majus 1. Begonia semperflorens Hort. Brassica oleracea 1. Callistephus chinensis (1.) Nees Cathranthus roseus (1.) G. Don Celosia argentea 1. Chelone obliqua 1. Clarkia amoena (Lehm.) A. Nels. & Macbr. Coleus blumei Benth. Cosmos bipinnatus Cav.

UNI more effective than PACLO Compact plant Height control Growth retardation Very sensitive, growth retardation UNI for effective size control Growth retardation, flowering Height control, flowering Growth control, UNI more effective Reduced height, increased branches and number of flowers Growth control Effective growth suppression Growth control Growth retardation Flowering Growth suppression Growth control

Dahlia variabiJis (Willd.) Desf. Dianthus caryophyllus L. Eustoma grandiflorum (Raf.) Shinn. Gypsophilla oldhamiana Miq. Hypocalymma angustifolium Schau. Hypoestes phyllastachya Bak. Impatiens wallerana (1.) Hook. F.

Reference Barrett and Nell 1989 Farthing and Ellis 1990 Whipker et al. 1994a Song et al. 1991 Barrett and Nell 1989 Barrett and Nell 1989 Beattie et al. 1990 Anderson and Hartley 1990 Barrett and Nell 1989 Ahmad et al. 1990 Whipker and Hammer 1997 Foley and Keever 1991 Whipker et al. 1994b Song et al. 1991 Day et al. 1994 Foley and Keever 1992 Barrett et al. 1994a

Lantana camara 1. cv. 'New Gold' Lythrum salicaria L. Osteospermum ecklonis (DC.) Nod. Pelargonium zonale (1.) L'Her ex Ait. Petunia hybrida Hort. Vilm-Andr. Physostegia virginian a (1.) Benth. Pimelea ferruginea Labill. Pimelea rosea R. Br. Rudbeckia hirta 1. Salvia farinacea x longispicata Benth. Salvia splendens F. Sellow ex Roem. & Schult. Tagetes erecta 1. Trifolium dubium (L.) Sibth. Verbena rigida K. Spreng. Viola x wittrockiana Gams. Zinnia elegans Jacq.

co

w

Growth and flowering Growth and flowering Growth inhibition and flowering Growth control UNI more effective Growth and flowering Growth, flowering Growth, flowering Shoot growth control, time to flower increased Growth and flowering UNI more effective in growth control Growth and flowering Growth in hydroponics Post-production growth and flowering Growth and drought tolerance Growth and flowering

Ruter 1996 Song et al. 1991 Olsen and Anderson 1995 Farthing and Ellis 1990 Barrett and Nell 19a9 Beattie et al. 1990 King et al. 1992 King et al. 1992 Kessler and Keever 1997a Rodriguez et al. 1993 Barrett and Nell 19a9 Chen et al. 1993 Morgan et al. 1994 Davis and Anderson 19a9a Keever and Foster 1991 Chen et al. 1993

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Hypoestes aristata R. Br. and Ruellia macrophylla Yah!. with PBZ, and in Strobilanthes dyerianthus M. T. Mast. and Cuphea hyssopifolia HBK treated with UNI. The after-effects lasted 6-12 months. In a study on the effect of PGRs on growth and flowering of bedding plants indigenous to Korea, PBZ reduced height only in Lythrum salicaria 1. and Callistephus chinensis 1. Nees, while UNI reduced height in these species and in Gypsophila oldhamiana Miq. Number of flowers per plant decreased by PBZ and UNI in L. salicaria and C. chinensis. None of these compounds significantly affect height in Dendranthema indicum, Aster yomena (Kitamura) Honda, Dianthus sinensis 1. and D. superbus 1. cv. longicalycinus (Song et al. 1991). An important issue in bedding plant production relates to their postproduction performance. It is expected that the bedding plants should grow and flower rapidly upon transplantation to the landscape beds. However, in triazole-treated plugs, growth suppression may continue after plants are transplanted into the landscape. Keever and Foster (1991) evaluated growth, flowering, and drought stress responses of geranium, marigold, pansy, impatiens, and salvia to UNI applied at the seedling stage at the end of production, and 5-7 weeks after transplanting geranium, impatiens, and salvia into the landscape. Although the response varied with species, sampling date, and the dosage, growth of all species was suppressed, and the stress tolerance of all species, except marigold, increased with increasing concentration of UNI. At high concentrations, growth was suppressed excessively and flowering was delayed. Also, depending upon the bedding plant species and the dosage, the effect of UNI persisted for five or more weeks. In seed geranium, triazoles cause distinct alterations of growth and flowering processes, and the incidence of these changes varies among cultivars (Starman et al. 1994). Latimer and Baden (1994) studied the effects ofPBZ in growth of seed geranium in the greenhouse and the subsequent growth and performance of treated plants in the landscape. Like chlormequat, very low concentration of PBZ provided growth regulation of seed geraniums in the greenhouse, while permitting unchecked growth in the landscape. High concentrations of PBZ excessively reduced seedling growth. These results point out that growth retardant dosage must be selected based on both growth responses during production as well as persistence in the landscape to tailor plants that meet size requirements for complete fill of landscape beds without compromising the aesthetic quality. In commercial applications, the triazoles may be comparatively more difficult to use than other PGRs, primarily due to poor translocation after foliar sprays and a large variability in optimum concentration for dif-

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ferent crops. Drench applications through drip irrigation or directly in the water of sub-irrigation system has been quite effective, although unplanned contamination of the irrigation system could be problematic (Barrett et al. 1994a,b). Several factors contribute to the utility of a specific growth regulator in production of bedding plants. These include ease of application, spectrum of acceptable dosage range, stability, and their effect on post-production performance and economics. Unfortunately, bedding plants are often perceived as a small, specialized market. Therefore, it is not likely that PGRs will be developed solely for bedding plant production (Davis and Curry 1991). 2. Potted Plants. Like chlormequat, daminozide, and ancymidol, triazoles have also been extensively tested to manipulate the shape, size, form, and aesthetic quality of many floricultural crops (Barrett and Nell 1989, 1990; Barrett et al. 1994a,b; Whipker and Hammer 1997). These growth retardants are commonly applied to containerized plants that tend to be disproportionately large relative to the container. Both PBZ and UNI have provided effective size control in many floricultural crops (Table 3.4) corresponding to the standard of marketable potted plants. However, it soon became evident that the application technique plays a crucial role in obtaining the desired growth control. For instance, in chrysanthemums, PBZ was reported to be more effective when applied only to stems or to the medium than when applied to leaves. High spray volumes, providing thorough coverage of plant stems and greater solution entering the medium, increased the efficacy ofPBZ (Barrett and Nell 1990). Similarly, the efficacy of UNI increased with increased stem coverage and with the amount of chemical reaching the medium, which was achieved with high spray volumes (Barrett et al. 1994b). Experiments with caladiums, croton, brassaia, and poinsettia determined the effectiveness ofPBZ in solid spike form as compared to media drench application (Barrett et al. 1994a). PBZ drenches and spikes were effective for all crops tested, with similar concentration response for all, except that drenches had greater efficacy than spikes in rapidly developing caladiums. In tuberous-rooted dahlias [Dahlia variabilis (Willd.) Desf.] substrate drench treatments of PBZ, UNI, and ancymidol effectively controlled growth to the desired level (Whipker and Hammer 1997). However, the decision to use PGRs should be evaluated on the basis of the response of the cultivar and the cost of the chemical. In dahlia, the desired control of growth for the lowest cost was obtained by using PBZ at the cost of about 8-17 cents per pot for 'Golden Emblem' and 4-8 cents for 'Red Pigmy' which was 25-81 % less expensive than ancymidol. The commercial success of PGRs in floricultural crops, as in bedding

00

OJ

Table 3.4.

Important

tl"WLH'lnrf

Species

Acacia MilL spp. Achillea x 'Coronation Gold' L. Achimenes longiflora DC. Aconitum nupellus 1. Anthurium andreamzm Linden Aster novi-belgii 1. Boronia megastigma Nees ex BartL Brassaia actinophylla EndL Caladium x hortulanum Birdsey Clerodendrum speciosum Dombr. Cordyline terminalis (1.) Kunth. Dendranthema x movifolium Ramat. Euphorbia pulcherrima Willd. ex Klotzsch. Exacum affine Bulf. f. Fuchsia x hybrida Hort. ex Vilm. Hibiscus rosa-sinensis 1. Hydrangea macrophylla (Thunb.) Ser. Impatiens wallerana Hook. f. Kalanchoe blossfeldiana PoeHn. Leucospermum cordifolium (Salisb.) ex Knight Lotus uliginosus 1. Matthiola incana (1.) R. Br. Pilea cadierei Gagnep. & Guillaum Rosa 1. sp.

responding to triazoles. Response

Reference

Cut foliage and flowers, growth and flowering Growth control, increased market quality Plant height, branching, flowering PBZ-dip to tubers to control growth and flowering Promotion of shoot-inducing effect of cytokinins UNI more effective than PBZ, growth control Flowering Drench and spike application for growth control No effect Hardening to chilling Compactness increased, oval leaves instead of elongated Resistance to desiccation improved during transplantation Growth control Growth control Growth and flowering Growth control, earlier tlowerjna Summer application results in Growth control Uniform growth retardation in ebb and flood irrigation Flowering

Parletta and Sedgley 1995 Kessler and Keever 1997b Vlahos and Brascamp 1989 Abd-Elrahem et aL 1993 Werbrouch and Debergh 1996 Whipker et aL 1995 Day et aL 1994 Barrett et al. 1994a Barrett et al. 1994a Tamari et aL 1992 Hagiladi and Watad 1992

Barrett et al. 1994a Barrett and Nell 1989 Davis and Anderson 1989b Andrasek 1989 and Clark 1992 Barrett et al. 1994a; Klock 1998 Adriansen 1989 Brits 1995

Growth control, increased seed yield Growth, flowering Growth control, dark green leaves Compact growth, more flowers.

Tabora and Hill 1992 Ecker et al. 1992 Kim et al. 1993 Kaminski 1989

Roberts and Mathews 1995

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

87

plants, depends upon availability of an easy application method, consistent responses, and the economics related to PGR usage (Davis and Curry 1991). The effect of a growth retardant application during production, and subsequent post-performance shelf life and longevity also has an important bearing in its commercial success. Two major obstacles to the further development of growth retardants for floricultural crops are chemophobia and the perception of the agrichemical industry that floriculture represents a relatively small and specialized market. 3. Foliage Plants. Increasing demands for interiorscapes in mega settings, such as shopping malls, airports, and luxury hotels, have opened up new avenues for utilizing large-leafed plants (Stone 1994) that were usually not preferred in smaller settings. Interiorscaping requires the use of plants that will tolerate low-light environments. The excessive spindly shoot growth, which occurs in many species under low light, is known to be effectively controlled by triazoles (Davis et al. 1988). Additionally, by controlling abscission and retarding senescence, they may effectively prolong the life of foliage plants in interior landscapes, thereby reducing the frequency of replacement. It has been observed that triazoles can greatly reduce water use in several foliage plants, such as Araucaria heterophylla (Salisb.) Franco., Dieffenbachia maculata (Lodd.) G. Don, Epipremnum aureum (Linden & Andre) Bunt, and Polyscias fruticosa (L.) Harms, under low light indoors (Poole and Conover 1993; Conover and Satterthwaite 1996). New or unusual features of commonly used plants could enhance interiorscape designs and create an increased market for these plants (Conover and Satterthwaite 1996). Golden pothos is a tropical vine native to southeastern Asia. This foliage species is one of the most favored by consumers in the United States. However, the pothos plants grown by the nursery industry typically have small leaves. PBZ or UNI applied as a drench were effective in suppressing stem elongation of golden pothos. Although the leaf production rate was reduced, the treated plants produced larger leaves than the control following 10 weeks in an interior environment (Wang and Gregg 1994). Even the cuttings collected from stock plants previously treated with PBZ or UNI produced longer-stemmed shoots, more and largerleaves, and heavier fresh weights of shoot than cuttings from the non-treated plants. Experiments have also been conducted to determine the concentrations of PBZ that would optimize leaf size, vine length, and quality of golden pothos cuttings on totem poles (Conover and Satterthwaite 1996). Overall, these results indicate that triazoles (Table 3.5) may be useful in maintaining foliage plant quality and improving interior performance

00 00

Table 3.5.

Important foliage plant species responding to triazoles. Species

Araucaria heterophylla (Salisb.) Franco. Codiaeum variegatum (1.) Blume Cordyline terminalis (1.) Kunth Dieffenbachia maculata (Lodd.) G. Don Epipremnum aureum (Linden & Andre) Bunt Epipremnum pinnatum (1.) Engl. Ficus lyrata Warb. Ficus benjamina 1. Hypoestes phyllostachya Bak. Philodendron Schott. 'Red Emerald' Pilea cadierei Gagnep. & Guillaum Plecthranthus australis R. Br. Polyscias fruticosa (1.) Harms. Schefflera actinophylla J. R. Forst & G. Forst Spathiphyllum floribundum (Linden & Andre) Syngonium podophyllum Schott. Zebrina pendula Schnizl.

Response Water use indoors decreased Growth control Compactness increased, oval leaves instead of elongated Water use under low light indoors decreased Optimum leaf size and vine length Water use indoors decreased, vine growth and leaf size optimized, larger leaves Growth altered Indoor quality enhanced Growth control Growth suppression Growth, leaf color Plant height, growth Water use under low light indoors decreased Growth control Growth control Growth control Improved quality indoors

Reference Poole and Conover 1993 Wang and Blessington 1990 Hagiladi and Watad 1992 Poole and Conover 1993 Conover and Satterthwaite 1996 Poole and Conover 1993; Wang and Gregg 1994 Poole and Conover 1988 Davis et al. 1988 Foley and Keever 1992 Bazzochi et al. 1990 Kim et al. 1993 Wang and Blessington 1990 Poole and Conover 1993 Wang and Blessington 1990 Poole and Conover 1988 Wang and Blessington 1990 Davis et al. 1988

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

89

under low light by controlling excessive shoot elongation, shortening internode length, increasing foliage size, and reducing leaf abscission (Davis et al. 1988). 4. Woody Ornamentals. There is considerable need to control growth of woody ornamentals during nursery production as well as in outdoor landscape settings. Numerous studies have evaluated the responses of woody ornamentals to triazoles (Table 3.6). Warren (1990) studied the effect of foliar spray and medium drench application of UNIon 13 container-grown landscape plants. Except in golden privet (Ligustrum x vicaryi Rehd.), Russian-olive (Elaeagnus angustifolia 1.), and waxleaf privet (Ligustrum lucidum Ait.), growth decreased with increasing rates of UNI regardless of method of application. However, degree of growth reduction varied by species, rate, and method of application. Generally, drench applications were more effective than the foliar sprays (Keever et al. 1990). UNI can provide effective height control as either a drench or spray for container-grown woody landscape plants. However, Warren et al. (1991) observed that growth was reduced in only four of the six species tested, suggesting that UNI may not be equally effective in all species. UNI reduced plant height of Ligustrum x ibolium E. F. Coe, Photinia x fraseri Dress. and Pyracantha koidzumii (Hayata) Rehd. 'Wonderberry' (Norcini and Knox 1989). Plants pruned at the time of UNI application exhibited more desirable growth habits than unpruned growth-regulated plants. The effect of PBZ on growth, leaf morphology, and water use of Feijoa and Ligustrum was evaluated in two disparate climates, humid temperate Georgia and arid Arizona (Martin et al. 1994; Ruter and Martin 1994). Leaf morphology and water use was differentially affected by PBZ at these two locations. PBZ offered more potential benefit to growers in Georgia for restricting container growth and/or decreasing water use because of increased growth and higher plant water use at this location. UNI and PBZ effects were found to be evident for at least 12 months following application in Photinia x fraseri, a fast-growing woody landscape shrub with strong apical dominance that requires several prunings per growing season to obtain a desirable shape (Owings and Newman 1993). Both dikegulac-sodium and PBZ also increased lateral branching. PBZ suppressed bypass shoots development of two azalea cultivars, while minimally affecting the bloom size, although flowering was slightly delayed (Keever 1990). Application made just prior to or immediately after cooling were equally effective. Thus PBZ may be used for controlling bypass shoots on late-season azalea cultivars. PBZ and UNI

CD

o

Table 3.6.

Important woody ornamental species responding to triazoles. Species

Abelia grandiflora (Andre) Rehd. Abies fraseri (Pursh) Poir. Astilbe Buch.-Ham ex D. Don x 'Avalanche' Berberis thunbergii D.C. Buddleja davidii Franch. Camellia sasanqua Thunb. Ceanothus thyrsiflorus Eschsch. cv. 'repens' Chamaecyparis lawsoniana (A. Murr.) ParI. cv. 'Ellwoodi' Choisya temata HBK Clematis 1. 'Capitan Thuj]]eaux' Coreopsis verticj]]ata 1. Crataegus 1. spp. Cuphea hyssopifolia HBK Delphinium 1. 'Blue spring' Elaeagnus angustifolia 1. Feijoa sellowiana O. Berg. Forsythia intermedia Zab. Gelsemium sempervirens (1.) Ait. (1.) Griff. Hedera helix 1. Hibiscus rosa-sinensis 1. Hydrangea macrophylla (Thunb.) Ser. Ilex crenata Thunb. Jasminum nudiflorum Lindl. Juniperus chinensis 1.

Response

Reference

Growth control in container Reduced shoot elongation, fewer lateral buds, needle discoloration Growth suppression Growth suppression in container Growth and flowering Flower number increased Growth retardation Compacting effect

Warren 1990 Hinesley et al. 1998 Holcomb and Beattie 1990 Warren 1990 Ruter 1992 Keever and McGuire 1991 Joustra 1989 Grzesik 1991

Growth Growth Growth retardation Retardation of new shoots, more flowers Growth suppression Growth retardation Growth retardation in container Changed leaf morphology in disparate climate Growth inhibition in container Growth control in container No effect Root formation in cuttings Growth retardation Growth retardation, forcing Growth retardation Growth control in container Growth reduction, enhanced root qualities

Joustra 1989 Joustra 1989 Holcomb and Beattie 1990 Huang et al. 1992 Runkova and Shakhova 1992 Holcomb and Beatttie 1990 Warren 1990 Martin et al. 1994 Warren 1990 Warren 1990 Runkova and Shakhova 1992 Geneve 1990 Joustra 1989 Joustra 1989; Bailey and Clark 1992 Warren et al. 1991 Warren 1990 Ruter 1994

Lagerstroemia indica 1. Liatris spicata (1.) Willd. Ligustrum ibolium E. F. Coe Ligustrum japonicum Thunb. Ligustrum lucidum Ait. Ligustrum ovalifolium Hassk. Ligustrum vicaryi Rehd. Ligustrum vulgare L. Mandevilla Lindl. sp. 'Alice du Pont' Malus Mill sp. Pachystachys coccinea (Aubl.) Nees Photinia x fraseri Dress. Pieris floribunda (Pursh ex Sims) Benth & Hook. Pieris japonica (Thunb) D. Don ex G. Don 'Debutante' Platycodon grandifloTUS (Jacq.) A. DC. Pyracantha coccinea M. J. Roem. Pyracantha koidzumii (Hayata) Rehd. 'Wonderberry' Rhododendron calendulaceum (Michx.) Torr. Ruellia macrophylla Vahl. Strobilanthes dyeranus M. T. Mast. Viburnum tinus L. Weigela florida (Bunge) A. DC.

co r-"

Growth suppression in container Growth suppression Chemical pruning, plant quality Leaf morphology changed Growth suppression in container Growth, water status, chemical pruning Growth retardation in container Chemical pruning Growth suppression Growth suppression No effect Chemical pruning, plant quality Growth retardation

Warren 1990 Holcomb and Beattie 1990 Norcini and Knox 1989 Martin et al. 1994 Warren 1990 Norcini 1991 Warren 1990 Norcini 1991 Hocomb and Beattie 1990 Joustra 1989 Runkova and Shakhova 1992 Norcini and Knox 1989 Warren et al. 1991

Growth retardation

Joustra 1989

Growth inhibition Growth retardation in container Chemical pruning

Holcomb and Beattie 1990 Warren 1990 Norcini and Knox 1989

Growth inhibition

Warren et al. 1991

Growth suppression Growth suppression Growth inhibition Compactness

Runkova and Shakhova 1992 Runkova and Shakhova 1992 Joustra 1989 Grzesik 1991

92

R. FLETCHER, A. GILLEY, N. SANKHLA, AND T. DAVIS

are known to promote flowering of field grown Rhododendron catawbiense Michr. and Kalmia latifolia L. (Wilkinson and Richards 1991; Ranney et a!. 1994; Gent 1995). However, PBZ and UNI also reduce stem elongation and it persists for a considerable period. The persistent inhibition of growth by triazole growth regulators could be a problem when woody ornamentals are transplanted into the landscape. For instance, in Rhododendron and Kalmia, a dose that promotes flowering may severely inhibit stem elongation for several years and delay establishment of the plant in the landscape. Furthermore, for PBZ, the dose per plant that inhibited stem elongation half as much as a saturating dose was ten-fold that for UNI (Gent 1997). For both chemicals, the dose response coefficient decreased exponentially with time after application and the experimental time constant was found to be about two per year. Based on these results, the doses per plant for Rhododendron and Kalmia in the second year from propagation would be less than O.5mg of PBZ or O.05mg of UNI when applied as a spray application to wet the plant completely. Vining plants such as Mandevilla Lind!. are used as horticultural annuals in temperate areas where it flowers over a long season on arbors or other support. In the landscape, its rigorous growth rate is valued, but can be troublesome during production and marketing. Multiple application of UNI, coupled to an appropriate application interval, effectively restricted vegetative growth of 'Alice du Pont', the most widely available cultivar of l\!landevilla Lind!. (Deneke et al. 1992). The final application can be timed so that manageable plants can be marketed with flower buds. Asiatic jasmine [Trachelospermllm asiaticllm (Siebold & Zucc.) Nakai], a twining evergreen vine, is one of the most widely used ground covers in U.S. southern landscapes. Similarly, Carolina jessamine (Gelsemillm sempervirens) (L.) Ait. is also a vigorous, twining vine. Both these crops are labor intensive and require frequent hand pruning during production. UNI applied foliarly suppressed growth of these plants for several weeks (Keever and Foster 1995), although higher rates may induce severe stunting. Currently, Bonzi (PBZ) is labeled for use on 15 woody species, while Sumagic (UNI) is labeled on 5 species grown in the greenhouse or shade house (Keever 1997). 5. Geophytes. Geophytes are plants that propagate and survive by a variety of underground storage organs such as bulbs, corms, or tubers (Rees 1972). In these plants, enhanced storage organ development in vitro could result in significant scale-up micropropagation and shortening the requirement for several seasons of growth to reach flowering size (Ziv et al. 1995). In liquid and bioreactor cultures, PBZ induced profuse prolif-

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

93

eration of bud clusters and reduced the incidence of leaf malformation (Ziv and Ariel 1991). Treatment with triazoles also enhanced bud and/or protocorm formation in Aechmea and gladiolus (Ziv 1986, 1990; Steinitz et al. 1991). Bulblet formation, cormlet proliferation, and rhizome multiplication following application of triazoles has now been reported in several other geophytic species (Table 3.7). In Nerine x mansellii, a member of Amaryllidaceae, production of embryogenic tissue in liquid shake or bioreactor culture using PBZ appears to be a potentially viable commercial method for mass production of bulbs (Ziv et al. 1995). In Lilium speciosum Thunb. and Dioscorea opposita Thunb., triazoles have also been reported to reduce the dormancy of bulblets and promote the sprouting of half-dormant bulbils (Gerrits et al. 1992; Tanno et al. 1995). PBZ and UNI effectively control growth in tuberous-rooted dahlias to the desired level to produce marketable plants (Whipker and Hammer 1997). In gloxinia (Sinningia speciosa [Lodd] Hiera.), a single drench application of PBZ resulted in reduced canopy growth and increased tuber size (Borochov and Shahar 1989), although the flowering was only slightly affected. In this chilling-sensitive species, PBZ also reduced the effects of chilling injury. 6. Orchids. In recent years, the demand for moth orchid (Phalaenopsis amabilis (L.) Blume x P. mount kaala 'Elegance') has increased consid-

erably. Most moth orchid cultivars with large flowers have long inflorescences, which results in high shipping costs. Therefore, in commercial trade, compact selections of this orchid are preferred. Wang and Hsu (1994) have assessed the effect oftriazoles for controlling orchid inflorescence length. Also, because moth orchid is a perennial and often reblooms, the long-term impact of these growth retardant solutions or sprays during inflorescence elongation was also evaluated. PBZ dips did not affect the bloom date but effectively restricted inflorescence growth below the first flower. Flower size, flower count, and stalk thickness were unaffected by the treatment and foliar applications were less effective than dipping. High concentrations oftriazoles caused plants to produce small, thick leaves. During the second bloom season, inflorescence and bloom date were progressively delayed by increasing concentrations of PBZ and UNI. Foliarly applied PBZ before inflorescence emergence restricted stalk growth more effectively and its effect progressively decreased when treatment was delayed. PBZ was also found quite effective in reducing leaf length, width, and thickness in Cymbidium sinense Willd. (Pan and Luo 1994). The treatment increased chlorophyll content, promoted the development of new buds, and increased the number of flowers per inflorescence.

CD

fi::>

Table 3.7.

Important geophytes responding to triazoles. Species

Response

Reference

Aechmea fasciata (Lindl.) Bak. Alstroemeria 1. x Beta vulgaris L. Caladium hortulanum Birdsey Colchicum autumnale L. Dahlia variabilis (Willd.) Desf. Dioscorea opposita Thunb. Freesia x hybrida 1. H. Bailey Gladiolus grandifloTUs Hort. x G. tristis L. Hyacinthus orientalis L. Lapageria rosea Ruiz & Pav. Ulium lancifolium Thunb. Ulium speciosum Thunb. Nerine x mansellii Hort ex Bak. Sinningia speciosa (Lodd) Hiern. Tulipa gesnerana L. Zantedeschia rehmannii Engl.

Protocarm proliferation Rhizome multiplication In vitro acclimatization Growth control Cormlet growth Growth control Bulbil sprouting Forcing as pot plant Cormlet proliferation Bulblet formation Rhizome bud multiplication Growth retardation, flowering Dormancy of bulblets reduced Induction of meristematic clusters Growth decreased, tuber size increased Growth, flowering Growth suppression

Ziv 1986 Bond and Alderson 1993 Ritchie et al. 1991 Barrett et al. 1994a Ellington et al. 1997 Whipker and Hammer 1997 Tanno et al. 1995 De-Hertogh and Milks 1990 Steinitz et al. 1991 Bach et al. 1992 McKinless and Alderson 1993 Bearce and Singha 1990 Gerrits et al. 1992 Ziv et al. 1995 Borochov and Shahar 1989 Rebers 1992 Carr and Widmer 1991

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

95

B. Turf and Amenity Grasses

The use of the PGRs as "chemical mowing agents" was envisioned many years ago because of the tremendous economic benefits for reducing labor, fuel, and equipment costs (Davis and Curry 1991). Additional potential benefits include improved color, fewer clippings, faster mowing, increased green spread, deeper roots, fewer seedheads, less time spent in trimming and edging, less scalping, and reduced liability for dangerous mower sites (Johnson 1997). The potential disadvantages are leaf burn, reduced turf recuperative ability, increased weeds, and increased disease incidence. Two types of PGRs have been identified for use in turf (Johnson 1997). Type I PGRs inhibit and suppress turfgrass growth by rapidly stopping cell division [e.g. Embark (mefluidide), Limit (amidochlor), MH-30 (maleic hydrazide)]. Type II PGRs mainly reduce cell elongation through the interference of GA-biosynthesis [triazoles, flurprimidol (Cutless), trinexapac-ethyl (Primo)]. Generally, type I PGRs are effective in suppressing vegetative growth as well as seedheads, while the type II PGRs reduce vegetative growth better than seedheads. Results of some of the relatively current investigations on use of triazoles have been summarized in Table 3.8. In many cases, PGRs such as PBZ and UNI do not inhibit seedhead formation and may reduce the visual quality by inhibiting blade growth, thereby rendering seedheads more visible. Johnson (1997) has provided results of a comparative study of several PGRs on tall fescue, centipedegrass, and bermudagrass. In tall fescue, a single application of triazole plus mefluidide effectively suppressed both vegetative growth and seedheads. In 'Tifway' bermudagrass, Primo suppressed seedheads but not Cutless or triazoles. Pursuit (imazethapyr) suppressed seedheads of common bermudagrass. The highest consistent suppression was obtained when PGR treatments were followed by timely mowing. Primo [4-( cyclopropyl-alpha-hydroxy-methylene )-3, 5 -dioxocyclohexane carboxylic acid methyl ester], which inhibits the hydroxylation of GA zo to GAl' is a new generation of turf grass PGRs that is being used extensively to reduce turf grass growth (DiPaola and Shepard 1996). It can be applied to all major warm- and cool-season turf grasses in quality areas such as golf courses, residential and commercial lawns, and sod farms. Favorable effects of Primo include increased turfgrass density, darker green color, increased root and rhizome production, and a reduction in water requirement. Overall, it appears that a single specific PGR may not be as effective as combinations of PGRs in achieving satisfactory vegetative growth retardation and inhibiting seedhead formation. Further, timely mowing

CD

crJ

Table 3.8.

Important turf and amenity grass species responding to triazoles.

Species (Common name)

Agrostis palustris 1. (creeping bentgrass) Buchloe dactyloides (Nutt.) Engelm. (buffalograss) Cynodon dactylon (1.) Pers. (bermudagrass) Cynodon transvaalensis Davy. ('Tifway' bermudagrass) Eremochloa ophiuroides (Munro) Hack. (centipedegrass) Festuca arundinacea 1. (tall fescue) Lolium perenne 1. (perennial ryegrass) Paspalum notatum Flugge lbahiagrassJ Pennisetum purpureum Schumach. (napiergrass) Phalaris arundinacea 1. (canarygrass) Poa pratensis 1. (Kentucky bluegrass)

Response

Reference

Control of blight, mold and dollar spot Induction of female inflorescences, growth alteration Growth suppression to reduce mowing frequency, responses to multiple PGR treatments, phytotoxicity, varietal susceptibility Growth suppression, injury

Johnson 1990b, 1994, 1995

Growth responses, phytotoxicity, crop quality

Fry 1994; Johnson 1992b

Growth suppression, mowing, sward dynamics Reduction in lodging, reduced leaf rust, floral abscission prevention, increased seed yield Growth, crop damage Growth suppression, respiration in leaf blade

Johnson 1993; Spak et al. 1993 Mares-Martins and Gamble 1993a, 1993b, 1993c Johnson 1990a Ito et al. 1992

Reduced seed production Growth responses, sod enhancement, delay in senescence, alleviation of salt to stress

Ehlke 1992 Goatley and Schmidt 1990a, 1990b, 1991; Nabati et al. 1994

Burpee et al. 1990 Yin and Quinn 1994

Johnson 1992a

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

97

after PGR treatment may turn out to be the best option in controlling turf growth without compromising quality. Due to inconsistent results and phytotoxicity, use of PGRs on turf has thus far been limited, but the tremendous potential economic benefits related to PGR usage in turf definitely warrant further research. C. Arboriculture and Vegetation Management

In recent years, considerable interest has been generated about using PGRs on a wide spectrum of environmentally important plants. Potential use of growth retardants for the maintenance of trees under or near power lines is significant. In the mid-1980s, an estimated $800 million per year was spent by U.S. utility companies on tree trimming under or near power lines (Davis and Curry 1991). Therefore, there is an intense interest in growth regulators that reduce plant size and promote compactness (Keever 1997). Growth retardants registered for woody plants include UNI (Sumagic), PBZ (Bonzi), maleic hydrazide (Royal SloGro), chlorflurenol (Maintain CF125), mefluidide (Embark or Trim-cut), and flurprimidol (Cutless). Currently, none of these chemicals is in extensive use, with the possible exception of trunk or soil injection of PBZ and flurprimidol by utility arborists. Both PBZ (Bonzi) and UNI (Sumagic) have been quite effective in control of growth in a variety of trees (Table 3.9). An interesting aspect of triazoles on tree growth regulation relates to their action on juvenility and shortening of generation time (Griffin et al. 1993; Moncur et al. 1994; Hasan and Reid 1995). Application of triazoles promotes flower bud production and reduces generation time in several species of Eucalyptus (Table 3.9). Similarly, the juvenile phase in Hevea brasiliensis (Willd. Ex A. Juss.) Mull. lasts for 4-5 years, posing a constraint on breeder's efforts to shorten the breeding cycle of the tree. In trials on various H. brasiliensis clones, girdling or PBZ application induced precocious flowering and a combination of both treatments greatly enhanced the effect (Yeang et al. 1993). DowElanco has developed two products, Cutless (flurprimidol) and Profile 25SC (PBZ), for use in the electric utility industry as tree growth regulators (Davis et al. 1994; Edmondson and Stewart 1996). Cutless is applied to trees by inserting solid implants into holes drilled at intervals around the trunk to a depth of one inch below the bark. Transpiration flow is necessary to cause the disintegration of the implant and to translocate the tree growth regulator to the meristematic areas of trees. Profile 25SC is a liquid formulation of PBZ that is applied around the base of a tree using either a basal drench or soil injection technique in

r.o co

Table 3.9.

Important tree species responding to triazoles. Species

Acacia mangium Wild. Acer saccharinum 1. Castanea mollissima Blume Cupressocyparis leylandii (A. B. Jacks Dallim.) Dallim & A. B. Jacks Elaeagnus pungens Thunb. Eucalyptus globulus Labill.

&

nitens (Dean & Maiden) Maiden Euonymus fortunei (Turez.) Hand.-Mazz. Ficus macrocarpa Blume = F. punctata Thunb. Juglans nigra 1. Liriodendron tulipifera 1. Liquidamber styraciflua L. Metrosideros collina (J. R. Forst.) A. Gray Quercus 1. spp. Ulmus americana 1.

Response

Reference

Growth control in nursery, water relations Reduction in trimming line Bud break Growth control

Abod et al. 1994; Abod and Yap 1994 Mann et al. 1995 Arnold and Davis 1994 Keever and West 1992

Growth control Stimulated flower bud production, reduction in generation time, trunk injection, collar drenching, wood strength, elasticity Growth control

Keever and West 1992 Hetherington et al. 1992; Hasan and Reid 1995; GriffIn et al. 1993; Wasniewski et al. 1993 Moncur et al. 1994; Griffin et al. 1993

Chemical pruning Growth retardation to reduce pruning

Norcini 1991 Horowitz 1992

Growth inhibition, shade tree Shade tree, growth inhibition, wood strength and elasticity Growth retardation Growth effects Growth retardation Growth, water relations, water use efficiency

Sterrett 1989 Sterrett 1989; Wasniewski et al. 1993; Zillmer et al. 1991 Ammon et al. 1989 Clemens et al. 1995 Ammon et al. 1989 Ashokan et al. 1995

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS

99

order to expose maximum root surface to the active ingredient. Commercial use of these products (Edmondson and Stewart 1996) has yielded promising results on trees growing under utility lines, although the degree of growth control varies widely with the tree species and the time of treatment (Davis 1991; Davis et al. 1994). Limited use of PGRs by arborists is due to several problems. These include phytotoxicity, inconsistent results, timely and uniform delivery to the growing apex, insufficient information on optimal dosage needed to limit tree growth, lack of experience in proper use, concerns about cost effectiveness, and limited registration (Davis and Curry 1991; Keever 1997). Part of these problems may be related to the largedimension and complex vascular system of the target plant. With trees, application methods are not as simple as with other plants and there is still insufficient information on the underlying factors that contribute to variability in tree response to PGRs. Until this is better understood, it is unlikely that triazoles will be used extensively for controlling tree growth. However, since trimming of trees is an expensive and hazardous task, development of suitable PGRs for controlling tree growth may have tremendous potential for economic benefits. D. Agronomic Crops

One of the most significant uses of growth retardants in agriculture has been to control lodging of grain crops (Rademacher 1991). Chlormequat and ethephon are two of the most widely used anti-lodging agents. Although triazoles have also shown promise in controlling lodging (Morrison and Chilcote 1985; French et al. 1990; Kang et al. 1992), their commercial use for this purpose is limited by persistence in both soil and plant tissues. PBZ application increased wheat yields in Hungary using the shortest lodging-resistant winter wheat 'Martonvasari 13' (Jolankai 1988). Treatment of wheat seeds with UNI before sowing inhibited the elongation of leaf sheaths, bud sheath, and leaves in seedlings, but the treated seedlings produced more tillers and had increased number of tillers producing effective spikes, which resulted in an increase in yield (Sheng et al. 1993). Beckett and Van-Staden (1992) reported an interesting effect of a combined treatment of the synthetic cytokinin thidiazuron (TDZ), mineral supply, and PBZ on yield of wheat. Application ofTDZ during the early growth stages of wheat promoted tillering but reduced yield. On the contrary, if applied during flag leaf senescence, TDZ had little effect on yield. However, the yield of plants was increased at all levels of nutrient

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supply by treating plants with PBZ during the early growth stages followed by TDZ application during flag leaf senescence. In wheat, triazoles induce tolerance to heat and paraquat injury, freezing, and water logging (Gilley and Fletcher 1997). Although all the triazoles have the potential to act as stress protectants, PBZ was found to be the most effective in comparison to the fungicides propiconazole and tetraconazole in protecting wheat plants from abiotic stress (Gilley and Fletcher 1997). As with wheat, PBZ and UNI have also been shown to protect maize seedlings from drought, and high and low temperature stress (Pinhero and Fletcher 1994; Li and van Staden 1998a). It is unclear whether any of these effects are commercially significant. The genus Brassica contains many economically important oilyielding species. The improved cultivars of rape (Brassica napus L.), in particular, are currently extensively grown in Canada, China, Europe, and India. Several investigators have shown that triazoles, especially PBZ, have potential in controlling growth, lodging resistance, cold hardiness, and yield (Armstrong and Nicol 1991; Baylis and Hutley-Bull 1991; Morrison and Andrews 1992). In Canada, in certain years, there is total crop failure of winter canola due to winter kill. This damage is prevented by planting PBZ-primed seeds (Fletcher and Kraus 1995). In China, PBZ is used commercially to raise stout and strong rape seedlings that consistently produce more than the weak and less vigorous seedlings (Zhou and Xi 1993). The seedlings of oil seed rape are raised in beds and then transplanted to the field (Zhou and Xi 1993). This is due to the fact that the seed yield is much higher if vigorous seedlings are used in oil production. Application of PBZ produced shorter plants with more branches, significantly increased chlorophyll content and photosynthetic rates, prolonged leaf longevity, and increased green pod area and seeds. PBZ, applied as foliar spray, was also found to be quite effective in influencing growth and yield of an Indian cultivar of rape (Brassica carinata A. BL). PBZ significantly reduced plant height and modified canopy structure by enhancing the number of branches and the angle of insertion of primary branches on the main axis (Setia et al. 1995). The seed yield per plant also increased due to an increase in the number of siliquae per plant. Leaves of the Indian cultivar exhibited higher chIorophy11 content and remained intact on plants for a longer period than the control, as was also found in a Chinese cultivar (Zhou and Xi 1993). The oil content, erucic acid, and glucosinlate content of rapeseed represent important quality characters. PBZ significantly increased oil content, but did not affect erucic acid and glucosinolate content of seed (Zhou and

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Xi 1993), while in B. carinata, the seeds from PBZ-treated plants contained higher levels of proteins, starch, and total soluble sugars, but less seed oil content. The effect of PBZ has also been evaluated on growth and development of some important legume crops such as soybean, lentil, and peanut. In soybean, PBZ delayed reproductive development, reduced the number of branches/plant, branch dry weight, and the number of vegetative and reproductive nodes per branch, thereby increasing grain and pod dry weight and pods per stem (Merlo et al. 1987). In lentil, PBZ modified the growth, yield, and lodging response exhibited in the presence of different phosphorus levels (Gill and Singh 1993). In young seedlings of peanut (Pan et al. 1988), treatment with PBZ resulted in decreased leaf area, stomatal resistance, and evapotranspiration, and increased drought resistance (Li and Pan 1988). In some Spanish-type peanut cultivars, PBZ reduced vegetative growth and increased leaf chlorophyll concentration (Lin et al. 1987). Mature pod dry weight also tended to be higher with the later applications. Although the treatment also increased the number of large seeds in 'TN-g', the number of empty pods also increased. Like PBZ, even TRIAD has been reported to retard growth, and increase root shoot ratio, chlorophyll content, activity of RuBP-carboxylase, and photosynthetic rates in peanut (Yan and Pan 1992), and has been shown to have profound potential to increase the yield. As with grain legumes, PBZ is effective in suppressing the growth of fodder legumes such as alfalfa and clover (Niemelainen 1987; Kamler 1991; Boelt and Nordestgaard 1993) and in increasing seed yield. E. Vegetable Crops

Maleic hydrazide, the first commercially significant growth regulator, has been used since the 1950s to inhibit sprouting of onions and potatoes during storage. Growth retardants have also been tested on several vegetable crops to alter assimilate partitioning in favor of increased production efficiency (Davis and Curry 1991). However, the effects have been quite variable depending on the dosage, timing, method of application, and cultivar. In Canada and USA, processing tomato crops are established in the field with greenhouse grown plug treatments. Daminozide had been used for several years to aid in the production of healthy, stocky transplants. After the prohibition of the use of this compound in the late 1980s, based on concerns regarding possible carcinogenic effects, tomato transplant producers have been actively seeking other alternatives for

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growth control of tomato seedlings. Both PBZ and UNI have shown promise for use in tomato transplant production. Initial research indicated that seed treatment with triazoles had the potential to protect the plants from a variety of environmental stresses (Fletcher and Hofstra 1990). Tomato seedlings that were grown in Ontario, Canada, from PBZprimed seeds, were able to withstand early transplanting in May. They were protected from frost injury, which included chlorotic and necrotic patches in the control leaves. In addition to the observed protection, the PBZ-treated plants yielded 30% more than the controls for three consecutive years (Fletcher and Kraus 1995). Similar protection from frost damage was observed in PBZ-treated tomato seedlings in Pennsylvania, USA (Orzolek 1986). Following this observation, the effect of seedpriming with triazole growth regulators coupled to nutrient loading on tomato seedling quality and harvest yield has now been extensively evaluated in a program involving greenhouse and field research (SouzaMachado et al. 1996) with encouraging results. PBZ-primed seeds followed by post-emergence nutrient loading greatly enhanced seedling quality. The primed seedlings were sturdier, with intense green foliage due to high chlorophyll, and the plug had heavier rooting. Higher root/shoot ratios in the primed seedling may be one of the traits that helps the seedling better cope with transplanting shock/environmental stresses. Triazoles have not been tested widely in improving the yields of root crops such as carrot, onion, sweet potato, or radish, but the available evidence indicates that these growth regulators may influence the development of sugar beet and potato (Davis and Curry 1991). These chemicals may increase partitioning of assimilates to economically important plant parts such as roots or tubers, and by restricting shoot elongation may indirectly improve yield per unit land area by permitting increased crop density. In 'Dianella' potato, PBZ and sucrose-induced improved tuberization was found to be positively correlated with the level of conjugated GAs (Simko 1994). PBZ has also been shown to prevent excessive elongation and poor growth, and improve survival of potato seedlings after transplanting. The greatest effects on plant quality and survival were observed with GA and BA in combination with PBZ (Li and Zhu 1994). In 'Norland' potato grown under greenhouse conditions, PBZ nearly doubled the number of usable tubers per plant without affecting total tuber yield and prolonged tuber dormancy by approximately three weeks (Bandara and Tanino 1995). These results suggest that PBZ treatment could be effective in enhancing potato minituber production under greenhouse conditions.

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F. Spice Crops The effect of triazole growth regulators on growth, flowering, and yield of crops such as allspice, fenugreek, garlic, and pepper has also been investigated. Haldankar et al. (1994) studied the response of 12-year-old allspice [Pimenta dioica (1.) Merrill.] trees to PBZ application in Maharashtra (India). PBZ induced earlier flowering and a higher percentage of flowering. PBZ treatments increased the total number of panicles, the number of flowers per panicle, and the yield of green berries. The effect of seed-soaking treatment with PBZ was studied in fenugreek (Trigonella foenum-graecum 1.). Soaking of seeds in PBZ resulted in inhibited germination, but leaf pigmentation and shoot branching was increased. Spraying 4 to 6-week-old plants in the greenhouse delayed flowering, but both flower number and seed yield increased (Shahine et al. 199Za,b). In Bangladesh, late-planted garlic often fails to develop due to exposure to high temperature prior to bulb initiation and during bulb development. The effect of soaking garlic cloves in PBZ and planting them at various day/night air temperatures was studied on advancement ofbulbing in garlic (Rahim and Fordham 1990). Dry weights of bulb, leaf, and root tissue, as well as clove number, were highest under the lowest air temperature (15°/10°C). The highest soil temperature (Z5°/Z0°C) regime produced the most rapid growth. Pre-cooling at 5°C accelerated bulb initiation, development, and maturity. Both PBZ and chlormequat were effective in accelerating crop development and increasing bulb yield. PBZ enhanced the total yield of 'Quiteria' garlic in Brazil (De-Resende et al. 1993). Currently, peppers (Capsicum spp.) are the prime spice ingredients throughout the~orld. The popularity of peppers, both as a spice and vegetable crop, has soared to new heights, resulting in an unprecedented demand for their production. Both PBZ and UNI effectively decreased the height and number of axillary shoots in several pepper cultivars (Park and Lee 1989). In 'Hanbyul' pepper the number of fruits decreased, but the weight of fruits was increased. In ornamental peppers (Starman 1993) later applications, i.e. 10 weeks after sowing, increased red fruit percentage. Both PBZ and UNI alleviated chilling injury in green and red bell pepper fruits during storage (Lurie et al. 1995). PBZ also encouraged spring regrowth from topped plants left to overwinter. G. Fruit and Nut Species 1. Pome Fruits. In apples, a delicate balance exists between vegetative growth and fruiting. Frequently, this balance is disrupted due to

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sub-optimal weather or errors of orchard management resulting in vigorous growth that is detrimental to fruit quality, productivity, and profit margins (Greene 1996, 1997). The use of growth regulators to control fruit tree growth began in the early 1960s when daminozide was used in apple trees. The challenge in use of PGRs to control growth in fruit trees relates to tailoring the application methodology to the prevailing orchard conditions, which are often inconsistent and less than ideal (Davis and Curry 1991). Since daminozide is no longer available for use on fruit crops, a search is on to find replacement chemicals for this important market niche. Over the years, several triazoles have been evaluated for efficacy in controlling vegetative growth of vigorously growing apple trees. These include BAS 111, TRIAD, tripenthenol, and UNI. Of these, TRIAD appears to be least effective and UNI the most effective. PBZ is registered in seven countries, excluding the U.S., to control vegetative growth of apple and pear trees by foliar application (Davis and Curry 1991). In general, multiple low-dosage foliar applications have proved more effective in several apple cultivars (Curry 1989; Greene 1991). The calcium concentrations in fruits of treated plants were higher and therefore both fruit softening and storage disorders linked to calcium deficiency, such as bitter pit and internal breakdown, were reduced by PBZ application (Greene 1991). PBZ has also been found to be quite effective in controlling growth, and promoting flowering and fruit set in several cultivars of pear (Davis and Curry 1991). It appears that PBZ promotes axillary flower initiation by decreasing apical dominance (Browning et al. 1992). In Asian pear, PBZ treatment promoted lateral shoot growth, resulting in slender, horizontally spread shoots in contrast to the upright growth on controls (Huang et al. 1989). In chili pear (Pyrus bretschneideri Rehd.), PBZ reduced terminal shoot growth and individual fruit weight, but increased fruit sugar content and firmness, and the activities of phenylalanine ammonia lyase (PAL) and peroxidase, and phenolic and lignin content (Ju et al. 1993). Fruits harvested from winter pears after soil drench of PBZ were also firmer, but had less titratable acidity than controls due to reduction in malic acid (Chen et al. 1989). However, after storage, changes in the content of extractable juice during ripening were found similar for all fruits, indicating that the fruits from the treated trees were capable of ripening normally after cold storage. In pear, the occurrence of watercore in fruits appears to be related to high temperature and GA content during the early period of fruit development, as well as to exposure to low temperature during summer. In Japanese pear (P. pyrifolia Nakai cv. Hosui), PBZ sprays, after full bloom, significantly suppressed the occurrence of watercore (Sakuma et al. 1995).

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In a recent study (Zimmerman and Steffens 1995) on micropropagated apple trees at three planting densities, PBZ and UNI controlled the growth of 'Gala' more readily than 'Delicious'. Tree size of both cultivars was inversely related to the planting density and both triazoles were effective in controlling tree size as the planting density increased. Triazoles also tended to stimulate flowering of young 'Delicious' trees, although the increases were not sustained. Triazoles also increased the yield efficiency of 'Gala' by three, and of 'Delicious' by one, because they reduced the final trunk cross-sectional area more than they reduced per tree yield. Although bienniality indices were higher for PBZ-treated 'Gala' trees, only UNI increased the bienniality index of 'Delicious' trees when applied as trunk paint. Although triazoles are among the most powerful growth retardants, their commercial use is limited due to their residual activity. In fact, a new generation of GA inhibitors that affects the dioxygenases and allegedly has no risks with regard to its residual, toxicological, and ecotoxicological properties-prohexadione-calcium (KIM or BAS 125 W)is being developed for registration in the U.S. by BASF Corporation and Kumiai Chemical Industry Co. (Basak et al. 1996). Due to its low persistence, such a compound can be used flexibly for site-specific growth control. The potential benefits of prohexadione application to apple include reduction in terminal growth and canopy density, reduced pruning costs, improved fruit color, size and packout, increased return bloom, increased fruit set, and reductions of fireblight incidence and severity. Growth retardation caused by this compound in 'McIntosh' apple trees allowed better light penetration, which resulted in more red color and a higher percent of US Extra Fancy fruit. The trees also required less time to prune (Greene 1997). 2. Stone Fruits. Growth control in stone fruits is generally more difficult to achieve than in pome fruits. Therefore, heavy pruning, both in summer and winter, is required to keep the trees within a given shape and to procure a good yield. Trials with triazoles, such as PBZ and UNI, have proven to be quite effective for both controlling growth and increasing fruiting. The increase in yield of stone fruit trees in response to triazoles has generally been more consistent than for pome fruits. Several methods of application have been evaluated for treatments with PGRs. In peaches, very low rates of PBZ via drip irrigation have been reported to be successful in regulating growth (Davis and Curry 1991), while in both cherries and peach, trunk application with organic solvent carrier was also suitable. Soil drench, or application to hydroponically grown plants of peach and nectarine, effectively suppressed

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shoot growth (Zhou and An 1993; Avidan and Erez 1995). Furthermore, PBZ induced a rapid increase in root diameter, with increased root branching upon recovery from the inhibitory effect. Reduced top growth also reduced water consumption (Avidan and Erez 1995). In a recent study (Grochowska and Hodun 1997), one-year-old potted trees of plum, sour and sweet cherries, apricot, and pear were treated with a single application of PBZ to the collar tissue (the vascular transition region at the root-stem connection). Over three consecutive years, this treatment significantly reduced shoot growth in all five species. The plum 'Early Prune' and 'Ujfehertoi Furtos' sour cherry were found most responsive to collar application ofPBZ. Fruiting of plum trees increased four-fold and an advance in flowering was clearly evident in pear trees. These results indicate that the collar tissue is a sensitive region of the trunk and a suitable location for influencing tree growth and cropping. The most important aspect of this study is that PBZ was applied to the tree, and not to the soil, in small amounts much before fruiting in subsequent years, and therefore, poses less hazard to the consumer or the environment. In peach, PBZ significantly reduces canopy volume and summer pruning requirements and increases floral bud initiation, fruit set, fruit weight, and yield efficiency. In Australia, PBZ significantly reduced the competing spring shoot growth and resulted in an earlier maturity of a greater crop of larger, better quality fruits of the low-chill 'Flordaprince' (Allen et al. 1993). In China, PBZ increased fruit set, hastened ripening, and increased fruit weight and fruit sugar content of peach. Fruit calcium and proline concentration were also increased but nitrogen and nitrogen:calcium ratio decreased (Zhang 1990). In contrast, in a study in New Zealand (Klinac et al. 1991), although soil-applied or post-flowering spray of PBZ effectively reduced the vegetative growth, total fruit number and yield per tree were reduced. The intensity of the effect varied in different cultivars, but in 'Hosui', PBZ increased the physiological "bud jump" floral bud disorder and consequentlya serious reduction was observed in fruit production. PBZ also reduced the growth in almond [Prunus dulcis (Mill.) D.A. Webb] seedlings (Koukourikou-Petridou 1996). PBZ has also been reported to have a beneficial role in acclimatization of Prunus scrotina Ehrh. plants in vitro (Eliasson et al. 1994). The treatment significantly reduced shoot growth but increased/improved the quality and coloration, reduced percentage water loss of leaves, and improved survival. Incorporation of PBZ in vitro better enables Prunus plants to withstand the stresses associated with acclimatization. Beneficial effects of PBZ application have been reported for plum. In

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a 10-year trial on plum high-density plantings in Israel, soil application ofPBZ controlled excessive vegetative growth and promoted flower and fruit production in plots of Japanese plum (Gaash et al. 1989). In India, PBZ application increased average fruit length, diameter, and weight of Japanese plum, and the combined treatment of triacontanol and PBZ resulted in the greatest fruit firmness, total soluble solids, total sugars, and anthocyanin pigments (Chandel 1991). Both PBZ and UNI increased fruit weight, size, and peel color development in 'Red Rosa' plum, and the trees bloomed earlier the following year than the untreated control trees (Lurie et al. 1997). Both PBZ and UNI at the low dose did not affect the storage ability of the plums, but the high doses enhanced gel breakdown in fruits, indicating a potential for deleterious effects when applied in excess. Overall, triazole growth regulators have proven extremely effective in controlling growth and enhancing fruiting in peach, nectarine, cherry, apricot, and plum. Triazoles may become the plant growth regulators of choice in controlling growth and cropping in stone fruits if the problems related to retention of these chemicals in the soil and fruits can be resolved. Currently, excluding the U.S., PBZ is registered for use in stone fruits and olive in 11 countries throughout the world (Davis and Curry 1991). 3. Nut Crops. In nut species such as pecan [Carya illinoensis (Wangenh.) C. Koch], as tree density continues to increase, tree size control becomes a major problem. Triazole growth regulators have been shown to effectively control growth in pecan, although the intensity of response may depend on cultivar and age of the trees. Unfortunately, triazoles are not available for use on nut species in the U.S., but wherever registered, have increased precocity and improved tree and labor efficiency in addition to increasing yields (Davis and Curry 1991). Triazoles have not been tested extensively for controlling the growth of nut species such as hazelnut (Corylus avellana L.) and walnut (Juglans regia L.). In Italy, foliar and soil application ofPBZ on hazelnut has been reported to reduce vegetative growth and promote flower bud induction (Tombesi et al. 1994). The leaves ofPBZ-treated plants had more chlorophyll and carbohydrates, and indicated more active photosynthesis in comparison to the control. High concentrations of PBZ reduced vegetative growth and increased yield of walnuts, but better results were obtained if PBZ was applied twice as a soil drench (Zhu et al. 1994). 4. Small Fruits. Treatment of strawberry with PBZ reduced the number

of runners, decreased runner length, and increased the number of lateral

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crowns and flower clusters (Deyton and Cummins 1991; Nishizawa 1993). The yield, however, was less consistent and it either increased or decreased or remained unchanged depending on the cultivar. Thus, in greenhouse-grown 'Nyoho', PBZ-treated plants had more ripe fruits and higher fruit yield in the first year (Nishizawa 1993), but in 'Maria' and 'Elista', regardless of the application date, there was a decrease in yield (Eftimov 1988). In 'Cambridge Favorite', 'Pantagruella', and 'Hapil', in the year following application of PBZ, the percentage of larger fruits by weight was reduced (Beach et al. 1988), but an increase was recorded in percentage of unmarketable fruits. In the second cropping season, yield increased, but there were few large fruits, more unmarketable fruits, and the median harvest date were delayed. Similarly, in greenhouse grown 'Shuksan' and 'Totem', PBZ increased the number of achenes per fruit but decreased yield by delaying fruit ripening (McArthur and Eaton 1988). 5. Viticulture. Grapevines are pruned frequently in order to manage vine vigor. Vigor can also be regulated by the use of rootstocks, training systems, fertilizer and irrigation management, and the use of plant growth regulators. In the past, inconsistent results have been obtained with MH, chlormequat, dikegulac, and daminozide. PBZ has proven to be a potent vegetative growth inhibitor in both table and vine groups, although its effect on berry quality depends on species, cultivars, and the stage of growth at the time of application. Currently, it is registered for use on grape only in Spain (Davis and Curry 1991). In 'Seyval Blanc' grape, PBZ reduced vegetative growth and leaf area, but delayed senescence, and increased leaf weight and retention of basal leaves (Hunter and Proctor 1992). No effect was observed on fruit set or berry size. In hedged 'Riesling' grape, PBZ had no effect on vine vigor during application, but reduced it after one year of application (Novello and Roberts 1992) together with yield. The berries increased in Brix and pH, but titratable acidity was reduced. The significant reduction in shoot growth advanced fruit maturity and improved winter hardiness. An increase in fruit yield was observed in 20-year-old 'Fiano' grape treated with PBZ and UNI (Forlani and Coppola 1992). However, the treatment also increased the compactness of fruit clusters, which could make them more susceptible to Botrytis. The treatment also decreased the sugar content of the berries. In muscadine grape (V. rotundifolia Michx.), a foliar spray of PBZ at full bloom reduced vegetative growth, increased the number of clusters/ vine and the number of berries/cluster, and improved fruit quality. PBZ and GA increased fruit weight and size, and increased yield (Basiouny 1994).

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Trained and trellised grapevines often produce more foliage, which results in dense, undesirably shady interiors. Canopy shade is a major factor in reducing grape yield and quality. Multiple foliar applications of PBZ of 'Riesling' grape in Virginia reduced shoot growth, annual cane pruning weight, canopy density, berry weight, but increased berries per cluster in the second year of application (Wolf et al. 1991). PBZ also reduced juice volume, NH4-N, titrable acidity, and malic acid, but increased total phenols and flavonoid phenols. No significant changes in fruit pH, color, potassium, or glucose-fructose ratio were observed. PBZ also prevented incidence of fruit rot (Zoecklein et al. 1991). 6. Tropical and Subtropical Fruits

Mango. Mango is now widely available as a fresh fruit or as frozen/ processed products, not only in the tropics and subtropics, but also year-round in Europe, Japan, and North America. PBZ applications have provided promising results on several cultivars of mango. Kulkarni (1988) observed that soil application ofPBZ effectively reduced growth in 1-8 year veneer grafted trees on a seedling rootstock. In young trees, it caused precocious flowering, and promoted flowering in bearing trees. Axillary flowering and cauliflory was also observed in the treated trees and flowering was advanced considerably. Depending upon the cultivar and the dose used, this advancement of flowering exhibited variable results (Tongumpai et al. 1991; Burondkar and Gunjate 1993). In 'Alphonso', PBZ induced 3-4 weeks early and profuse flowering, and 2-6 times more annual yield. Impressive yield increases were also achieved in several other Indian cultivars (Burondkar and Gunjate 1993; Kurian and Iyer 1993). A significant earliness in flowering was also observed in 'Tommy Atkins' mango under rainfed conditions of the Pacific coast of Mexico (Salazar-Garcia and Vazquez-Valdivia 1997). This may have potential for stimulating off-season harvest, as was observed earlier (Tongumpai et al. 1989; Burondkar and Gunjate 1993). Although the effects of PBZ on fruit size and quality may be variable (Kulkarni 1988; Rowley 1990a; Burondkar and Gunjate 1993; Kurian and Iyer 1993), an increase in fruit set has been observed in several cultivars (Burondkar and Gunjate 1991; Goguey 1992). In general, soil application of PBZ has proven more effective than foliar sprays for growth control. PBZ, applied as a soil drench or foliar spray prior to flower bud differentiation, was also reported to reduce the incidence of floral malformation and enhance yield and fruit quality in 'Dusehri' (Singh and Dhillon 1992). Under rain-fed conditions, PBZ application has to be done during the

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rainy months (Salazar-Garcia and Vazquez-Valdivia 1997). A high application rate (10 g/tree) gave desired effects for at least two years in 5 year-old bearing trees, while lower rates (1.25-2.5 g/tree) were safe and effective for 1- to 2-year-old trees (Kulkarni 1988). In 'Valencia' mango, 5 g/tree PBZ applied for two consecutive years followed by a year without application resulted in an increase in fruit size and an early harvest. In 'Tommy Atkins' mango, it is advisable to leave trees untreated for one year after two consecutive years of PBZ application. The use of 10 g/tree may have a dual effect in reducing tree size and inducing early flowering. In mango, reproductive buds are initiated at the apex of mature vegetative shoots and can be purely floral or mixed. During the cool winter months, the growth flush produces panicles, while warm temperatures promote initiation of vegetative buds and "flushing." In containerized 'Tommy Atkins' mango with chilling alone, 74% of buds initiated were floral, whereas chilling plus PBZ/UNI sprays caused more than 90% of the buds to be floral (Nunez-Elisea et al. 1993). Furthermore, bud initiation occurred nearly 3 weeks earlier than with chilling alone. Under south Florida conditions, UNI was not efficacious unless the trees were pruned (Davenport 1994). However, all new growth from pruned scaffold limbs was severely stunted. Thus, major permanent losses in productivity may arise if trees that have been treated with UNI are severely pruned. Currently, application of Cultar (PBZ) to control size and stimulate early flowering in mango is being tested widely in southeast Asia, Australia, Africa, Mexico, and other areas of the tropics and Central America (Kulkarni 1988; Burondkar and Gunjate 1991, 1993; Charnvichit et al. 1991; Voon et al. 1991; Vuillaume 1991; Winston 1992; Davenport 1994; Salazar-Garcia and Vazquez-Valdivia 1997). Early flowering is a major objective of mango growers because it provides precious earlyseason fruit harvests and increased yields.

Citrus. Lured by earlier returns, citrus growers are increasingly moving toward high-density plantings. However, an efficient and acceptable method of controlling growth in citrus is currently unavailable. Therefore, several trials have been conducted to evaluate the role of triazoles for this important use (Davis and Curry 1991). Calamondin (Citrus reticulata Blanco x fortunella sp.) and lime (G. aurantifolia Christm) have considerable potential as dwarf ornamentals, whereas sour orange (G. aurantium 1.) serves as an important rootstock for citrus throughout the world. Fucik and Davilla (1991) observed that UNI delayed the germination and decreased the growth of

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sour orange seedlings. However, it did not affect shoot growth in lime and calamondin. Leaf growth and development was also not affected by UNI for any of these citrus cultivars. Yelenosky et al. (1995) found that all the cultivars of citrus rootstock were sensitive to PBZ, which caused a proliferation of shorter/thicker roots, shorter internodes, and lower dry weight. The leaves of the treated plants showed higher N, Ca, Fe, and Mn. GA stimulated growth and increased carbon supply in shoots of citrus rootstock seedlings, whereas PBZ delayed growth, reduced sucrose, and enhanced storage sugars (Mehouachi et al. 1996). PBZ did not enhance flooding and freezing tolerance in citrus root-stock seedlings (Yelenosky et al. 1995). Thus, although PBZ may be potentially useful in dwarfing citrus, it does not appear to enhance flooding and freezing tolerance. Both PBZ and UNI were found effective in suppressing internode length and increasing the branch angle in 'Cleopatra' mandarin seedlings and mature trees of 'Hamlin' orange (Wheaton 1989). The resultant drooping growth was more important in reducing tree size than were shortened internodes, and the effect persisted for two years. Okuda et al. (1996) have studied the effects of soil application of PBZ at the beginning of maturation, on sprouting, shoot growth, flowering, and carbohydrate contents in roots and leaves of satsuma mandarin (c. unshiu Marc. cv. Okitsu). During the season following PBZ application, flowering was enhanced, but new shoot sprouting and growth decreased. PBZ increased total carbohydrates in the roots of the current season but decreased them in the leaves. In an earlier study, Ogata et al. (1995) observed that multiple applications of all the GA-inhibitors (PBZ, UNI, prohexadione) during autumn were most effective in promoting flowering in satsuma mandarin. Even single or double applications were sufficient for a significant increase in flower bud formation. In China and Japan, fruit set and yields were also increased following PBZ treatment (Kawase et al. 1992). In a study carried out in Cuba on 'Valencia' orange (c. sinensis L.), the number of floral buds as well as the number of fruits per plant was significantly greater in plants treated with PBZ (Acosta et al. 1994). In Australia, 29% ofGA-treated fruits on 'Valencia' were retained to maturity, while PBZ caused 100% fruit drop within 35 days of application (Turnbull 1989). Regulation of GA levels may, therefore, be a means to control alternate bearing. Initial and final fruit set were also increased in 'Lisbon' lemon during the year ofPBZ application (Smeirat and Qrunfleh 1989). In Israel, PBZ was found to have potential for increasing flowering and restricting vegetative growth in citrus (Greenberg et al. 1993). However, problems arising from its long-term persistence were evident.

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In Texas 'Rio Red' grapefruit (C. paradisi Macf.), both PBZ and UNI increased the percentage of small-sized fruit, and lowered juice quality by increasing acid and decreasing soluble solids and the sugar/acid ratio. Overall, both chemicals retarded fruit growth and maturation (Fucik and Swietlik 1990). In rough lemon (G. jambhiri Lush.), PBZ inhibited the activity of PEP-carboxylase, suggesting that organic acid metabolism could be affected by this growth retardant (Vu and Yelenosky 1988). On the basis of the above results, it is apparent that in citrus more studies are needed to fully evaluate the role of these compounds in growth, photosynthetic partitioning, induction of cold hardiness, and the interactions with endogenous PGRs.

Pineapple. Favorable climatic conditions can accelerate the growth rate of pineapple suckers to such an extent that the incidence of precocious fruiting can be extremely high. In a study in Australia, Cultar (PBZ) was found to be less effective than Fruitone CPA in reducing the incidence of precocious fruiting (Scott 1993). On the other hand, in Thailand, PBZ significantly increased the sucker number (Suwunnamek 1993). The induced suckers were rather stumpy and heavier than those of controls. In pineapple, ethylene plays a primary role in floral initiation, whereas GA may be involved in inflorescence development. It is quite common to force inflorescence development with ethephon. However, environmental induction of inflorescence development, caused possibly in response to naturally produced ethylene or due to the plant's sensitivity to it, may severely disrupt scheduling of fruit harvest and cause significant losses if plants are induced when they are too small. It was observed that GA 3 , AOA, AVG, daminozide, and STS were ineffective in influencing induction, while CPA, PBZ, and UNI delayed or inhibited growth, reduced ethylene production by basal-white leaf tissue, and inhibited the activity of ACC oxidase (Min and Bartholomew 1993, 1996). Thus, UNI and PBZ may have potential in inhibiting environmental induction of inflorescence development, although effects on yield needs to be carefully analyzed. Avocado. Low fruit set greatly limits production in avocado. Vegetative shoots in avocado emerge shortly after anthesis in the spring and again during the summer, and apparently compete with fruit setting. PBZ, applied as soil drench, reduced growth, changed assimilate partitioning, and induced flowering in 'Hass' avocado (Symons et al. 1990). In highdensity orchards in South Africa, PBZ application resulted in significantly smaller trees of 'Hass' avocado. PBZ also increased flowering and subsequent yield and yield efficiency the year following application.

3. TRlAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS 113

However, in the second year, controls had the highest yield (Kohne and Kremer-Kohne 1990). In Mexico, higher rates ofPBZ increased the number of flowers in spring and autumn for 'Hass' (Obando et al. 1992), but had little effect on vegetative growth and fruit set. Low sensitivity of avocado to foliar sprays of PBZ and high tree to tree variability and differences in responses of various cultivars (Davis and Curry 1991) indicate limited utility for triazoles in avocado production.

Banana. Few studies have been done to study the effect of triazole growth regulators on banana. In Morocco, bananas are cultivated almost exclusively in plastic greenhouses. Growth of banana under these conditions is vigorous. Both foliar sprays and soil application of PBZ applied prior to flowering significantly reduced vegetative growth (EIOtmani et al. 1992). However, residues in the fruit were detected 18 months after application, and yield, especially of younger plants, was reduced in the second cycle due to reduction in bunch length and number of fruits per hand, although the number of hands per bunch was not affected. Effective tree size control was achieved by soil application of PBZ in China, but wider variations occurred in yield, and residues were detected in the aril of the fruit (Liang et al. 1994). Kiwifruit. Kiwifruit vines, due to excessive vegetative growth, would seem to be a prime candidate for PBZ application. However, in general, the response has been highly inconsistent and disappointing (Davis and Curry 1991). In a recent study in New Zealand, however, soil application of PBZ was reported to increase fruit size by 7% in the season of treatment but had no effect in subsequent years. This in no way matches the reported increase in yield by 12-62% with hydrogen cyanamide (Henzell et al. 1992). In Actinidia spp., PBZ and UNI were reported to significantly increase cold hardiness (Tafazoli and Bey11992, 1993; Zhang and Beyl 1997). If this response is consistent, it could be an important side effect of triazole regulation of growth in kiwifruit. Other Fruits. The effects of triazoles have also been evaluated on several other tropical fruit plants (Table 3.10). The reported responses range from growth retardation to enhanced flowering, yield, improved fruit quality, and better performance under environmental stresses. However, variable effects within a genus/cultivar grown in different countries were also apparent. Overall, triazoles appear to have potential beneficial effects on growth and yield of several tropical fruit trees. However, their large-scale use in the field may be limited until the problems related to persistency can be fully resolved.

,...,

,..., fl::.

Table 3.10.

Responses of some tropical fruits to triazoles.

Species (Common name)

Response

Reference

Carica papaya 1. (Papaya) Diospyros kaki 1. (Persimmon) Durio zibethinus J. Murr. (Durian) Malpighia glabra L. (Acerola) Litchi chinensis Sonn. (Lychee) Pistacia vera L. (Pistachio) Psidium guajava L. (Guava)

Growth control Increased fruit weight, accelerated fruit softening Earlier flowering Growth reduction, earlier and sustained flowering Growth reduction, increased flowering and yield Anthesis of staminate plants delayed Growth control, shortening of juvenile period, promotion in rooting from cuttings Fruit auality enhanced, stress-induced cracking prevented

Rodriguez and Galan 1995 Flohr et al. 1993; George et al. 1995 Chandraparnik et al. 1992 Michelini and Chinnery 1989 Rowley 1990b; Chaitrakulsub et al. 1992 Porlingis and Voyiatzis 1993 Rahman et al. 1991; Barrientos-Perez et al. 1992 Sankhla et al. 1989

Ziziphus mauritiana Lam. (Zujuba)

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS 115

VII. A NOVEL SEED TREATMENT TECHNOLOGY

A novel seed treatment technology that enables efficient use of triazoles has been developed recently (Fletcher and Hofstra 1990). The triazoles are administered via imbibition followed by acclimation, and these "programmed" seeds develop seedlings that express a high degree of resistance to a variety of environmental stresses. Depending on the species, seeds were imbibed for a period of 2 to 16 h at room temperature. Cereals, tomato, and pepper were imbibed for as long as 16 h, whereas canola and soybean, which tend to split, were imbibed for shorter periods (1 to 4 h). The imbibed seeds were dried, stored, and germinated when required. Addition of potassium to the triazole solution and exposure of the seeds to a heat shock (acclimation) during the imbibition period further enhanced the PGR effects and improved the efficacy of the triazole-induced protection against various stresses (Fletcher and Hofstra 1990). Compared to other conventional methods of application, including seed coating, foliar spray, or soil drench, the seed treatment procedure has several advantages, which include simplicity, cost effectiveness, reduced chemical concentration, little or no persistence, and minimal spread of the chemical in the environment. This procedure also eliminates conventional fungicide seed coating treatment since the triazoles themselves are potent fungicides. The treated seeds may be stored as long as one year without any loss in efficacy (longer periods have not been tested). The triazoles likely induce molecular and biochemical changes during imbibition that are preserved and manifested during and after germination; this accounts for the use of the term "programmed" seed. Priming of seeds with triazoles not only precludes the contamination of the environment, but, on analysis, insignificant traces of PBZ were detected in the seedlings for 2-3 weeks and then were not detectable at all (R. A. Fletcher 1998, unpublished). Therefore, for further trials, PBZprimed seeds have now been sent to several countries such as Bangladesh, China, Ghana, and India. The overall results of the trial have been quite exciting and encouraging. For instance, in Kerala (India), rice crop is followed by vegetables crop under irrigated conditions and the use of triazole-treated seeds has reduced the irrigation needs in cash crops such as cucumber and tomato. By far the most extensive trial, as reported earlier in this communication, has been conducted in Ontario (Canada), where growers are using the procedure for early planting with tomato (V. Souza-Machado 1997, pers. commun.). The method, as reported for tomato, is equally applicable to sweet corn. 'Supersweet' sweet corn, where sweetness is based on shrunken 2

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gene (Tracy 1997), is also chilling susceptible. Planting the primed seeds of this cultivar before the danger of frost is over in early May resulted in greatly increased seedling survival (Fletcher and Kraus 1995). VIII. SUMMARY Triazoles, the most potent group of growth retardants, have pleotropic effects. They act primarily by inhibiting GA biosynthesis, followed by secondary modulation of ABA, ethylene, cytokinins, and polyamines. Other related reactions, especially those related to stress protection, may also be affected and add to the benefit of growth inhibition. The morphological changes induced by triazoles include reduction of plant height, a higher root to shoot ratio, and modified leaf morphology. Physiological changes induced by these chemicals include improved water status, enhancement of flowering, and protection from various abiotic and biotic stresses, including fungal pathogens, drought, salinity, air pollutants, and low and high temperatures. At the biochemical level, triazoles enhance activity of antioxidant systems to effectively scavenge free radicals and thus enable plants to better cope with suboptimal environmental constraints. Results from field studies, conducted under different environments, suggest that the triazoles are highly effective as plant stress protectants, especially under hostile environmental conditions. The degree of protection is greater in stress susceptible cultivars, and triazoles allow this potential to be expressed. These highly active PGRs hold considerable promise for a number of applications in horticulture and agriculture. For many ornamental plants, the application of triazoles is a standard practice to manipulate the shape, size, form, and esthetic quality, as well as to extend the marketing period. Triazoles alone, and in combination with other PGRs, have also shown some promise in controlling turf growth and for maintenance of trees under or near power and utility lines. They have been useful in controlling growth, lodging resistance, cold hardiness, and yield of some important crops such as rape and tomato. Their role in scaling-up the production of geophytes and potato via tissue culture has become a commercial possibility. Triazoles have also shown potential use in regulating the growth and yield of several fruit and nut species, including pome fruits, stone fruits, nut species, and some tropical and subtropical fruits. Legitimate concerns have been raised regarding the use of these highly potent chemicals due to their relatively high stability and low metabolism, which results in persistence over a long span of time. However, bet-

3. TRIAZOLES AS PLANT GROWTH REGULATORS AND STRESS PROTECTANTS 117

ter methods of targeting and application can greatly reduce the risks emanating from their high persistency. Therefore, more imaginative methods of application need to be devised to reduce the risk of environmental contamination. A promising start in this direction has already been made. For instance, presoaking seeds in low concentrations oftriazole solutions prior to germination has proved to be an efficient way of treating plants to minimize the spread of the chemical to the environment and enhance the potency of these chemicals. In field trials with triazole-primed seeds, yield increases have been reported for several crops in Bangladesh, China, India, and Canada. In some fruit species, a single application of PBZ to the collar tissue significantly reduced shoot growth, advanced flowering, and increased fruiting. The significance of these results is that PBZ was applied to the tree, and not to the soil, and the minute amounts applied were separated in time and space from fruiting in subsequent years and, therefore, any hazard to the consumer or the environment is minimized. In light of the above, more definitive studies are urgently needed to resolve the extent of the triazole residue problem. Scientific data are needed to fully assess the impact of triazole residues on the environment and, in the case of edible crops, on consumers. With the current advances in analytical techniques, our ability to detect chemicals at concentrations of attograms (10-18 ) and milliatograms (10-21 ) is now feasible. Thus, the mere detection of a chemical, in a plant or plant parts, in reality means very little. The relevant question is: How do these residues affect the environment and human health? In addition, there is always an open question about interpretation of risk and the trade-off between benefits and risk. The triazoles are highly effective growth regulators, and therefore their exclusion from use simply on the basis of few reports on their persistency is not supported by good science. In practice, chemicals such as triazoles continue to be in use in food and fruit crops in several countries, especially developing countries. Growth regulators in general have long been tagged with the label "minor agrichemicals." Although their total sales are certainly only a fraction of the large pesticide market, this should not diminish their importance to industries that use them. Because of this, there has been a trend for smaller, specialized chemical companies to take on the marketing of growth regulators. Fortunately, these companies appear to be well suited for meeting the needs of the industries that rely on PGRs to produce their commodities. Therefore, it is imperative that PGRs should continue to play an important role in current agriculture and horticulture. The role of chemicals such as triazoles in elucidating basic mechanisms of plant growth and development is also vital and cannot be

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underestimated. While the primary action of triazoles is in the inhibition of GA biosynthesis and subsequent modulation of hormonal balance, it is still not clear what changes are involved in plant protection to stress. Biochemical changes include increases in antioxidants and activities of free radical scavenging enzymes. The morphological changes induced by these chemicals are more akin to those observed with xerophytic species. GA mutants of several species have some of these characteristics and should, therefore, provide valuable insights into the possible mode of action of triazoles. The information obtained, using genetic mutants, should be useful to confer stress protection and thus enhance crop yield globally. LITERATURE CITED Abbas, S., and P. H. Zaidi. 1997. Chemical manipulation through triadimefon against ultra violet-B induced injury to membrane components of Cicer arietinum 1. Plant Physiol. Biochem. 24:64-66. Abd-Elrahem, A. A. Watad, R Ecker, and A. Barzilay. 1993. Aconitum as a flowering pot plant. Scientia Hort. 56:175-179. Abod, S. A., K. 1. Cheong, and S. A. Abod. 1994. Effects of a growth retardant and shoot pruning on the growth of Acacia mangium seedlings. J. Trop. For. Sci. 6:239-248. Abod, S. A., and S. W. Yap. 1994. Effects oftwo different growth regulators on the growth and water relations of Acacia mangium seedlings. J. Trop. For. Sci. 6:489-501. Acosta, J. F., J. Gonzalez, R Rodriguez, and W. Leon. 1994. Effect of growth regulator applications on the juvenile period of Valencia oranges (Citrus sinensis 1.). Centro Agricola 21:51-56.

Adriansen, E. 1989. Growth and flowering in pot plants soaked with plant growth regulator solutions in ebb and flood benches. Acta Hort. 251:319-327. Aguirre, R, and A. Blanco. 1992. Pattern of histological differentiation induced by paclobutrazol and GA 3 in peach shoots. Acta Hort. 315:7-12. Ahmad, M., and G. Shanker. 1990. Effect ofpaclobutrazol on growth and flowering of cosmos (Cosmos bipinnatus Cav.). Punjab Hort. J. 30:200-202. Allen, P., A. P. George, R J. Nissen, T. S. Rasmussen, and M. J. Morley-Bunker. 1993. Effects of paclobutrazol on phenological cycling of low chill 'Flordaprince' peach in subtropical Australia. Scientia Hort. 53:73-84. Ammon, V. D., D. E. Griffin, and J. 1. Tate. 1989. Growth retardant effects of uniconazole on oak and sweet gum. Res. Rep. Miss. Agr. For. Expt. Sta. 14:3. Anderson, R G., and G. Hartley. 1990. Use of growth retardants on satin flower, Godetia, for pot plant production. Acta Hort. 272:285-291. Andrasek, K. 1989. Increasing the ornamental value of Hibiscus rosa-sinensis and P. hortorum cv. Springtime by using gibberellin inhibitor growth regulator. Acta Hort. 251-250.

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Ecologically-based Practices for Vegetable Crops Production in the Tropics * Hector R. Valenzuela Department of Horticulture University of Hawaii at Manoa Honolulu, Hawaii 96822-2279

I. Introduction A. Vegetable Production in the Tropics B. Ecologically-based Pre-harvest Technology for the Tropics C. Scope of the Review II. Integrated Cultural Management A. Stand Establishment B. Productivity of Monocultures and Semi-perennial Monocultures C. Polycultures 1. Polyculture with Legumes 2. Polyculture with Solanaceous Crops 3. Crop Breeding for Polycultures 4. Polycultures and Experimental Design III. Nutrient Management and Soil Conservation A. Soil Fertility B. Nutrient Calibration Studies C. Organic Amendments D. Cover Crops and Green Manures E. Plastic, Organic, and Living Mulches F. Crop Residues and No-till Agriculture G. Polycultures and Resource Utilization H. Double-cropping Systems I. Rhizosphere Microbial Associations * Published as Journal Series No. 4303 of the College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI. Acknowledgment is made to Dr. D. Sanders, Dr. H. C. Wien, D. J. Janick, and an anonymous reviewer for critical review of the manuscript.

Horticultural Reviews, Volume 24, Edited by Jules Janick ISBN 0-471-33374-3 © 2000 John Wiley & Sons, Inc. 139

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IV. Ecologically-based Pest Management A. Introduction B. Nutrient by Pest Interactions 1. Diseases 2. Arthropod Pests C. Habitat Manipulation Techniques 1. Polyculture Systems 2. Cover Crops and Living Mulches 3. Trap Crops 4. Insectary Plants D. Cultural Management Programs 1. Organic Matter Buildup and Rhizosphere Biological Control 2. Physical Controls 3. Other Alternative Controls E. Integrated Pest Management Programs 1. Sampling and Monitoring 2. Timing of Control Practices 3. Multi-tactic Approaches V. Conclusions and Future Prospects Literature Cited

I. INTRODUCTION The science of agroecology employs a systems approach (Lewis et al. 1997) and ecophysiological research techniques to develop sustainable agricultural practices that are cognizant of local socioeconomical conditions (see Table 4.1). Goals of agroecological research are to increase long-term crop productivity, sustain the economic well-being of rural communities, minimize the dependence of rural communities on expensive external inputs, and reduce any detrimental effects of agricultural practices on the environment (Hecht 1987). While agroecological techniques are ideally "low-input" in terms of external material inputs brought into the farm, the resultant systems are "high-input" in terms of the use of biological and indigenous knowledge (NRC 1993) and in terms of the greater management skills required on the part of the farming community to develop and maintain area-wide integrated ecologically-based crop management programs. Vegetable crops represent only a small fraction, by volume, of all the agricultural items produced in the tropics. However, vegetables have for millennia been important staples in many impoverished rural and urban tropical locations (Valenzuela and DeFrank 1995), and impart a vital potential nutritional supplement to the millions of undernourished inhabitants in the tropics suffering from critical nutrient deficiencies such as from vitamin A. Also because of their relative high market value, vegetables represent an important

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Table 4.1.

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Definitions of some terms used in this chapter.

Agroecology: The "application of ecological concepts and principles to the study, design, and management of sustainable systems" (NRC 1993). For a perspective on the definition of agroecology and its social and environmental implications see: Hecht (1987), Lal (1987), and Altieri and Hecht (1990). Appropriate technology: The introduced technology should target a particular physical and biological environment to meet its desired goals on crop productivity (yields, profitability, subsistence, improved labor efficiency, etc.). The technology should also be relevant to the socioeconomical conditions of the particular region and farming system (Bradfield 1981; Harwood 1981; Lancini 1987). Crop ecophysiology: The science of crop interactions with the environment, including acclimation and adaptation processes (Luttge 1997; Prasad 1997). Luttge (1997) indicates that the "development of modern experimental technology which is increasingly well adapted to the use in field work in the tropics, is also allowing more and more detailed ecophysiological studies." Farming Systems Research and Development: "An approach to agricultural research and development that (1) views the whole farm as a system, and (2) focuses on the interdependence among the components under the control of members of the farm household and how these components interact with the physical, biological, and socioeconomic factors not under the households' control" (Shaner et al. 1982). Land Equivalent Ratio (LER): The LER is the ratio of the production area needed under monoculture to obtain the same yields as those obtained under polycultures. The LER ratio is obtained by adding the fractions of the yields of the intercrops relative to their respective monoculture yields (Francis 1986). Participatory approach to development: To assure development of appropriate technologies, farmers need to be involved in all stages of the technology development process, including problem diagnosis, research, and the technology transfer process. See Beets (1990), Hall (1978), Joshi and Witcombe (1996), and Shaner et al. (1982). Polyculture/lntercropping: The practice of growing two or more crops simultaneously in the same field (Francis 1986). For definitions of different intercropping variants see: Francis (1986). Sustainable agriculture: Involves farming systems that are environmentally sound, profitable, productive, and compatible with local socioeconomical conditions (Pesek 1994). Systems approach: To study "the system as an entity made up of all its components and their interrelationships, together with relationships between the system and its environment" (Shaner et al. 1982). Also see Lewis et al. (1997).

Tropics: Regions located between the lines oflatitude 23°27' north and south of the equator, i.e. between the Tropic of Cancer and the Tropic of Capricorn (Lal 1987; Luttge 1997).

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source of income to the millions of subsistence and small-scale farmers who grow them as the supplemental "cash-crop" component in their farms. Over the second part of the twentieth century, significant advances have been made in the field of agroecology (Hatfield and Karlen 1994; Valenzuela and DeFrank 1995). Even though much of this information was developed in temperate areas, the techniques are often applicable in tropical settings. Agroecological programs in the tropics, however, should build upon the rich indigenous agricultural knowledge that evolved through millennia to establish sustainable agricultural systems (NRC 1993; Ellis and Wang 1997). However, many traditional farming techniques (such as shifting cultivation) must be modified to increase yields, to restore degraded soils, and to provide economic security. A.

Vegetable Production in the Tropics

In the tropics, vegetables are typically grown in small-scale «2 ha) polycultures (Knott and Deadon 1967; Stelly 1976; Williams et al. 1991). For instance, small-scale vegetable polycultures are observed with leafy crops (Ikeorgu 1990; Olasantan 1992), maize/cowpea, maize/yams, maize/cassava, and tomato/maize-based systems in Nigeria (Remison 1978; Adelana 1984), in the bean/banana intercrops of several eastern African countries (Wortmann and Sengooba 1993), the bean/maize systems of South America (Altieri et al. 1978), the mixed vegetable cultures of Indonesia (Grubben and Asandhi 1992), and in the sorghum/pigeon pea-based systems of India (Rao and Willey 1980). The type of vegetable cropping systems found in the tropics (Norman 1979; Ruthenberg 1980; Webster 1980; Beets 1990) range from the subsistence level to the agro-export large-scale plantations, to the more highly intensive systems such as those found in areas of Singapore and Hawaii. In Singapore, for instance, a farm survey found annual yields of over 100 t/ha with the production of seven leafy crops and one fruit vegetable, resulting in profits of about US$ 25,000 in 1988 (Koay and Loh 1990). B. Ecologically-based Pre-harvest Technology for the Tropics For development of appropriate (Table 4.1) sustainable technologies, research needs to focus on productivity and marketability, and to evaluate the effect of production practices on environmental quality and on regional food security. A recent review of commercial fresh market solanaceous crop production technology revealed the following priority

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research areas: planting technology and stand establishment; fertilization and irrigation; fumigation technologies to manage soil-borne pests; management of other major pests through conventional and alternative methods; and postharvest market quality (Cantliffe et al. 1995). These reflect some of the major challenges that currently exist for the development of sustainable programs for vegetable crops production in the tropics. Site-specific socioeconomical conditions must be considered in research designed to improve agroecosystems. Lorenz and Errington (1991) developed a no-till-based rotational program that had lower gross and peak seasonal labor demands than conventional year-round production systems. This program better matched labor availability in agricultural settlements of Kalimantan, Indonesia. The lack of acceptance for exotic crop species or cultivars in traditional production areas in Nigeria also underscores the need to use locally available knowledge and production techniques (Olasantan 1992). Integrated vegetable production programs that included conservation tillage and reduced pesticide and fertilizer applications, evaluated in long-term trials in temperate areas (Steiner et al. 1986), demonstrated no yield declines and increased economic returns. However, these types of systems have not been evaluated to a large extent in the tropics. Furthermore, in complex agricultural systems it has been difficult to implement research programs with a holistic systems approach, an approach that would stray from the traditional replicated factorial research that focuses only on one or two variables at a time. However, an increasing trend exists in agroecology work to conduct multidisciplinary on-farm system-wide studies (Sheenan et al. 1991; Caporali and Onnis 1992). An example of large-scale "integrated-interdisciplinary" on-farm organic vs. conventional farming studies conducted in California was presented by Sheenan et al. (1991). Systematic research can be conducted effectively to develop integrated cultural management programs for important cropping systems (NRC 1993; Cantliffe et al. 1995). This approach in Louisiana led to recommendations for the intensive production of cucumber. These included the use of high-density staking schemes by reducing in-row spacing and application of 700-900 kg/ha of 13-13-13 N-PzOs,-KzO fertilizer to maximize yields (>39 t/ha) in soils with low nutrient levels. They also identified the need for supplemental drip irrigation and fertilization. The use of white or black plastic mulch, the planting of two successive cucumber crops to minimize trellising costs in areas with low pest pressure, plus establishment of a tomato-cucumber double-cropping program to minimize staking costs were also part of their production package (Hanna and Adams 1989, 1993). The use of a nematode-resistant tomato cultivar in

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the double-cropping system, plus the use of transplants, effectively reduced root-knot nematode pest levels and provided an alternative to the use of restricted nematicides (Hanna et a1. 1996). A similar integrated approach was successfully designed and implemented in India for the intensive commercial production of a host of vegetables through the use of improved cultivars, disease-specific-free seedlings, improved transplanting techniques, fertilizer recommendations, planting schedules and arrangements, rotations, and improved herbicide application technologies (Randhawa 1988). Similarly, research on tomato production in high-rainfall areas of Taiwan showed the potential benefits of incorporating several cultural techniques, including the use of 40 cm raised beds to reduce bacterial wilt incidence, improved cultivars, grafting on waterlogging tolerant eggplant or tomato rootstock, fruiting hormone treatments, and the use of rain shelters to obtain greater yields (Midmore et a1. 1997). The application of agroecological techniques to improve system pest suppression and internal nutrient cycling (NRC 1993) can lead to improved yields and economic benefits for low-input small-scale vegetable production, even for resource-poor farmers in developed countries, as was shown in the Salinas Valley of California (Altieri et a1. 1991). Examples of such projects in tropical regions are becoming more common, many of them led by non-governmental organizations (NGOs) in cooperation with government or international a.gencies. An example of a successful grass-roots integrated cultural management program that resulted in increased fruit yields and quality is the successful peach production program by the El Novillero Agricultural Cooperative in Solola, Guatemala (Williams et a1. 1992). Another successful program, targeting soil conservation and increased yields was conducted in the highlands of Ecuador, where a variety of crops are grown by subsistence farmers. The grass-root program, established to reduce high erosion levels of over 80 t/ha, consisted of using strip cropping, intercropping, rotations, contour cultivation, green manuring, planting of N-fixing trees in surrounding areas, establishment of terraces and terrace stabilization with cover crops such as alfalfa, bluegrass (Paa spp.), ryegrass, clover, and vetch (Vida spp.). After six years of implementation, about 4,000 ha had been incorporated into soil conservation programs and crop yields (for bean, garlic, onion, peas, and potato, among others) were increased by 47 to 475% (Nimlos and Savage 1991). To ensure farmer acceptance, new production techniques should be introduced following participatory concepts (Beets 1990; NRC 1993) promulgated by the "Farming Systems Research, Extension and Development" approach (Table 4.1; Shaner et a1. 1982; Joshi and Witcombe

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1996; Van Huis and Meerman 1997). An agroecological perspective was recently provided for the production of root and underground tuberous crops in small farms of the tropics (Valenzuela and DeFrank 1995). The economic and ecological benefits of adapting indigenous knowledge to implement environmentally compatible low-input technologies were illustrated in a case study with maize (Pimentel et al. 1989).

C. Scope of the Review Selected promising agroecological preharvest techniques evaluated for the establishment of sustainable crop production systems are highlighted in this review. Although an integral part of a farming systems approach (Table 4.1), topics not covered in this paper include emerging biotechnological techniques, postharvest management, farm management and marketing, and technology transfer programs. When data from tropical regions is scant, information is borrowed from work conducted in temperate countries. Data from work conducted with agronomic and fruit crops are also used in several instances to illustrate current technology or production systems that may be applicable for vegetables. Thus, the data presented hereinafter is illustrative of available production techniques, rather than being comprehensive. Because agricultural settings in the tropics are so diverse, this review only provides reference to general directions in terms of possible technological innovations, with a focus on the agroecological principles. Sustainable agriculture, from that perspective, is considered a goal and an ongoing process of technological innovation, rather than a specific set of agricultural practices (Hatfield and Karlen 1994). From that standpoint, progress toward sustainability is possible in all settings, ranging from the subsistence farmer, to the organic eco-farmer, to the large-scale capital intensive commercial vegetable farm. As such, the terms "lowinput" and "sustainable" become relative depending on the particular agricultural setting, and the particular techniques employed to target sustainability will thus vary accordingly. An overview is first presented of key cultural management programs that affect the productivity of vegetables. These include stand establishment techniques and strategies to improve the sustainability of crop monocultures. Because polycultures are predominant in the tropics (see: Section IA), an overview is then presented of polycultures with a focus on legumes and solanaceous crops. The two remaining sections of this review cover alternative practices to improve nutrient management, soil conservation, and pest management programs in tropical agroecosystems.

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II. INTEGRATED CULTURAL MANAGEMENT A. Stand Establishment Stand establishment and transplanting technologies have advanced significantly over the past two decades, offering the potential for uniformly high yields and quality under intensive vegetable crop production programs. In countries such as the United States, transplant production operations have grown increasingly in technical and marketing sophistication (Cantliffe et al. 1995). In Florida, for example, vegetable transplants are produced in 40 ha of plastic greenhouses, with deliveries made to over 30 states and to two countries (Vavrina 1992). In Florida, commercial transplant production exists for broccoli, cabbage, celery, collard, eggplant, lettuce, muskmelon, onion, pepper, squash, tomato, and watermelon (Vavrina 1992). The potential exists to transfer these transplant production/marketing techniques for intensive vegetable crop production to other sub-tropical and tropical locations and adapt them there. Promising stand establishment techniques include the use of pregerminated seed for planting (Hall et al. 1989) and seed priming (Parera and Cantliffe 1994) to improve germination in large-scale operations, or when planting is conducted under unfavorable environmental conditions (Khan 1992). Seed-priming techniques were developed for a variety of crops, such as leek (Parera and Cantliffe 1992a) and sweet corn (Parera and Cantliffe 1992b). Thermodormancy or germination under high temperatures was overcome through seed priming in carrot (Cantliffe and EIBalla 1994), celery (Parera et al. 1993), lettuce (Valdes and Bradford 1987), and tomato (Odell and Cantliffe 1986). Also, seedling emergence in problem soils and damping-off caused by Pythium were overcome through priming in sugarbeets (Rush 1992). However, under optimal conditions, pre-germination or seed priming may not improve seedling growth compared to untreated seeds, as observed in pepper (Stoffella et al. 1991). Fluid-drilling establishment of pre-germinated seed with such products as a potassium starch acrylamide gel mix also offer potentials for improved stand establishment of vegetables such as pepper (Schultheis et al. 1988). Fluid drilling was used to improve stand establishment in carrot, especially with the addition of commercially available humic and folic acid biostimulants, resulting in improved root growth compared to conventional sowing techniques (Sanders et al. 1990). Seed treatments with biological control organisms have proven effective for the management of several soil-borne diseases (Taylor 1990). For

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example, seed coating with Bacillus subtilis was as effective as fungicide treatments for control of Fusarium roseum in maize (Chang and Kommedahl1968). Another example is pythium damping-off control in chickpea with Penicillium oxalicum seed treatments (Kaiser and Hannan 1984). Furthermore, granular pellet formulations are being developed for the application of beneficial microbials, such as the use of alginate, carrageenan, and chitosan pellets or vermiculite/bran preparations (Lewis and Papavizas 1991). Other recently proposed seed treatments include the inoculation of bean (Buonassisi et al. 1986) and maize (Bjorkman et al. 1998; Mao et al. 1998) seed with microbial antagonists for the management of environmental stress or of important soil-borne diseases such as Fusarium and Pythium. For example, tomato and lettuce inoculated with Pseudomonas fluorescens resulted in improved seedling growth, indicating the potential to improve stand establishment through the application of growth-promoting soil microbes (Digat et al. 1990; Schippers et al. 1995). In Florida, tomato seeds were inoculated with both Trichoderma harzianum and with Glomus intraradix, for management of Fusarium crown and root rot (McGovern et al. 1992). Trichoderma inoculation alone resulted in enhanced subsequent seedling growth as compared to uninoculated seedlings. In combined Trichoderma/ Glomus inoculations, Trichoderma was a more aggressive colonizer, resulting in reduced Glomus growth rates, but otherwise the dual inoculation process was effective (McGovern et al. 1992). The potential thus exists for multi-species seed inoculation in vegetables to enhance seedling growth, stand establishment, and to resist rhizosphere pathogenic attack. Nutrient conditioning of transplants is another area that offers potential to increase stand establishment and vegetable yields. In nutrient conditioning, the adequate nutrient combination and amount is applied to seedlings to optimize stand establishment in the field after transplanting, resulting in subsequent high marketable yields. Advancements have been made with nutrient conditioning of vegetables in temperate and some warmer regions with asparagus (Precheur and Maynard 1983), cabbage (Wright and Smith 1989); cauliflower (Booij 1992); celery (Dufault 1985), lettuce (Kratky and Mishima 1981), muskmelon (Dufault 1986), pepper (Dufault and Schultheis 1994), tomato (Woltz and Jones 1972; Garton and Widders 1990; Widders and Garton 1992), and watermelon (Schultheis and Dufault 1994), but much cultivar and location-specific work is required in tropical and sub-tropical regions.

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B. Productivity of Monocultures and Semi-perennial Monocultures

High commercial yields are obtained in intensive vegetable crop production areas of the tropics, especially in areas where the crops are grown for export. For example, average commercial tomato yields in sub-tropical Sinaloa, Mexico, exceed 40 t/ha, with 50-60% of the volume exported to the U.S. (Anon. 1992). When resources such as prime agricultural land, capital, fertilizers, and irrigation are available, highdensity plantings are possible to increase crop productivity per unit area, as shown with Luffa cylindrica in the Philippines (Aurin and Rasco 1988), cucumber in Louisiana (Hanna and Adams 1993), and bell pepper in Puerto Rico (Unander et al. 1991). Cultural practices may be modified to improve the productivity of monocultures. For example, high-density plantings and pruning (Unander et al. 1991) in crops such as pepper and eggplant may increase the length of harvest and total yields. A drawback of monocultures that limits long-term sustainability is the frequent tilling of the soil for the production of short-growing-cycle crops. Short-growing-cycle (2-4 months) production monocultures and frequent soil disturbance often result in increased weed pressure, soil organic matter losses, and soil erosion. These drawbacks can be alleviated by developing alternative longer-duration monocultures. One such alternative is the semi-perennial growing of vegetables by extending the duration of harvest. This practice is common in many parts of the world where crops such as eggplant are kept in the field for several years. The longer growing cycle minimizes soil erosion and disturbance of the dormant weed seed pool by reducing the number of tillage operations. Ratoon cropping can also be practiced with short-term crops such as lettuce (Wagner et al. 1994). As with okra in the Philippines (Miranda and Rasco 1991) and eggplant in Hawaii (Valenzuela and DeFrank 1994), cultivars can be selected that are better adapted to pruning, ratooning, and thus to extended harvesting periods. Other cultural practices such as irrigation, fertilizer application rates, and pest management will need to be researched and modified to better complement the longer-duration monocultures. C. Polycultures

Polycultures (Table 4.1) are widely practiced in all tropical regions, but these traditional systems have received little attention from the international research community. Subsistence farmers follow intercropping practices for several reasons, and not just to maximize productivity.

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Additional benefits may include input substitution (e.g. labor for land), improved operation productivity, sequential decision making in response to market or climatic conditions, spread of labor demands, and minimizing the risk of crop losses (Francis 1986; Altieri and Hecht 1990; Shaxton and Tauer 1992). A relative index of crop productivity in polycultures compared to productivity of the same crops under monocultures is the Land Equivalent Ratio (Table 4.1). In general, in traditional subsistence production areas, polycultures show greater sustained productivity on an areal basis (Juo et al. 1995), as well as increased economic returns, than monocultures (Andrews 1972; Willey 1979a). For example, intercrops in southwestern Nigeria were more productive (Land Equivalent Ratio or LER of 1.5-3.9, indicating a 50-390% greater productivity) and economically viable when papaya planted at 2,500 plants/ha was intercropped with okra, watermelon, bush beans, and Solanum gila than in papaya monocultures (Aiyelaagbe and Jolaoso 1992). A 13-year experiment in Nigeria also showed a greater relative yield sustainability in a maize/cassava polyculture than maize monoculture (Juo et al. 1995). In developed countries, some studies conducted on small farms have shown similar economic returns in intercrop systems compared to conventional monocultures (Brown et al. 1985). Despite the many ecological and socioeconomical benefits reported for polycultures throughout the twentieth century, the trend has been toward increased monocultures. The trend for increased monocultures was promoted, in part, by national and international development agencies through tax-based subsidy and extension programs (Mountjoy and Gliessman 1988). This resulted in greater farmer dependence on external inputs such as fuel, fertilizers, pesticides, hybrid cultivars, external technical expertise, and often outside seasonal employment needed to afford the purchase of these external inputs. Research should thus challenge the assrnption that intensive monocultures are more productive and economIcally sound in tropical agroecosystems, and re-evaluate traditional production programs that may be more suitable for sustained productivity. To highlight relevant production factors, results from polyculture studies with legumes and solanaceous crops are discussed below. 1. Polycultures with Legumes. Benefits of polycultures, as outlined by Francis (1985), include improved resource (light, water, nutrient, labor) utilization (Willey 1979a), low to moderate pest and weed pressure, and lower economic risks (by reducing the risk of losing the entire crop) to the farm. Also, when vegetable or grain legumes are involved, the protein yields may be greater in polycultures than in monocultures

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(Francis et al. 1982a; May 1982). A legume-grain polyculture increased maize yields by 30% when compared to monoculture maize, but soybean yields were unchanged (Alexander and Genter 1962). In West Bengal, India, soybean-based polycultures resulted in greater soybean and component crop yields, compared to the corresponding monoculture yields. Optimal row-planting arrangements were 2 soybean: 2 maize; 1 soybean: 1 rice, and 1 soybean: 1 pigeon pea (Patra and Chatterjee 1986). An evaluation of 94 sorghum/pigeon pea intercropping studies by Rao and Willey (1980) showed greater yield stability over time in the polycultures than under monocultures. Furthermore, intercrop component species showed a lower incidence of crop failure during "disaster level" situations as compared to the failures incurred by the monocultures under similar environmental stresses (Rao and Willey 1980). In related sorghum/pigeon pea intercrop studies, sorghum showed little yield loss when intercropped with pigeon pea, thus reaching the farmer's goal of obtaining a "full" sorghum crop, while the initial pigeon pea crop growth reduction was compensated for by faster growth after the sorghum harvest (Natarajan and Willey 1986). Sorghum-based intercropping "also resulted in greater land equivalent ratio (LER) values, and maximum yields in a 2 sorghum: 2 intercrop row ratio, when intercropped with green gram (Phaseolus aureus), blackgram (Vigna mungo) , cowpea, and peanut (Singh 1981). However, in India, intercropping of sunflower, as the main component crop, with several legumes, including soybean, peanut, green gram, and cluster bean (Cyamopsis tetragonoloba) , did not result in any area-based yield increases compared to their respective monoculture yields, during two years of irregular rainfall. Greatest polyculture yields were obtained with the sunflower/green gram mixture, reaching an LER of 1.05, or practically equivalent to the yield of the component crops under monoculture (Narval and Malik 1985). Even though polyculture yields were low, overall productivity of these systems was deemed adequate to meet the vegetable oil and protein requirements of populations in northwest India. In Tanzania, millet grain yields were also increased by 13 to 27% when intercropped with green gram (May 1982). Optimal population densities and planting arrangements in polycultures will vary depending on the availability of resources such as rainfall and nutrients (Fisher 1977b; Siame et al. 1998). In Botswana, sorghum/cowpea polycultures showed areal yield advantages over their respective monocultures under abundant rainfall, but no yield advantages (LER <1) were observed during two drought years (Rees 1986b). Corresponding recommended monoculture sorghum populations would

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range from 10,000 plants/ha during dry conditions to 120,000/ha during moist conditions. Medium population densities resulted in optimal yield stability over several years (Rees 1986a). Specific planting arrangements may be established to develop complementary resource utilization patterns among component crops in intercropping systems. In replacement population studies conducted in Uganda, a two-thirds dwarf sorghum to a one-third bean combination resulted in the greatest combined yields on an areal basis, compared to the individual component crop yields under monoculture (Osiru and Willey 1972). The synergistic yield effect was attributed to the different (complementary) rooting depths and to their different growth cycles. In Nigeria, similar replacement studies with maize and cowpea showed that greater yields were obtained as the proportion of the highestyielding component crop (maize) was increased at up to 63% of total plant stands. Optimal cowpea yields, however, were obtained with a 50/50% mixture in plant stands (Osiru and Willey 1972). Cowpea cultivars with a greater leaf area and longer vegetative growth period were better adapted to the maize/cowpea system (Remison 1980). While ideal cowpea ideotypes for monocultures are deemed to be those with erect habits and little branching, ideal ones for polycultures would be those that climb and branch profusely. It is thus recommended that the productivity of both determinate and indeterminate cowpea types be evaluated in terms of yields and efficiency of resource utilization in polycultures, as was done for monocultures in India (Chaturvedi et al. 1980). In a maize/cowpea polyculture study conducted in Australia (Watiki et al. 1993), maize was also found to be the dominant, most competitive species. A LER of 1.1 was found in the polyculture only when maize represented a large proportion of the plant stands, at the higher maize population densities of 4.4 and 6.7 plants/m 2 • Cowpea yields varied greatly among the 15 cultivars tested, with regard to partial LER and yields. However, maize yields were not greatly affected by the cowpea intercrops (Watiki et al. 1993). Thus, specific intercrop species combinations need to be determined to maximize the productivity of polycultures and to maximize the complementary effects of resource utilization. For example, in studies with cowpea, pigeon peas, and bean, Enyi (1973) in Tanzania found that, of the three crops, bean had the least competitive effects on sorghum, while pigeon peas showed the least competitive effects as an intercrop with maize. Also, intercrops may provide a complementary structural benefit to companion crops. Thus, in Colombia, climbing beans reduced lodging in the companion maize crop, as compared to the lodging rates observed in the corresponding maize monocultures (Davis and Garcia 1987).

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Cultivars need to be selected that are adapted to growth under polycultures. However, in some instances, productivity under monocultures may be a good index to predict the performance under polycultures, as was found by Wortmann and Sengooba (1993) when they evaluated 16 bean cultivars under both monOCUltures and banana/bean polycultures in Uganda. Overall, the three-year study found the bean/banana system to be 60% more productive than the respective monocultures (Wortmann et al. 1992). In both Colombia and Zambia, economic returns were greater in maize/bean intercrops than in monocultures (Francis et al. 1982b; Siame et al. 1998). In Colombia, an economic analysis of 20 comparative experiments showed that with high investment inputs, bean monocultures showed the greatest potential for high economic returns, but also had the highest risk for failures. The probability of obtaining a consistent income with a minimal level of investment was overall greatest in the maize/ bean polycultures (Francis and Sanders 1978). From three-year studies in Guatemala, Kass (1982) found that among several vegetables evaluated, broccoli, carrot, and potato would be optimal intercrops for incorporation in the traditional bean-maize polycultures, by providing a supplemental cash income to subsistence communities. In addition, yields of these crops were only reduced by 40-60% in the bean/maize intercrops, while yields of maize, the staple crop, were little affected (Kass 1982). 2. Polycultures with Solanaceous Crops. Solanaceous crops, such as tomatoes, are popular components of polycultures in many tropical and sub-tropical areas, such as Taiwan (Gomez and Gomez 1983). In Nigeria, Adelana (1984) conducted six tomato/maize polyculture experiments in three Savannah sites. In the polycultures, maize yields were reduced by 12-30%, while those of tomato were reduced by 50%, as compared to their respective monoculture yields, indicating the greater competitive ability of maize in this system. Due to the seasonality of production, profits were highest in the tomato/maize combination, followed closely by tomato monoculture-but the intercrop provided a greater overall income stability to the farmer (Adelana 1984). Also in Nigeria, tomato yields of an improved cUltivar were drastically reduced when intercropped with okra, but yields of a local tomato cultivar were unaffected by the polyculture (Olasantan 1985). While the okra yields were reduced in the polyculture, overall crop yields on an areal basis, as estimated by the relative total yields, was greater in the polyculture compared to the respective crop monocultures. Monoculture and polyculture yields were 18.6 and 17.7 t/ha, respectively, for the local tomato

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cultivar, and 5.5 and 2.6 t/ha, respectively, for okra (Olasantan 1985). Additional work in Nigeria showed over 50% tomato yield reductions in a tomato/Corchorus olitorius system, compared to 31.5 t/ha tomato monoculture yields. Corchorus yields were actually increased by 74% when using a 1 tomato: 2 Corchorus row planting ratio (Ojeifo and Lucas 1987). However, maximum economic returns, for off-season tomato production when prices are highest, were obtained with a 2 tomato: 1 Corchorus row ratio. In Illinois, tomato yields in a tomato/cabbage intercrop were reduced by an average of 30% in two-year trials, but statistically lower yields were obtained only in one year. Yield comparisons were made based on crop productivity per unit area, whether the crop was grown in a polyculture or under monoculture. Thus the polycultures utilized a total of one unit area for both crops, while the monocultures utilized one unit area for each crop. Cabbage yields were unaffected by the intercrops. Overall, yields in the polycultures produced economic returns and yields that were considered "comparable" to those obtained by the respective monocultures (Brown et al. 1985), even though the polycultures only used half the amount of land. Similar tomato yield reductions were observed in two tomato/cucumber studies in Michigan (Schultz et al. 1982, 1987). Tomato monocropping may thus be advisable on a given year when market demand is high, but overall system stability should be considered for analysis of the cropping system on a long-term basis. In India, several tomat%nion intercrop combinations resulted in only a 20% tomato yield reduction compared to tomato monoculture, while a tomato/radish system resulted in 55% tomato yield reductions (Singh 1991). Among the systems tested, high relative tomato yields were obtained in intercrops with onion and fenugreek, Trigonella foenum-graecum, while the highest economic returns were also obtained with the tomat%nion system. It is still undetermined why overall crop productivity was greater under the polycultures, but improved resource utilization by the intercrops likely played an important determinant role. The inclusion of an N-fixing legume as an intercrop may improve tomato polyculture yields, and/or reduce the need for high fertilizer application rates, as was observed in tomato/cowpea and okra/cowpea studies in Nigeria (Olasantan 1991). For example, in Nicaragua, tomato yields were similar in tomato/bean systems compared to tomato monocultures (Rosset 1989), while bean yields were reduced by 25% in the polycultures. However, the LER of the tomato/bean system was 1.72 compared to the respective monocultures showing a 72% greater crop productivity on an areal basis in the polycultures. Profits were also

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greater with the tomato/bean system than with the respective monocultures (Rosset 1989). However, not all legume-based polycultures provide needed resources to obtain high companion crop yields. Thus tomato yields were reduced by 32% in a tomato/bean polyculture conducted in Maryland (Teasdale and Deahl 1987), even though the LER for the system (1.17) showed a 17% greater crop productivity in the polyculture than in the monoculture, due to a better use of the planted area by the intercrops. In Nigeria, tomato yields were also reduced by 55% if unfertilized or by 46% if supplemental-N (30 kg/hal was applied in a tomato/cowpea system, indicating the nutrient resource limitations in the legume-based intercropping systems (Olasantan 1991). In this experiment, the polyculture was established following several crops such as cassava, maize, and melon, indicating a possible nutrient depletion and thus greater competition for limited resources in the tomato/cowpea planting. In Northern India, three-year potato/sugarcane polyculture trials showed that an earlier-maturing potato cultivar and a potato double-row (1 row sugar cane: 2 rows potato) planting arrangement resulted in system optimization, compared to the use of a late-maturing potato cultivar and a 1 potato row: 1 sugarcane row planting arrangement. Overall polyculture potato in the double-row design had mean yields of large-sized tubers (>75 grams) of 10.1 to 15.9 t/ha that were similar to those obtained under potato monocultures (Verma and Yadav 1986). In Bhutan, potato polycultures with either maize or faba bean, Vida faba, at 2700 m elevation, resulted in LER values ranging from 1.03 to 1.49, showing 3-49% greater productivity for the intercrops than for the respective monocultures (Roder et al. 1992). The polycultures presented less risk of economic losses to the farmer, compared to the sole-crop plantings, but economic returns were 12-15% greater in the polycultures in only one of the two locations where the trials were conducted. Based on the research results, it was considered that optimal productivity would be obtained by using a potato cultivar that matured in 100-120 days in combination with a maize cultivar that matured in 170-190 days (Roder et al. 1992). 3. Crop Breeding for Polycultures. The study of crop breeding for polycultures is in its infancy (Willey 1979b). Francis (1985) described some of the challenges in this area, such as the complexity of interspecific competition, and the challenge of crop breeding and evaluation under different types of cropping systems. However, the selection of cultivars based on timing of crop maturity to match particular polyculture productivity patterns is an effective tool to increase crop yields

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and potential economic returns to the farm (Francis 1985; Valenzuela and DeFrank 1995). Selection traits for polyculture systems may include days to maturity, competitive ability, shade tolerance, water and nutrient uptake patterns, and canopy architecture (Wooley and Rodriguez 1987) to fit particular polyculture schemes (Francis 1985). In addition, activity and duration of the photosynthetic assimilating areas, root and top growth, carbohydrate partitioning (harvest index), and efficiency of resource utilization (Sashidhar et al. 1986) will be additional selection traits used when evaluating individual crops for their productivity under polycultures. When breeding legumes for drought tolerance, not only the yielding ability under water stress should be considered, but also the effectiveness of N fixation under the water stress conditions. Water stress-tolerant germplasm has been identified in cowpeas, but now higher N fixation capacity under these conditions should also be selected for (Walker and Miller 1986). A difficulty in the selection of improved cultivars is the significant season x cultivar and environment x season x cultivar x cropping system interactions that are prevalent in polycultures (Francis et al. 1978). A proposed methodology is to begin selection programs under monoculture, and later evaluate top promising cultivars under polyculture trials (Francis et al. 1978). Germplasm diversity among crops normally allows for breeding of desirable polyculture selection traits (Valenzuela and DeFrank 1995). For example Phaseolus lines exist that germinate, grow, and blossom at suboptimal temperatures (Dickson and Boettger 1984). This works well if beans are grown as understory crops in a polyculture, and/or at high elevations. 4. Polycultures and Experimental Design. In complex polycultures, it is

difficult to isolate variables that affect the yield response to particular cultural treatments and environmental conditions. Even in monocultures biological/environmental/temporal interactions such as nematode species x soil pH (Shafer et al. 1992), nematode x bacterial/fungal infection (Johnson and Powell 1969; Swarup 1990); leafminer x fungal infection (Chandler and Thomas 1991); temperature x aphid numbers (Valenzuela and Bienz 1989), and light x N rates (Valenzuela et al. 1990) need to be unraveled prior to developing an understanding of crop yield dynamics in response to a defined set of environmental parameters. For example, covariate analyses of asparagus trials in France involving seven cultivars grown in four locations over nine years (with missing data), resulting in 35 treatment combinations, revealed that temperature during the four months preceding initial harvest date had the major effect

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on the observed genotype x environmental interactions (Rameau and Denis 1992). Similarly, a two-year study in 26 subsistence farms in the northern highlands of Rwanda to determine factors affecting yields of potato and bean cultivars found, based on a least square methods procedure, that pH x bean fly pressure, elevation x K, bean fly pressure x K, and % silt x K were among the major variables affecting bean productivity, and that organic C x Mg, elevation x K, and elevation x % sand were important variables affecting potato yields (Burleigh et al. 1992). This type of research is necessary to understand underlying agroecological mechanisms that have an effect on pest pressure, fertility, and crop yields. Advancements in the design and statistical analysis of polyculture experiments (Hill 1973; Huxley and Maingu 1978; Willey 1979b; Pearce and Gilliver 1978, 1979; Yates and Dutton 1988; Finney 1990; Federer 1993) and in the evaluation of crop yields in intercrops vs. monocultures (Hiebsch and McCollum 1987) will facilitate the elucidation of mechanisms of yield dynamics, resource utilization, and pest dynamics in polycultures. An important aspect in the analysis of polycultures is the "yield stability" of particular systems over several seasons (Rao and Willey 1980), which is a primary concern for the grower-that is, what are the probabilities for crop failure in a given year. Advancements in the statistical analysis of genotype x environmental interactions through stability analysis (Westcott 1987) will also be helpful in the analysis of performance of cropping systems across seasons and locations. Sharma et al. (1986) used this analysis in Costa Rica to evaluate 50 soybean genotypes under monoculture and in soybeanl maize polycultures, as did Wooley and Rodriguez (1987), in an experiment consisting of 34 maize cultivars x 4 bean cultivars x 3 cropping systems x 2 locations. III. NUTRIENT MANAGEMENT AND SOIL CONSERVATION A. Soil Fertility

Subsistence farmers grow staple crops with little or no fertilizer applications (Aggarwal et al. 1992; Olasantan 1992; Sanginga et al. 1992). In Thailand, about 45% of farmers use no synthetic fertilizers, and the other 55% average 27 kg/ha (Jiraporncharoen 1993). Average application rates of inorganic fertilizers in Rwanda are 0.4 kg/ha (Drechsel et al. 1996). Low-input technologies available to build or maintain soil fertility in the tropics include legume-based polycultures, minimum tillage,

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organic mulch and organic amendment applications, rotations, slashand-burn agriculture, and agroforestry systems (Sanginga et al. 1992; Juo et al. 1995). In addition, more room exists for use of locally available materials as a source of organic fertilizers such as seaweed products (Crouch and Van Staden 1994) to reduce the dependence on subsidized and/or imported synthetic fertilizers. For example, Thailand, a country that imports 100% of its synthetic fertilizers (over 850 x 10 3 t NPK) , annually produces over 82 x 10 6 t ofbiowastes, which, upon development of appropriate distribution channels, could be applied on some of the 19 x 10 6 ha of nutrient-depleted agricultural soils (Jiraporncharoen 1993). Other Asian countries highly dependent on imported fertilizers (80-100% imported NPK) include Cambodia, Fiji, Laos, Nepal, Papua New Guinea, Sri Lanka, Thailand, and Vietnam (Ahmed 1994). Alternative nutrient management techniques need to be developed to maximize the use of locally available resources, to improve nutrient cycling budgets on a regional basis, and also to reduce the commercial farmer's dependence on external inputs such as pesticides and fertilizers. Paradoxically, in capital-intensive production systems, overfertilization is often more of a concern for both environmental and economic reasons. The common practice of over-fertilizing in intensive production systems not only results in economic losses to the farmer, from the purchase of unnecessary inputs, but may also result in nutrient leaching and in subsequent pollution of aquifers and aquatic habitats. The levels of potential nutrient losses from commercial production systems was shown by Lowrance and Smittle (1988) in Georgia. In rotational studies with squash, snap bean, and winter rye for a period of 15 months, a total of 388 kg/ha N was applied but only 90 kg N was removed with the crop harvest, leaving about 290 kg N available for potential leaching as nitrate (Lowrance and Smittle 1988). B. Nutrient Calibration Studies Soil fertility calibration studies for individual nutrients are required for individual production areas to optimize fertilizer applications, crop yields, and efficiency of nutrient utilization. However, such data, which is developed through multi-year and multi-location trials to determine soil nutrient analysis, crop cultivar nutritional status, and crop yields in response to fertilizer rate applications (Chapman et al. 1992) is lacking throughout the tropics (Fox and Valenzuela 1992). Furthermore, calibration work needs to be revisited on an ongoing basis, as new cultivars or cultural practices are introduced into important production areas (Hargrove et al. 1984; Chapman et al. 1992), such as with the calibration ofK

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rates in Florida for fertigation in strawberry (Albregts et al. 1996). Other examples of nutrient calibration work include tomato in Nigeria (Sobulo et al. 1977) and jicama, Pacyrrhizus erosus, in West Bengal, India (Sen and Mukhopadhyay 1989). Also, the use of nutrient sap analysis for crops such as broccoli (Kubota et al. 1997), lettuce (Alt and Full 1988), and tomato (Rhoads et al. 1996; Valenzuela and Riede 1996), based on appropriate calibration work, is an available rapid diagnostic technique to determine crop nutritional status.

c. Organic Amendments Long-term experiments are beginning to unravel the benefits provided by the maintenance of soil fertility through annual organic amendment applications. For example, in Germany, soil quality analysis after 100 years of annual 12 t/ha farmyard manure applications showed that improvements in soil organic matter were associated with the fine and medium silt fractions, and in an increase of lipid contents in the silt fractions as compared to untreated plots (Schulten and Leinweber 1991). The establishment of diagnostic techniques for assessment of soil quality will facilitate the evaluation of available soil improvement techniques for enhancement of sustainable crop yields. Manure, when locally available, is an effective nutritional supplement to obtain high crop yields. Additional benefits of manure applications include increase of soil pH in acid soils (Hue 1992); slower nutrient release over time, resulting in less nutrient leaching as compared to synthetic fertilizers; and improvements in soil quality through an increase in the soil organic matter content; and promotion of greater earthworm activity (Tiwari 1993). In Madurai, India, 50: 50 treatment combinations (totaling 100 kg/ha N) of several manure sources and urea resulted in greater eggplant yields than sole urea, sole manure, and controls. The slow-release nature of the manures likely explains why the compatibility of the manure and urea treatment meets the crop nutritional demands throughout the crop's growing cycle (Jose et al. 1988). No information was provided as to how N contents were assessed in the manures, duration of the harvest cycle, and other experimental details, which precludes making any definite conclusions on the effects of using manures as fertilizers on eggplant yields from this particular set of experiments. However, similar results were obtained by this research team (Abusaleha and Shanmugavelu 1988) in work with okra, showing greatest yields by using a 50: 50 synthetic N (ammonium nitrate) : manure combination as compared to using a sole synthetic N source, or sole manure applications.

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The use of composts as fertilizer amendments, which is typical in many temperate areas (Nishio 1996), has received little attention in the tropics but may have potential for the increased productivity of high-value crops as shown in five-year trials by H. R. Valenzuela and R. T. Hamasaki (unpublished data) in Hawaii. Composts are not only useful as nutrient amendments, but may also be useful by promoting soil microbial activity to improve nutrient cycling, and also to promote nematode, disease, and soil arthropod pest biocontrols. D. Cover Crops and Green Manures Cover crops and green manure applications were used by many ancient cultures to improve soil fertility and/or to break disease cycles. In temperate regions, cover cropping has also been an integral part of crop rotational programs and in orchards, for many years. In Hastings, Florida, summer and fall cover crops have been a feature of potato production programs for over 100 years, resulting in weed management, use of residual fertilizers, and in soil organic matter conservation (Davis et al. 1996). Cover crops provide several benefits to the agroecosystem, including use of residual fertilizers, increased soil C and N content, improved soil water content and drainage, improved soil tilth, reduced soil temperatures, reduced erosion, and enhanced weed control. Sainju and Singh (1997) found that cover crops reduced annual erosion levels to 2 t/ha compared to 18 t/ha in bare soils. Also, in the southeastern U.S., cover crops were estimated to reduce erosion levels by 62% in ultisols, and from 47-96% in alfisols, compared to bare soils (Sainju and Singh 1997). Extensive location-specific trials need to be conducted for selection of cover crop species and cultivars adapted to local growing conditions (Shribbs and Skroch 1986). Also, work is required to match individual cover crops with compatible vegetables to maximize yields, to minimize possible problems such as disease or pest outbreaks, and to synchronize the nutrient release of the cover crop with the nutrient demands of the cash crop (Doran et al. 1988; Sainju and Singh 1997). The selection of the adequate cover crop/cash crop combinations, and the proper timing of all cultural practices, will result in minimal competition for limiting nutrients (Shribbs and Skroch 1986), and in increased cash-crop yields. In Oklahoma, cover crops with potential for use in no-till systems included annual ryegrass, rye, barley, and wheat. These cover crops were selected based on the percentage ground cover (cover density), ability to suppress weeds, and ease of herbicide kill (Nelson et al. 1991).

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Green manuring can provide a significant portion of the following crop's nutritional requirements (Sainju and Singh 1997). In China, over 20% of land under rice production is green manured for this purpose. Desirable traits for green manure species include ease of establishment, high and fast biomass production rates, effective nodulation with indigenous rhizobia, ease of eradication, and inability to host important pests or diseases (Sanginga et al. 1992). Cover crop selection will also vary whether it is needed to supplement N-fertilization (legume) or to scavenge N from the previous crop (non-legume species) to serve as a nutrient reservoir for the following crop (Sainju and Singh 1997). The length of the growing season of the green manure species should match the cultural program of the cropping system. In the Philippines, shortduration cover crops such as mung bean (Vigna radiata) and cowpea, contributed 40-80 kg/ha N in a 45-day growing cycle, which in turn increased yields of the subsequent rice crop by over 2 t/ha (Morris et al. 1986). In separate work conducted at Los Banos in the Philippines, soybean used as a green manure in a maize/soybean system, and plowed under 42 days after sowing, contributed 28 kg/ha N to the companion crop, resulting in increased maize yields (Pandey and Pendleton 1986). Green manure crops that excelled in the Philippines, by contributing N to the subsequent rice crop, included, among the eight species tested, Sesbania cannabina and sunhemp, Crotalaria juncea (Meelu et al. 1992). Mung bean and cowpea, although less effective as green manures than Sesbania or Crotalaria, would be effective dual-purpose crops, serving as green manures plus providing an edible product. Location-specific work thus needs to be conducted to evaluate the potential nutrient contribution of different green manure species and cultivars. In India, Egyptian clover, Trifolium alexandrium, was the green manure that contributed the most toward the yield of the subsequent maize crop, compared to the yield contributions provided by Lathyrus sativus and by peas (Singh and LaI1985). In Maryland trials conducted over two years, a 5 cm layer of hairy vetch, Vida villosa, mowed in the spring after fall planting, contained an average of 182 kg/ha N (Teasdale and Abdul-Baki 1997). A tomato crop transplanted over the mulched plots and treated with 66 kg/ha N produced greater yields compared to plastic mulch treatments that received 112 kg/ha synthetic N, showing the potential nutritional contributions of green manure cover crops to the subsequent cash crop (Teasdale and AbdulBaki 1997). Five-year trials in Oregon also showed that the use of leguminous cover crops alone or in combination with cereal cover crops plus moderate N treatments (at one-fourth to one-half of recommended rates) resulted in yields that were similar to those obtained with conventional

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synthetic N fertilizer applications rates (Burket et al. 1997). Similar work showed that rhizobium inoculated Leucaena, contributed the equivalent of 70 kg/ha N to the subsequent crop after six months of growth (Sanginga et al. 1986), Mean N production by several leguminous green manure crops during the summer in Florida was (in kg/hal: Crotalaria (170); hairy indigo (Indigofera hirsuta) (220); jointvetch (Aeschynomene americana) (170); mungean (40); pigeon pea (250); and velvetbean (Mucuna pruriens) (190) (Reddy et al. 1986a). Dry biomass produced by these crops, when determined prior to seeding, normally exceeded 10 t/ha. However, N contributions to the subsequent crops planted in the fall were estimated to range only between 2 to 23 kg/ha N, indicating that any N accumulated in the soil will be released over a longer period of time (Reddy et al. 1986a). Effective green manure species that were evaluated in Africa included Centrosema pubescens, Mucuna pruriens, Pueraria phaseoloides, and Stylosanthes guianensis; among the results were improved soil bulk density, soil moisture retention, and protection against erosion (Sanginga et al. 1992). The use of cover crops/green manures may be limited to relatively affluent small- or large-scale farms, and may have less potential in areas with high population pressure and where subsistence agriculture is prevalent. For example, multi-year developmental programs in Rwanda, which has average population densities of 380 persons/km 2 , and where densities of 700 persons/km 2 are common, failed to successfully introduce the use of cover crops and green manures to small-scale farmers. Factors that prevented acceptance of cover crops or use of hedgerows in Rwanda included acute land shortages, lack of consistent results (in terms of biomass productivity, and on nutrients returned to the soil), and insufficient cover crop growth in nutrient-depleted fields (Drechsel et al. 1996). E. Plastic, Organic, and Living Mulches Black paper mulches were used for commercial pineapple production in Hawaii around 1914, resulting in greater soil temperatures, moisture preservation, weed control, and corresponding improved pineapple yields (Ekern 1967). Commercially implemented in several areas in the 1960s (Bryan and Dalton 1974; Johnson 1987), plastic mulches are now popular for the production of crops such as sweet corn, cucumber, eggplant, muskmelon, pepper, squash, and tomato (Johnson 1987). Yield increases from the use of plastic mulches occurred in bell pepper (Monette and Stewart 1987), chili pepper (Vos et al. 1995), collard

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(Hochmuth et al. 1992), cucumber (Farias-Larios et al. 1994), sweet corn (Hochmuth et al. 1990), tomato (Schalk and Robbins 1987; Wien and Minotti 1987), and watermelon (Hochmuth and Hochmuth 1994). However, economic analyses need to be conducted to assess the profitability of these capital-intensive systems. An environmental assessment may also be required to determine options for disposal/reuse of these plastic materials, and to conform with any local regulatory laws regarding the use/disposal of plasticulture materials. The use of organic mulches is an effective way to utilize locally available resources, return nutrients to cultivated land, protect fields from soil and nutrient erosion, and smother weed growth. Benefits provided by organic mulches and other organic amendments also include improved soil structure, soil aggregation, water infiltration, water-holding capacity, water content, aeration and permeability, increased rooting depth, decreased soil crusting and bulk density, increased soil pH (with exceptions, see Tindall et al. 1991), and cooler soil temperatures, as observed in plantain, Musa AAB (Obiefuna 1991), and in tomato (Tindall et al. 1991). The degree of protection from soil erosion and water runoff is in general proportional to the percent residue cover and to the amount of residue in the field (Lal1998). In Punjab, India, the rate of soil losses in maize ranged from 357 kg/ha with 98% mulch residue cover, to 586 kg/ha with 85% cover, to 2,258 kg/ha of soil loss with a 0% residue cover (Sur et al. 1992). In addition, the mulched treatments grown with maize overall had 30% less soil losses than the uncropped mulched treatments, further stressing the relationship found between residue and vegetative ground cover and low soil erosion rates The use of organic mulches to minimize soil erosion is especially promising in sloped lands, as shown by Wu and Yamamoto (1996) in Taiwan. Erosion soil losses of 300-400 t/ha, in monsoon area slope lands, were reduced to less than 5 t/ha through the use of Gliricidia/ napier (Pennisetum purpureum) hedgerows in combination with organic mulches and no-till planting. However, limitations to the adoption of organic mulches includes the high labor demand, mulch availability, the lack of appropriate mechanization, and the need to re-apply the mulch on a frequent basis in areas with high rates of debris decomposition, e.g. 3 months in Taiwan with Bahia grass (Paspalum notatum) biomass applications rates of 4.4 t/ha (Wu and Yamamoto 1996). Organic mulches lower soil temperatures in warm areas such as Nigeria and promote the activity of soil fauna decomposers such as earthworms, ants, and millipedes (Tian et al. 1993), which further promotes internal system nutrient cycling and improved soil tilth. In addition, with a better understanding of how specific crop residues affect the population dynamics of particular pests, residues from selected crop

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species may be used to either promote or suppress specific soil arthropod populations (Tian et al. 1993) that may have an effect on soil fertility or on the level of soil pests. Improved nutrient cycling and soil tilth from organic residue mulch applications may result in greater crop yields. Greater wheat yields were obtained in India with organic mulch applied at rates of 5 and 7.5 t/ha after wheat sowing (Mittal et al. 1986). Similar results were observed elsewhere in India under dryland growing conditions (Prihar et al. 1979). When organic mulches were applied to maize prior to harvest, or during a fallow period in a maize-wheat double-cropping system, both maize and wheat yields were increased, with corresponding improvements in water conservation and use efficiency. Overall, mulching resulted in 16% increased maize yields, while wheat yields increased by 864 to 1,023 kg/ha following maize, and by 1,323 to 1,513 kg/ha when following a mulched fallow period (Prihar et al. 1979). Mulching also increased yields of a mung bean-rapeseed, B. campestris, double-cropping system during dry growing conditions (De and Giri 1978).

In Nigeria, the use of 80-90 mm of grass organic mulches resulted in increased tomato growth and greater yields. Further yield increases were obtained with mulching plus staking (Olasantan 1985). Improved tomato cultivars showed the greatest yield response to both mulching and staking, compared to local cultivars. Mean 'Roma' tomato yields (t/ha) from two-year trials were non-staked controls (15.8), staked alone (20), mulched alone (26.1), and mulched plus staked treatments (35). Overall, mean crop yields of the three cultivars tested were more than doubled in the mulched plus staked treatments, compared to the controls (Olasantan 1985). Mulching up to a depth of 10 cm also resulted in 20-39% increased tomato yields, larger fruit size, and reduced incidence of disease and arthropod pests in Malawi (Kwapata 1991). Differential yield and pest tolerance responses from determinate and indeterminate tomato cultivars were observed in response to the mulching and irrigation frequency treatments. In Georgia, the application of approximately 2.5 t/ha straw mulch in tomato, which provided cooler soil temperatures, resulted in greater yields compared to plastic mulch treatments. The straw mulch treatments also resulted in improved soil quality parameters, which were conducive to improved root growth, nutrient uptake; and subsequent crop yields (Tindall et al. 1991). In Puerto Rico, yields of bell pepper grown under organic mulch were similar to those obtained under several plastic mulch treatments (Goyal et al. 1985). In southeastern Nigeria, 2 t/ha straw mulch applications were optimum for improvement of soil quality characteristics conducive to crop growth enhancement (soil-water sorptivity, transmissivity, and

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infiltration-rate), compared to no-mulch controls (Mbagwu 1991). However, greatest yield increases of 80 and 67% over controls were obtained with 4 t/ha mulch applications for maize and cowpea, respectively (Mbagwu 1991). Optimal mulch application rates, in this case, ranged between 3 to 4 t/ha straw mulch, depending on access to mulching materials and on labor demands involved with the mulch application process. Organic mulches can improve crop nutrient uptake. In Costa Rica, this was observed in bean and maize, following Erythrina poeppigiana- and Gliricidia sepium-based mulch applications. Mulching rates of 5 t/ha dry weight increased P tissue levels and yields, compared to unmulched treatments (Haggar et al. 1991). The use of living mulches (sod culture) is common on orchards (Shribbs and Skroch 1986), but less work on this area has been conducted with vegetables and other annual crops. Decreased crop yield due to competition from living mulches was reported for several crops (Nicholson and Wien 1983; Andow et al. 1986; Neilsen and Anderson 1989). However, in a selection study of eight living mulch species, three (Festuca rubra, Poa pratensis, and Trifolium repens) were identified as having no adverse effects on main crop yields (Nicholson and Wien 1983), indicating that some species can suppress weeds without decreasing yield of the main crop. Also, competitive cash crop cultivars may be selected for improved performance under living mulches, as shown in eggplant by Valenzuela and DeFrank (1994). Living mulch systems introduced in the Kalimantan agricultural settlements of Indonesia showed that yields could be maintained on a sustainable basis, due to an improved soil fertility and nutrient availability. Conversely, in conventionally tilled systems, yields would decline after six cropping seasons (Lorenz and Errington 1991). Legume living mulches (hairy vetch; barrel medic, Medicago truncatula; and black lentil, Lens culinaris) , interseeded on a stand of chili pepper, were also established successfully to produce a range of 20-136 kg/ha N, without affecting chili pepper yields (Guldan et al. 1996). Bulb onion, cucumber, eggplant, head cabbage, and zucchini were also grown successfully under a rhodes grass, Chloris gayana cvr. Katambora, living mulch in on-farm trials in Hawaii 0. DeFrank, unpublished data). F. Crop Residues and No-till Agriculture

Crop residues left in the field and conservation tillage practices offer potential in the tropics to improve soil quality, to minimize soil erosion, and to sustain yields (Akobundu and Deutsch 1983; Elliot and Papendick

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1986; LaI1998). Potential benefits of no-till farming include time saved by the farmer due to less cultivation and fewer farming operations (Lorenz and Errington 1991), improved soil water retention, reduced evaporation, improved soil tilth, energy conservation, less N losses through surface run-off with residue rates of 750 kg/ha (Mostaghimi et al. 1991, 1992), reduced sand "blasting" and subsequent improved product quality (McKeown et al. 1988; Boydston 1995), improved use of crop residues, and equal or greater yields as compared to conventional tillage systems (Gallaher 1981; McKeown et al. 1988; Hoyt et al. 1994). Work conducted to date indicates that no-till farming may result in up to a 50-fold reduction in soil losses in highly erodible soils, compared to conventional moldboard plowing, while reducing fuel and labor demands by 60-75% (Buhler 1995). For example, a ten-year experiment in a sub-tropical location showed greater C (67%) and N (66%) to a 10-cm soil depth, in no-tilled than in conventionally plowed plots (Wood and Edwards 1992). Similarly, improvements in soil quality determined after a continuous 12-year no-till experiment in Iowa included soil aggregates with greater stability in water and with a greater total carbon, microbial activity, and ergosterol concentrations than conventionally plowed treatments (Karlen et al. 1994). The greater soil-strength and bulk density that may result from no-till farming may be offset by greater earthworm numbers and burrowing activity, as observed in Australia (Rovira et al. 1987) and Iowa (Karlen et al. 1994). Earthworm activity in undisturbed soils, such as in the Lamto savannas of the Ivory Coast, which consists of annually ingesting 800-1100 t/ha soil, may also result in organic matter conservation and build-up in no-till soils (Martin 1991). The contribution of earthworms in undisturbed soils with respect to decreased soil bulk density, and increased soil organic matter content, infiltration rate, pH, and soil moisture was shown in a 20-year study conducted in England (Clements et al. 1991). Earthworms also effectively re-distribute nutrients such as K (Basker et al. 1992) among the different soil profiles, increasing nutrient availability in the rhizosphere (Sanginga et al. 1992). A three-year experiment with a maize/cowpea rotation in Guayana (Simpson 1992) showed no yield differences in response to tillage or notillage treatments. However, establishment of crops under no-till conditions may be more difficult if low soil temperatures and excessive soil moisture occur (Simpson 1992), a condition which lowered no-tilled snap bean yields in Virginia (Bellinder et al. 1987). Evidence from tropical (Roth et al. 1992) and temperate regions (Holland and Coleman 1987) indicates that no-tillage results in greater organic matter and nutrient conservation, as compared to plowing-under

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of similar residue biomass. A greater fungal to bacterial decomposer population ratio, plus slower litter decomposition rates, contributed to the greater organic matter conservation rates under no-till compared to the plowed plots in temperate climates (Holland and Coleman 1987). Another beneficial result of no-till practices may be to homogenize the soil microenvironment, especially during periods of stressful growing conditions due to high temperatures or water stress. For example, greater wheat yields were observed under no-till than under conventional tillage under moderately dry growing conditions, plus the maximum soil temperatures at a 5 cm soil depth "vere 3°C lower under the no-till treatments (Izaurralde et al. 1986). Similar no-till yield responses under drought growing conditions were obtained with maize (AI-Darby and Lowery 1986) and snap beans (Bellinder et al. 1987). Greater water-use efficiency may be found with no-till systems under drought conditions through a combination of greater root growth in the lower soil layers (below 30 cm), and through greater water conservation under no-till compared to conventional tillage, as shown in maize by Newell and Wilhelm (1987). In terms of crop productivity, no-till experiments in temperate areas showed increased or no yield reductions, as compared to conventional tillage in tomato (Zehnder and Linduska 1987; McKeown et al. 1988), snap bean (Bellinder et al. 1987; Abdul-Baki and Teasdale 1997), and asparagus (Boydston 1995). Vegetables that in temperate areas were successfully grown under no-till, in part due to the availability of registered post-emergence herbicides, included asparagus, bean, beets, cabbage, carrot, lima bean, onion, peas, potato, spinach, sweet corn, and tomato (Hoyt et al. 1994). However, the following challenges exist for the further implementation of no-till systems in the tropics: land preparation and cover crop establishment; effective methods to kill the cover crop prior to planting; development of appropriate planting tools and machinery; effective preemergence and postemergence weed control; control of those pests promoted by the no-till system; nutrient calibration; scheduling considerations under rainfed systems; cooler soil temperatures during the winter or at high elevations; stand establishment, especially for direct sowing of small-seeded crops; and demand for greater farmer management skills and expertise, especially for mechanized no-till farming (Akobundu and Deutsch 1983; Hoyt et al. 1994). G. Polycultures and Resource Utilization

The interactions that occur between plants from different species growing in close proximity is complex, and can involve both competitive and

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"beneficience" (mutualism and commensalism) interactions (Hunter and Aarssen 1988). Beneficience effects on the physical environment may include providing an improved microclimate (such as providing shade or lower temperatures in the understory), physical support (such as maize providing support to vining intercrops), rhizosphere enrichment (by adding organic matter or stimulatory allelochemicals); and possible direct or indirect intra-species nutrient transfers via proximal associations, as shown in a rice/mung bean intercrop (Aggarwal et al. 1992) or in mycorrhizal associations (Hunter and Aarssen 1988). Improved resource utilization was shown for polycultures as compared to monocultures, for example, with N, K, and Ca (Willey 1979a; Zobisch et al. 1995). Models indicate less nutrient leaching in some polycultures with an estimated 56 kg/ha nitrate losses in monocultures vs. 1 kg/ha losses in perennial polycultures (Seyfried and Rao 1991). In the highlands of central Kenya, P and K nutrient runoff losses in a maize/bean polyculture were about 9% of the losses obtained under bare-fallow, 35% of maize monoculture, and 65-70% of bean monoculture (Zobisch et al. 1995). The improved resource utilization observed with root and underground tuberous crops based polycultures in the tropics was reviewed by Valenzuela and DeFrank (1995). Spatial arrangements and total plant populations are key variables that need to be considered to understand yield dynamics in polycultures. In monocultures , the yield dynamics of agronomic crops in response to planting pattern arrangements is also being investigated, building on a research base that spans over 60 years (Duncan 1986). Considerable polyculture work has been conducted in the area of planting arrangements with agronomic crops (Willey 1979b; Ssekabembe 1991), but little work has been conducted with vegetables. Legumes are common in traditional polycultures, as observed with cowpea in Nigeria (Andrews 1972; Remison 1978), perhaps due to the contributions provided to the system because they are N-fixing plants. The contribution that N-fixing nodulating legumes provide to companion non-legume crops in polycultures was shown by Elmore and Jackobson (1986) in a soybean/ sorghum system and by Aggarwal et al. (1992) in a mung bean/rice intercrop. Sorghum yields were increased by 9% when intercropped with a nodulating soybean cultivar, compared to yields obtained when intercropped with a non-nodulating isoline of the same cultivar. This study also showed that proximity to the legume was an important variable, as the N transfer from the legume to the sorghum intercrop occurred with 0.4 m row spacings but not with the 0.8 m soybean row spacings (Elmore and Jackobson 1986). Similar results were obtained in India in maize/ legume systems, where soybean and blackgram were identified, among

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the legume species tested, as being the most effective in increasing Nfixing bacterial populations in the maize rhizosphere (Singh et al. 1986). Nitrogen-fixing bacteria populations were 40-50% greater in the polycultures than in the maize monoculture, by 60 days after sowing. The maize N uptake was increased by 15-200/0 and the maize grain harvested from the polyculture had 30-35% more N than the respective monocultures. Overall polyculture maize yields were increased by 15-20% (significance obtained only in one of two years) over monoculture maize, and the total N contribution by the legume intercrops was estimated at 80 kg/ha N (Singh et al. 1986). Polyculture systems with agronomic crops have shown additional yield advantages over monocultures under water stress growing conditions (Natarajan and Willey 1986). Water use efficiency was also shown to improve in a mustard, Brassica juncea/ chickpea intercrop than in the respective monocultures (Kushwaha and De 1987). However, little work is available in the area of efficiency of resource utilization and corresponding yield responses in vegetable polycultures. While overall yields on an areal basis may increase in polycultures as compared to monocultures, higher fertilizer rates are generally required to meet the nutrient demands of the more productive cropping system (Kurtz et al. 1952), as shownin standard monoculture N x population density studies (Dweikat and Kostewicz 1989); in early fruit tree/cover crop studies (Goode and Hyrycz 1976); and in polycultures involving okra/cowpea and tomato/cowpea (Olasantan 1991); root and underground tuberous crops (Valenzuela and DeFrank 1995), and bean/maize intercrops (Siame et al. 1998). In tomato/cowpea and okra/cowpea studies, tomato and okra polyculture yields were increased by 34 and 21 %, respectively, by applying 30 kg/ha N, compared to the unfertilized treatments. Thus additional resources such as water (Schultz et al. 1987) and N and P (Remison 1978; Siame et al. 1998) may be required in polycultures, especially at the higher population densities (Fisher 1977b). This feature of polycultures was observed with respect to water utilization in maizebased polycultures conducted over several seasons in Kenya. During seasons of low rainfall the maize-bean and maize-potato systems yielded less than their respective monocultures, but the polycultures overyielded the monocultures during high-rainfall growing seasons (Fisher 1977a). The main polyculture yield reductions were attributed to the decreased maize yields as competition with the intercrops was increased, which reflects the fact that highly productive agroecosystems need the resources (nutrients, light, and water) necessary to match any expected yields. Another critical aspect dealing with crop productivity and efficiency of resource utilization in polycultures is the need to select crop cultivars

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that have nutrient demands that match the potential productivity of particular agroecosystems. Germplasm with differential nutrient uptake exists for most studied plants (Barker 1989; Marschner 1995), including okra (Emebiri et al. 1992), peanut (Branch and Gascho 1985), pumpkin (Swiader et al. 1991), tomato with respect to P (Coltman and Kuo 1991) and N (Valenzuela and Riede 1996), and tropical underground tuberous and root crops (Valenzuela and DeFrank 1995).

H. Double-cropping Systems Double cropping is a Widespread practice with some agronomic crops such as the wheat-soybean system in the southeastern U.S. (Hargrove et al. 1984), and has been used for decades in several vegetable cropping areas such as south Florida (Bryan and Dalton 1974; Palada et al. 1979), resulting in improved nutrient and water utilization (De and Giri 1978; Albregts and Howard 1985), improved soil quality, and reduced pest outbreaks. However, few studies have been conducted with double cropping for the production of vegetable crops in the tropics. In a wheat/peanut double cropping study conducted in India, P applied to wheat left sufficient residual P levels for the subsequent peanut crop (Pasricha et al. 1980). Similarly, residual P from an initial 75 kg/ha P application in snap bean also improved yields of two subsequent crops (tomato followed by okra and cabbage) in a triple-cropping system evaluated over two years in Bangalore, India (Prabhakar et al. 1987). Supplemental P applications on cabbage, during the growing season, only increased yields when initial soil available P levels were very low (below 6 kg/ha P). A plasticulture experiment conducted in Louisiana showed an effective double-crop system consisting of a spring tomato crop, followed by cucumber. The cucumber crop was produced with savings in installation of the plastic mulch, drip systems, and stakes (Hanna and Adams 1989). In addition, cucumber required no fertilization since adequate yields were obtained by utilizing the residual nutrients that remained after the first tomato planting. Tomato-tomato and tomato-cucumber two-year studies in Florida also showed minimal response (yield increases only observed with applications of 75 kg/ha N) to fertilization of the second crop (Everett 1978). Other effective double-cropping systems evaluated in Florida were fall-planted strawberry followed by spring planting of sweet corn, squash, cucumber, or snap bean (Albregts and Howard 1985). The crops following strawberry responded to modest (35-70 kg/ha N) fertilizer rates in two of three years, when rainfall or overhead irrigation caused nutrient leaching.

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In Florida, broccoli-tomato or cucumber-broccoli triple-cropping system studies were conducted to evaluate crop sequence, soil type, irrigation system, and supplemental fertilizer application effects on crop performance (Clough et al. 1990). The second and third crops in the system responded to supplemental fertilizer treatments (135 to 270 kg/ha N) especially in drip-irrigated plots and also on the fine sandy soil plots, attributable to nutrient leaching effects. The drip-irrigated mulched beds maintained an overall greater moisture accumulation in the root zone throughout the growing season, which may have been more conducive to nutrient leaching below the root zone, compared to over-head irrigation. However, in the heavy soils, tomato showed no response to supplemental NK applications when following a fall broccoli planting that received the higher (270-404 kg/ha ofNK) fertilizer rates. Also, in the heavy soils, over-head irrigated squash did not respond to supplemental fertilization, showing similar yields in treatments grown with residual fertilization alone, in accordance with the strawberry-squash studies conducted by Albregts and Howard (1985). Drip irrigation resulted in greater moisture uniformity and less water used than overhead irrigation. A weekly fertigation program could be implemented to reduce possible nutrient leaching under drip irrigation, especially in sandy soils. Overall, the triple-cropping system optimized use of the plastic mulch, fertilizers, fumigant, fuel, and labor (Clough et al. 1990). 1. Rhizosphere Microbial Associations

Intensive associations are typically established between roots and the rhizosphere microflora. The enhanced plant growth that is often observed as a result of these associations is attributed in part to the release of plant hormones (plant growth regulators) by the associated microflora. The production of plant growth regulators (abscisic acid, auxin, cytokinins, ethylene, and gibberellins) by Azotobacter, Azospirillium, Rhizobium, and mycorrhizae, and the possible mechanisms for enhanced plant growth was recently reviewed by Arshad and Frankenberger (1998). Arbuscular myccorrhizae (AM) fungi, which develop symbiotic associations with most terrestrial plants, aid plants in the uptake ofP, K, Zn, Cu, Mn, Fe, and S. Effects brought about from these beneficial AM associations include enhanced root, seedling, micropropragated plantlet (Chang 1996), and plant growth; seedling survival in asparagus and strawberry (Chang 1996); improved soil structure through rhizosphere aggregate stabilization (Burns and Davies 1986); drought tolerance in sorghum (Sieverding 1986; Chang 1996); salt-tolerance in cucumber (Rosendahl and Rosendahl 1991); resistance to soil-borne fungal and bacterial disease

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infection (Chang 1996), as shown for asparagus (Wacker et al. 1990) and with Pythium and bacterial biomass in cucumber (Rosendahl and Rosendahl 1990; Christensen and Jakobsen 1993); and earlier fruiting in strawberry (Chang 1996). In the tropics (Zaire), mycorrhizal associations often contribute significantly toward the productivity of traditional subsistence vegetables such as cassava, Dioscorea, onion, sweetpotato, and tomato (Khasa et al. 1992). Information is required on cultivar responses and cultural practices that maximize effective AM mycorrhizal associations with vegetables. For example, AM-bean associations were affected by both cultivar and by tillage (soil compaction) treatments (Mulligan et al. 1985). Differential cultivar response to G. fasciculatum inoculation was also found in cassava and sweetpotato in terms of inoculation rate and increased yields (Sivaprasad et al. 1989). In general, greater AM root colonization levels occur in organic than in conventional farms (van Bruggen 1995), which is attributable, in part, to the greater P fertilizer applications in conventional farms. The relationships between effective AM associations and soil P status were elucidated for a number of crops, including potato (McArthur and Knowles 1992). In low P soils from Hawaii, yields in bell pepper were increased when inoculated with Glomus aggregatum (Waterer and Coltman 1989). Organic matter amendments promoted greater microbial activity and release of available P in soils with high Pfixing rates (Nishio 1996). Similarly, selection of efficient Glomus AM isolates adapted to the Dharwad district of India increased the beneficial mycorrhizal association effects on chili pepper (Sreenivasa 1992). In legumes such as soybean, a synergistic interaction appears to exist between N-fixing mycorrhizae and AM populations that increase nutrient uptake, N-fixation rates, and yields (Pacovsky et al. 1986). This yield enhancement will most likely be seen in low Nand P content soils or in soils with high P-fixing capacity. Synergistic Rhizobium x Azospirillum interactions were observed in winged bean and in soybean (Iruthayathas et al. 1983). In the tropics, AM are not yet widely available from commercial or public suppliers. However, in Taiwan, the Chiayi Agricultural Experiment Station annually supplies to growers AM fungal inoculum for muskmelon production in about 500 ha, and AM-inoculated strawberry for commercial growers is also available in Brazil (Chang 1996). A limitation to a more widespread use of AM in production fields is the low inoculation rates in soils high in phosphorus, but inoculation of seedlings prior to transplanting may effectively overcome this obstacle, as shown from preliminary work in Taiwan (Chang 1996). Charcoal applications, which are used commercially in Japan to promote root-AM

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associations, resulted in more effective AM associations, improved nutrient uptake, and yields (Nishio 1996). Elsewhere, fertilizer products with microbial inoculants are available in the market, such as the 'biosuper' pellets in Australia, which consist of rock phosphate, sulfur, and sulfuroxidizing bacteria (Nishio 1996). Other soil microorganisms that enhance root growth include Azospirillum spp., which resulted in increased plant growth in spinach (2.6-fold increased fresh leaf weight), Chinese cabbage (46%), Chinese mustard (11 %), and cucumber (31 %) (Garno 1991). Trichoderma improved the growth of radish (Paulitz et al. 1986). A greater understanding of plant-microbial interactions, characterization of soil microflora species, and an understanding of their response to cultural practices could lead to the increased trend of using microbial inoculants for crop growth enhancement (Arshad and Frankenberger 1998).

IV. ECOLOGICALLY-BASED PEST MANAGEMENT A. Introduction Over the past 50 years, pesticides have become a dominant feature of pest control programs in capital-intensive crop production systems throughout the world. In the tropics, pesticide-based crop production programs are mainly focused toward the production of export-oriented crops (NRC 1993). For instance, Latin America is considered to be among the fastest-growing regions for agrochemical sales (US $2.6 billion annually), accounting for about 10% of world sales, and for onethird of pesticide global sales growth. The increase in pesticide sales is mainly for use on maize, soybean, fruits, and vegetables (PAN 1996a). Environmental and human-health concerns exist about the indiscriminate use of pesticides, especially in tropical areas where pesticides that are banned in developed countries continue to be used. For example, in Brazil, every year over 300,000 cases of human pesticide poisoning are reported by the Ministry of Health (SEJUP 1996). Farm surveys in Nigeria showed that pesticides that are banned in the U.S., such as lindane, DDT, aldrin, and dieldrin, are all used and are readily available (PAN 1998a). This and other concerns (Pimentel et al. 1991; PAN 1998b), including the high cost of pesticide production and registration, has resulted in a call for alternative pest control methods. For this reason, several countries, such as South Korea (PAN 1996b), Sweden (Weinberg 1990), Denmark, Norway, and the Netherlands (Pettersson 1994), have developed goals for pesticide use reduc-

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tion in agriculture (>50% initial reduction), goals which are also deemed realistic for implementation in the U.S. (Pimentel et al. 1991). Furthermore, low-input pest controls are necessary for the vast majority of farmers throughout the tropics who have little or no access to the capital needed to purchase pesticides or other high-cost capital inputs (such as fertilizers, hybrid seed, and machinery). However, for the development and implementation of alternative pest management practices, a greater knowledge is required of pest biology, environmental interactions, and consequently greater management skills for implementation (McSorley 1996). B. Nutrient by Pest Interactions 1. Diseases. Healthy and vigorous crop growth often promotes tolerance to pest attack (Marschner 1995). Plants exposed to an unbalanced

nutrient supply may become more susceptible to arthropod pest, disease, and/or weed infestations. For example, excessive N applications have resulted in a greater incidence of rust, powdery mildew, and bacterial diseases (van Bruggen 1995). Also, in Florida, high P application rates resulted in a greater incidence of Helminthosporium in sweet corn (Stoner 1951). Nutrient x disease interactions, being affected by microorganisms, environmental variables, and the host, are consequently highly dynamic and complex, and thus are poorly understood. However, a better understanding of host nutrition and its effects on disease development offer the potential for modification of fertilizer application programs for management of important diseases (Huber 1981). Nutrients such as N, K, P, Ca, and others have been reported to improve plant tolerance to disease attack (O'Rourke and Millar 1966; Canaday and Wyatt 1992; Yamazaki and Hoshina 1995). For example, modifications in the application rates of complete fertilizers resulted in less phytophthora root rot, soybean mosaic virus, and stem canker in soybean (Pacumbaba et al. 1997), and in a reduced incidence of early blight, Alternaria solani, in tomato (Jones and Jones 1986). Calcium fertilizer levels affect plant tolerance to disease, not only in the field, but also during the postharvest phase (Conway et al. 1994; Yamazaki and Hoshina 1995). Along these lines, the literature covering crop disease x nutrient interactions is extensive, but due to the many variables involved it is still difficult to make generalizations. Thus location-specific nutrient x disease interactions need to be evaluated on a case-by-case basis (Huber 1981).

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Nutrient applications may enhance plant tolerance to pest attack (Marschner 1995). In some cases nutrients may directly inhibit pathogenic growth, or even promote activity of biocontrol organisms, as was shown with zinc for the biological control of tomato crown and root rot, Fusarium oxysporum (Duffy and Defago 1997). Nutrient foliar sprays may also induce systemic resistance to disease, as shown with phosphate salts, resulting in powdery mildew suppression and enhanced growth in cucumber (Reuveni et al. 1993). The mechanism for the induced disease resistance is unknown, but the potential exists for expanded use of these disease control approaches, as the control mechanism becomes better understood and as the application technology is adapted to commercial growing conditions (Huber 1981). 2. Arthropod Pests. Nutritional imbalances and excessive fertilization may result in increased arthropod pest numbers in plants. For example, artemisia plants fertilized with ammonium nitrate showed greater phloem- and seed-feeding insect numbers than unfertilized plants (Strauss 1987). Examples of other pests that showed increased numbers in response to higher N application rates include the corn leafhopper, Dalbulus maidis (Power 1987); the potato leafhopper (Roltsch and Gage 1990); caterpillar pests in head cabbage (H. R. Valenzuela, unpublished data; Jansson et al. 1991); and increased aphid population growth rates in potato (Jansson and Smilowitz 1986). In soybean, high P application rates resulted in greater larval velvetbean caterpillar, Anticarsia gemmatalis, numbers than at the lower Prates. Southern stink bug, Nezara viridula, levels were also greater with the higher P rates, but only in one of two years (Funderburk et al. 1991). The fertilizer source and farming technique may also affect arthropod pest levels. In New York, collards that received organic amendments had less arthropod pest numbers than plants receiving inorganic fertilizers (Culliney and Pimentel 1986), a result also observed in sorghum (Strauss 1987). In Ohio, soils from organic farms showed biological "buffering," resulting in fewer European corn borer oviposition levels, compared to comparable soils from conventional farms. Lower pest levels were found in plants grown in the soils from organic farms than from conventional farms even after application of several fertilizer sources and despite similar plant growth in both treatments (Phelan et al. 1995). However, additional work is necessary to evaluate the effect of organic amendments on arthropod pest pressure, to better understand existing ecological interactions that explain the observed results, and to corroborate the tentative data available from the few studies conducted to date in this area.

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C. Habitat Manipulation Techniques 1. Polycultures Systems

Diseases. Some polyculture designs may reduce disease pressure. This was first reported in Europe in 1767 with the use of non-host aphid intercrop barriers, which reduced virus transmission on the cash crop (Batra 1982). This assumption was further backed up by early work with agronomic crops that showed less disease in fields planted with both multiline disease resistant and susceptible cultivars (Leonard 1969). Since then multi-line cropping was established in several areas for management of diseases such as Puccinia, Septoria, Rhynchosporium, and Pseudocercosporella in cereals (Wolfe 1990). This approach also showed potential in Colombia, as observed in Phaseolus, with respect to Uromyces rust (Panse et al. 1997). Commercial examples of this approach are seen in India as well as in Germany, where over 350,000 ha of multi-line spring barley is grown, resulting in less fungicide applications (Wolfe 1990). Polycultures may also be designed to minimize crop disease outbreaks. Work in Kenya showed less halo blight, common mosaic, anthracnose, common blight, and angular leaf spot in polyculture beans than in monocultures (Francis 1985). However, Boudreau and Mundt's (1992) studies showed reduced Uromyces bean rust pressure in bean/maize polyculture than in bean monocultures in some cases, but not in others, indicating the importance of season and site-specific effects. The mechanism for reduced disease levels found in some polycultures, rather than in monocultures, is yet to be unraveled. However, intimate interactions may exist between plant species grown in close association. For example, fungal isolates obtained from the zoysiagrass, Zoysia tenuifolia, rhizosphere enhanced growth and induced systemic resistance in cucumber to anthracnose attack (Meera et al. 1994). Arthropods. Research initiated in the 1940s showed reduced arthropod pest pressure (Willey 1979a; Batra 1982; Risch et al. 1983; Francis 1985; Risch 1987; Andow 1991a), a lower probability of exceeding economic injury levels (Andow 1991b) and thus a decreased incidence of arthropod (Risch 1987) pest outbreaks in some diversified systems than in monocultures. However, many exceptions do occur, as observed with the higher pod-sucking bug, Clavigralla spp., levels found in cowpea intercropped with maize, than in cowpea monoculture (Gethi and Khaemba 1991). In the U.S., strip intercropping was practiced before the advent of pesticides, to fight pest attack. Mixed plantings to prevent pest outbreaks

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is also a common practice in forestry (Batra 1982). However, knowledge gaps are extensive in the area of habitat manipulation concerning pest population dynamics. From a regional perspective (Ricklefs 1987), little is understood about how larger landscape biotic and abiotic factors, such as those caused by extensive monocultures, affect smaller microhabitat biodiversity (Altieri and Letourneau 1984; Dennis and Fry 1992). Interplanting insect resistant and susceptible cultivars may also result in less damage to susceptible cultivars, as observed with ground arthropod pests in sweetpotato (Schalk et al. 1992), and with respect to Liriomyza leafminer damage in potato (Midmore and Alcazar 1991). By selecting compatible early- and late-maturing potato cultivars with differential tolerance to pest attack, overall pest pressure was reduced, and resource utilization apparently improved, resulting in a 20% overall increased productivity in the multi-line plots than with the singlecultivar treatments (Midmore and Alcazar 1991). The high level of potential biological interactions that exist in polycultures makes it difficult to explain pest dynamics resulting in lower pest levels or in possible pest outbreaks. The high degree of arthropod interactions observed in unsprayed crop monocultures alone (Ellington et al. 1997) give an indication of the challenge to develop a systematic understanding of the underlying mechanisms for possible pest management through enhanced biocontrol in polycultures. The level of complexity in monocultures, focused on the population dynamics of a single pest species on an area-wide basis, was shown by Brazzle et al. (1997), who evaluated ten cultural management practices that affected whitefly population dynamics in 56 commercial cotton fields in the Imperial Valley, California. Whitefly numbers were affected by planting dates, number of pesticide applications, plot size, and proximity of muskmelon fields. Similarly, with regard to black rot disease management, a survey of 27 strawberry farms in New York found several cultural factors that were correlated with disease severity, including soil compaction, soil texture, flat-bed culture, herbicide treatments, and years under monoculture (Wing et al. 1995). This intricate level of complexity rapidly increases, even in simple polycultures (Andow 1991b). Thus, a polyculture consisting of 2 crop species, 6 herbivore species, and 6 beneficial arthropod species results in a system with 91 potential two-way and 364 potential three-way ecological interactions (Andow 1991a). Similarly, the many facets that need to be evaluated, dealing with crop growth alone, in the highly complex agroforestry systems typically found in many tropical areas was described by Huxley (1985). Reduced pest loads in diverse systems may result due to the presence of alternative hosts, alterations of the abiotic environment such

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as physical barriers or shade (Roltsch and Gage 1990), release of chemical signals that disrupt or delay host identification (Teulon et al. 1993; Dobson 1994), presence of alternative preys, as well as lower host numbers (resource concentration hypothesis), and increased natural enemy activity, as compared to monocultures (Risch 1987; Hunter and Aarssen 1988), as well as a combination of several factors. As reviews indicate (Risch et al. 1983), broad generalizations cannot be made with regard to arthropod population dynamics in response to habitat manipulation, but instead an understanding should exist of the pest's dispersal and demographic behavior, prior to making predictions regarding response patterns in polycultures (Kareiva 1987). Both plant species and the plant structural diversity existent in polycultures may explain the greater arthropod diversity and correspondent lower pest damage levels reported for some polycultures. Altieri (1984) evaluated this supposition in brussel sprout density and polyculture studies. Doubling the planting density alone did not alter the arthropod fauna, but increasing the plot complexity by intercropping brussel sprouts with either faba beans or wild mustard did increase the diversity of the arthropod population in the intercrops, compared to the monocultures. A more diverse arthropod community may have been supported by the greater plant structural diversity in the polycultures, and not by increased plot biomass production alone, since simply doubling the planting density did not increase the monoculture arthropod faunal composition as observed in the polycultures. The decreased pest pressure observed with many arthropod pests in polycultures is attributable, in part, to a greater activity of beneficials (Altieri and Letourneau 1984). For example, in Texas, relay intercropping of cotton with wheat, mustard, and sorghum resulted in predator conservation during fallow periods and in respective higher predator numbers and lower cotton aphid, Aphis gossypii, levels in the polycultures than in the isolated cotton monocultures (Parajulee et al. 1997). Also, greater generalist predatory spider numbers were found in squash/ cowpea/maize polycultures than in squash monocultures, supporting Root's "enemies" hypothesis, while specialist leafhopper Anagrus parasitoids showed no response to the habitat manipulation treatments (Letourneau 1990a). However, Letourneau's work (1990a,b) indicates that variables other than response to species diversity and density may be involved in arthropod response to polycultures. Additional variables such as recruitment, tenure time, oviposition patterns, reproduction, emigration rates, short-distance movements, in response to aspects of polycultures (such as structure, color contrasts, volatiles, and vegetation texture) other than species richness itself, need to be identified, so that

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specific polyculture schemes may be designed that will result in greater natural enemy activity and in corresponding decreased pest levels (Letourneau 1990b). A greater activity of beneficiaIs is not always the mechanism responsible for the lower pest numbers found in polycultures, as determined with the lower Empoasca potato leafhopper numbers found in a tomato/ bean system compared to the numbers found in bean monocultures by Roltsch and Gage (1990) in Michigan. This latter study, which supports the resource concentration hypothesis, showed lower leafhopper numbers as the tomato density increased, thereby decreasing relative bean densities, plus the higher tomato densities may have also decreased the "quality" of the bean foliage, as indicated by parallel N-nutrient greenhouse studies (Roltsch and Gage 1990). Therefore, non-host intercrops may provide a physical barrier, or modify the microenvironment in other manners, disrupting the motility of target pests. For example, in Nicaragua, two-year replicated and large-scale on-farm (2 ha plots each for tomato/bean and tomato monoculture plots) validation trials, a tomato/bean system also resulted in lower pest levels in tomato with respect to several pests including Heliothis fruit worms, and Liriomiza leafminers, while a trend was observed toward lower Spodoptera armyworms numbers (Rosset 1989). The mechanism of action for the reduced pest numbers was not determined in this study, but presumably the bean plants provided a physical barrier, resulting in less pest oviposition on tomato. Natural enemy activity was minimal in these plots, perhaps due to the history of intensive pesticide applications in the area (Rosset 1989). In another example, shade and wind protection provided by maize plants in a bean/maize system were the likely causative factor resulting in greater aphid dispersal rates (lower residence-time) from the bean plants in the polycultures as compared to the bean monocultures (Bottenberg and Irwin 1991). The greater dispersal rates from bean plants may result in less virus transmission in the polycultures, since the next landing site for the departing aphids may be a non-host, either a maize plant in the polyculture or another plant outside of the production area (Bottenberg and Irwin 1991). The extensive chemical volatile richness that exists in monocultures (Charron et al. 1995) such as celery (Van Wassenhove et al. 1990) and squash (Peterson et al. 1994), is increased in polycultures (Dobson 1994). This is significant, considering the role that volatiles and alkaloids play in insect and plant interactions (Roltsch and Gage 1990; Peterson et al. 1994; Charron et al. 1995). Many of these volatiles attract beneficials and can also possibly delay, detract, or prevent pests from finding their

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target hosts in polycultures (Dobson 1994), and thus could represent yet another tool for pest management through habitat diversification. Even though lower pest numbers have been reported in polycultures than in monocultures, little is understood about possible yield advantages resulting from the reduced pest loads in polycultures (Andow 1991b). Plant compensatory response to pest damage and the resulting effects on inter- and intra-species competition in the polycultures may confound yield effects of component crop species, which precludes making any broad generalizations.

Nematodes. Even though the evidence is scant, polycultures may also be effective for suppression of nematode pests, as observed in cowpea/ maize systems in Nigeria (Bridge 1996). Also, in the West Indies, intercropping carrots reduced M. incognita levels by 16% with onion, 26% with cabbage, and 27% with chives compared to the counts found under carrot monoculture (McDonald 1985). Weeds. Polycultures may be designed to effectively manage weed populations in small-scale production systems. In a two-year study conducted in Nkpolu, Nigeria, optimal weed control and greatest maize yield were obtained in monoculture maize with herbicide applications. However, overall economic returns were highest when weeds were managed through a combination of one hand-weeding in a maize/sweetpotato polyculture or one hand-weeding with a maize/peanut system than in the respective monocultures (Zuofa and Tariah 1992). In California, polyculture combinations of lettuce, faba bean, and pea had less weed biomass than the respective monocultures (Sharaiha and Gliessman 1992), but some crop combinations and planting patterns were more effective in smothering weeds than others. Most polyculture combinations also had an LER greater than one (Sharaiha and Gliessman 1992). The selection of the crop cultivar used may also have an effect on the level of weed control obtained in polycultures, as do other cultural factors such as N and irrigation application rates, as shown in a pea/barley/weedy mustard system (Liebman 1989). 2. Cover Crops and Living Mulches. Cover crops, which provide another mechanism for enhanced vegetational diversity in the farm, showed a positive effect on beneficial arthropod populations, resulting in less arthropod pest fruit damage in Mediterranean and temperate climate orchards (Fye 1983; Altieri and Schmidt 1985). The cover crops were effective in serving as a refuge for beneficial organisms, and also as a source of alternate prey for the target pests. In Georgia, several cover

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crops enhanced predator populations in muskmelon (Bugg et al. 1991a) and in pecan orchards (Bugg et al. 1991b; Bugg and Dutcher 1993). It is plausible that similar mechanisms to build up beneficial populations through the use of cover crops may be established in annual tropical cropping systems. An understanding of pest population dynamics in response to cover cropping practices will be useful in the design of cover crop and residue management techniques that will minimize the incidence of pest outbreaks. For example, incorporated legume cover crop residues resulted in greater seedcorn maggot, Delia platura, numbers in soybean (Hammond 1990). However, gradually lower maggot numbers were found with incorporated grasses, and with unincorporated (no-till) legume and grass species, with no enhanced maggot numbers in the latter two treatments, as compared to the bare-ground soybean treatments (Hammond 1990). Legumes were effective as living mulches in Brassica plots to reduce pest incidence of Brevicoryne brassicae, Pieris rapae, and Erioischia brassicae. The reduction in P. rapae was attributed to an increase in the activity of the predacious ground beetle Harpalus rufipes (Altieri and Letourneau 1984). In other studies clover living mulches resulted in a 34% increase in cabbage root fly, Delia brassicae, predator levels. Similar results were obtained in one experiment by Ryan et al. (1980). In addition Ryan et al. (1980) observed lower root fly numbers in all experiments, and increased cabbage yields in experiments when water was not a limiting factor. In similar studies, fewer aphids were found in collards intercropped with lana vetch living mulches, compared to collard monoculture plots (Altieri and Letourneau 1984). In work with maize and tomato, plots with clover living mulches had a larger number of natural enemies than bare-ground plots, including more ground predators (Carabidae, Staphylinidae, and spiders) (Altieri et al. 1985). Broccoli grown in clover living mulches also had fewer cabbage aphids and flea beetles than monoculture broccoli, but differential broccoli growth in these treatments confounded the arthropod pest data (Altieri et al. 1985). Follow-up work, in which clover living mulch growth was suppressed through mowing, to minimize competitive effects, also showed fewer aphids in the living mulch treatments than in the broccoli monocultures (Costello 1994). Living mulches, by providing a barrier to insect movement, or through other undetermined mechanisms, may also be effective in delaying the onset of aphid-transmitted viral diseases, as observed in a zucchini experiment with buckwheat, mustard, and buckwheat/mustard living mulch treatments in Hawaii (Hooks et al. 1998).

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An important feature of living mulches is their weed suppression characteristics (Ilnicki and Enache 1992), as observed in living mulch trials with eggplant in Hawaii (Valenzuela and DeFrank 1994). Living mulches may thus be used to reduce pest outbreaks, in addition to their contribution to soil fertility and to enhanced weed control (Enache and Ilnicki 1990). Cover crop rotational programs may also be effective for disease management. For example, a Sudan grass, Sorghum vulgare, cover crop effectively reduced verticillium wilt and increased potato yields, compared to fallow treatments and cover cropping with other species (Davis et al. 1996). The lower verticillium levels caused by the Sudan grass cover crop were attributable to an increased activity of biological control organisms in the rhizosphere. Similarly, cover crops can effectively be used in rotation programs for management of important nematode pests. A list of plant species with reported resistance or tolerance to particular nematode species buildup is presented in Table 4.2. 3. Trap Crops. Arthropods often show feeding preference among cultivars or crop species, a response which may be used to develop trapcropping systems as a tool for pest management (Hokkanen 1991). For example, Liberty Hyde Bailey observed that the squash vine borer, Melittia cucurbitae, oviposited more readily on some squash species than on others (Robinson 1992), an observation also recorded by J. DeFrank and H. R. Valenzuela (unpublished data) concerning melon fly (Dacus spp.) oviposition preference in cucurbits. Other examples include strawberry cultivar susceptibility to spider mites (Poe and Howard 1971), cucurbit cultivar response to Diaphania pickleworm damage (Peterson et al. 1994), and Brassica cultivar susceptibility to caterpillar damage (Valenzuela 1993; Mays and Kok 1997). Trap-crop systems have been used for pest management in several forestry and agronomic crops (Hokkanen 1991), such as the use of an alfalfa trap crop, which effectively reduced the green mirid, Creontiades dilutus in cotton (Mensah and Khan 1997). Commercial applications of trap crops, as part of an overall pest management program, were established in temperate areas for vegetables such as potato and cauliflower, and in tropical regions with field crops such as cotton and soybean (Hokkanen 1991). In Oklahoma, systemic insecticide-treated 'Lemondrop' squash plantings representing <1 % of the total area planted effectively acted as a trap crop for the Acalymma cucumber beetle and Diabrotica squash bug, in muskmelon, squash, and watermelon. Followup studies showed that more cucumber beetles were attracted to 'Blue Hubbard' than to 'Lemondrop' squash (Pair 1997).

Q:l """"

N

Table 4.2. Selected list of plant species with reported tolerance or resistance to nematode infestations for potential use as cover crops in rotational cropping systems. z Nematode species

Cover crop Species

Agrostis alba Amaranth us hybridus Hordeum vulgare Cannavalia Cassia fasiculata Ricinus communis Centrosema pubescens Dendranthema x grandiflorum Vigna unguiculata Crotalaria juncea Crotalaria spectabiIis Desmodium triflorum Digitaria decumbens Festuca rubra Panicum maximum Jacq. var. trichoglume Indigofera hirsuta Aeschynomene americana Zea mays Tageles erecta Mucuna deeringiana B. campestris

Common name Agrostis Smooth pigweed Barley Horsebean Partridge pea Castor Butterfly pea Chrysanthemum Cowpea Sunhemp Showy crotolaria Three-flower beggarweed Digitaria Chewing'S fescue Green panic Hairy indigo Jointvetch Maize Marigold Velvetleaf Mustard,

Lesion Y'x

M. incognita x, w

M. arenaria x , w

M. javanica x , w

Reniform/spiral x

Sting x

Cyst X

T T (Spiral) T R3 R1,3 R2 T T R2 T

T T T

T T T

T T

T (Spiral)

T T

T

T

R2 R2

T (Spiral)

T T T (P.b.)

T,R1 T

T T

T

T T (P. P & b) T(P.b.)

T T (R1-3) T

T T

T (Ren)

A vena sativa Arachis hypogeae Cajanus cajan Chloris gayana Lolium perenne Lolium multiflorum Sesamum indicum Macroptilium atropurpureum Sorghum bicolor Glycine max Sorghum vulgare Sorghum bicolor Triticum aestivum

Oats Peanut T Pigeonpea Rhodes grass Ryegrass, perennial T T Ryegrass, Sesame Siratro Sorghum Soybean Sudan grass Sudex Wheat

T (R1-3) T

T

T (Ren) T (Spiral) T(Ren)

T(R1) R1

T

T T

T (R1)

T T

T

T

T

T(Ren) T (Ren)

T T

T T (R1,3) T

ZT = reported tolerance or resistance to that particular nematode species. Tolerancelresistance is often cultivar specific, not necessarily for all cultivars of each reported plant species. YFor lesion nematode P.p.

= P. penetrans,

and P.b.

= P. brachyurus.

XLesion nematode, Pratylenchus penetrans (Cobb) Sher & Allen, see: MacDonald and Mai (1963); and P. brachyurus (Godfrey) Goodey, see McSorley and Gallaher (1992). Root-knot nematode, Meloidogyne incognita (Kofoid and White) Chitwood, see: Sharma et aI., 1980; Wilson and Caveness, 1980; Reddy et aI., 1986b; McSorley and Gallaher, 1992; Gallaher and McSorley, 1993; McSorley et aI., 1994b; McSorley et aI., 1995; Bridge, 1996; and Nogueira et aI., 1997. M. arenada, see: Khan and Esfahani, 1992; Stanton and Stirling, 1993; Rodriguez-Kabana et aI., 1992; and McSorley et aI., 1994a,b. M. javanica see: Sipes and Arakaki, 1997; and McSorley et aI., 1994b. M. hapla see: Roberts, 1992. Reniform, Rotylenchulus reniformis, see: Ko and Schmitt, 1993; Sting nematode, Belonolaimus longicaudatus Rau, see: Reddy et aI., 1986b; and Weingartner et aI., 1993. Cyst nematode, Heterodera glycines Ichinoe, see: Ross, 1962; Rodriguez-Kabana et aI., 1990; and Rodriguez-Kabana et aI., 1992. WRoot-knot nematode, X = general resistance reported for M. Meloidogyne spp.; R1-R3, resistance reported for either ofraces 1, 2, or 3. Within cultivated crop species root-knot nematode resistance is reported for 'Iron' cowpea, 'Nemared' tomato, chili pepper, 'Nemagreen' limabean (M. incognita), and soybean (M. incognita) (Malo, 1964); for 'California Wonder' bell pepper (M. javanicaJ, and 'Charleston Gray' watermelon (M. hapla) (Khan and Esfahani, 1992); and for pea and sweetpotato (Bridge, 1996).

I-'

():)

w

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Alternative flowering Brassica weedy hosts were identified in California to have potential as trap crops for control of Phyllotreta flea beetles in collard. Flea beetle numbers were greater in the B. campestris intercrops than in the collard main crop. In addition, collards sprayed with wild mustard plant extracts or with allysothiocyanate emulsions showed greater flea beetle numbers than collard sprayed with water alone (Altieri and Schmidt 1986). Differential feeding preference of seedlings was also shown by flea beetles among nine Brassica species in Canada (Palaniswamy and Lamb 1992). Evaluations of other effective trap-crop systems include the use of snap bean to attract the Mexican flea beetle, E. varivestris, in soybean; the use of wild Brassica spp. to attract flea beetles, P. cruciferae, in collards (Altieri and Gliessman 1983); and the use of marigold cv. Golden age to trap the Helicovera fruit borer in tomato (Srinivasan et al. 1994). The effectiveness of trap crops, as in polycultures, will be affected by the population dynamics of the target pest. Highly motile arthropods pests and natural enemy species may be unamendable to small-scale habitat manipulation techniques, while less motile ones may be more affected by small-scale vegetational diversity (Corbett and Plant 1993). This was indicated by the lack of diamondback moth population response to establishment of canola/cabbage border trap crops in head cabbage fields in Hawaii (Luther et al. 1996) and by the similar parasitism rates found on the Plathypena green cloverworm in diversified and monoculture soybean plots in Ohio (Pavuk and Barrett 1993). 4. Insectary Plants. Considerable natural enemy activity is typically

found in conventional agricultural settings. For example, surveys of commercial strawberry fields in California found 10 insect and 9 phytoseiid mite species preying on the two-spotted spider mite (Oatman et al. 1985). Despite the standard intensive pesticide-based management practices followed in commercial cotton production, which in the Imperial Valley, California, consist of more than 10 "high potency tank" mix insecticide applications per growth cycle, the natural enemy populations (esp. Chrysopa, Encarsia, Eretmocerus, Geocoris, Hippodamia, and Orius sp.) component was still found to represent about 20-23% of the total variance that affected whitefly populations, among the seven other cultural and management factors investigated (Brazzle et al. 1997). However, standard pesticide treatments can dramatically reduce beneficial populations, as shown in comparative studies of unsprayed (or organic) vs. sprayed apple orchards in Connecticut (Maier 1982), Massachusetts (Wisniewska and Prokopy 1997), Michigan (Strickler et al. 1987), and West Virginia (Brown and Adler 1989). Similarly, in Florida commercial

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vegetable fields, the mean percent parasitism of Liriomyza leafminers found on weedy hosts ranged from 0.16% in fields sprayed weekly, to 14.4% in fields sprayed once monthly, to 46.7% in unsprayed fields (Genung et al. 1978). Arthropod surveys in commercial fields show that weedy habitats within the agroecosystem often harbor significant populations of beneficials (Schuster et al. 1991). Therefore, an alternative to increased habitat complexity is to manipulate weed populations by selective weeding (Power 1987; Andow 1991b) or by the actual establishment of selected weedy species, with the goal of maximizing the population of beneficial organisms in the agroecosystem (Altieri and Whitcomb 1979; William 1979; Wyss 1996), a suggestion made by Genung et al. (1978) after observations of intensive beneficial insect x weed interactions in commercial celery fields in Florida. The Florida surveys found >50% Liriomiza leafminer parasitism levels in 20 weed species and 100% parasitism in 11 species (Genung et al. 1978), overall reaching similar levels (91 %) in the weedy hosts as those found in unsprayed vegetable crops. High populations of leafminer parasitoids were also found on weeds in Florida tomato fields (Schuster et al. 1991). Elsewhere, weedy collard plots resulted in fewer green peach aphids and in greater numbers of beneficials (Coccinellidae, Syrphidae, and Chrysopa), than in weed-free plots (Horn 1981). Predator population dynamics were also manipulated in soybean by modifying weed populations (Naranjo and Stimac 1987). The greatest predatory attacks were observed in soybean/pigweed, Amaranthus hybridus, combinations, indicating the potential for enhanced arthropod biological control through mechanistic habitat manipulations. Ecological studies demonstrated the dependence of several introduced biological control agents on the presence of specific weedy species (Altieri and Whitcomb 1979), but extensive research is needed to evaluate the effect of specific habitat manipulation techniques that would favor the presence of specific weed species and their subsequent effect on arthropod faunal populations. Examples of reduced crop pest pressure in weedy habitats include less leafhopper numbers in maize (Power 1987), flea beetles in collard (Altieri and Schmidt 1986), and greater predator and alternative prey numbers, resulting in improved pest control in apple (Wyss 1996). The establishment of diversified vegetative windbreaks, hedgerows, field margins, or "insectary" patches within production fields has also been effective for the build-up of generalist predator populations (Altieri and Letourneau 1984; Wratten and Thomas 1990; Dennis and Fry 1992; Rodenhouse et al. 1992; Dix et al. 1997). The diversity of beneficial organisms that may visit established "insectaries" was illustrated with

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a survey of two flowering herbs proximal to organic vegetable gardens (Maingay et al. 1991). A weekly aerial survey of small 0.5 x 1.0-m plots of a 12-week flowering sweet fennel, Foeniculum vulgare, and of 4-week flowering spearmint, Mentha spicata, found a range of 277-497 arthropod specimens representing 53-195 predatory taxa and 33-105 parasitic taxa. Potential insectary crops identified for pecan orchards among the warm-season cover crops evaluated by Bugg and Dutcher (1993) included buckwheat, hairy indigo, Sesbania exaltata, and showy partridge pea, Cassia fasciculata. An additional list of potential insectary plant species was compiled by Valenzuela (1994). A possibility for enhancing insectary patches is the incorporation of plants species with extra-floral nectaries such as cowpea and faba bean (Koptur 1992). The contribution of extra-floral nectaries for attraction of beneficials, by providing a food supplement when prey levels are low, was shown in monoculture comparisons of nectaried and nectariless cotton genotypes (Treacy et al. 1987). Another alternative available to increase populations of beneficials is the artificial application of honeydews in the field. In Australia, this tactic increased predator populations of beetle bugs and increased lacewings numbers for management of Helicovera spp., while the standard pesticide controls resulted in the virtual extermination of predator populations (Mensah 1997). Additional benefits brought about by the establishment of diversified "patches" or "corridors" within agricultural fields include creation of microclimates conducive to plant growth and protection from water and wind soil erosion, which may contribute toward enhanced and sustained crop yields (Rodenhouse et al. 1992). D. Cultural Management Programs

Integrated cultural management programs may be the last resort for management of some pests or diseases for which there are no registered pesticides available for remedial control. Crop management techniques that may have an effect on pest levels include rotations, planting and harvesting, crop diversification, planting arrangements, and the use of trap crops, among others. Over the past 30 years, many of these cultural techniques were implemented as part of the overall pest management programs for agronomic crops such as cotton. Area-wide implementation of many of these practices broke the heavy reliance that cotton had on pesticides for pest control (Summy and King 1992). The goal of these practices could be to break pest cycles; to modify the crop microclimate, making it unfavorable for pest development; to prevent the development

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of pest outbreaks; or to manipulate aspects of the pest's behavior, resulting in less crop damage (Watson 1979). For management of the devastating Rhizomonas corky root disease in lettuce, cultural management options may include a combination of crop rotation (Alvarez et al. 1992), to minimize inoculum levels, resistant cultivars, and the use of transplants to minimize duration of exposure to the disease (Van Bruggen and Rubatzky 1992). Alternative pest control practices such as the use of rotations (Bullock 1992), habitat management, deep plowing (Green 1958), alternative irrigation patterns (Bell et al. 1998b), the use of cover crops, and other organic matter building techniques may become viable choices to manage the yield "declines" observed in several regions with the continuous monoculture of many crops such as asparagus (Grogan and Kimble 1959) and strawberry (Wing et al. 1995). 1. Organic Matter Buildup and Rhizosphere Biological Control

Biological Control in the Rhizosphere. Soils with antagonistic properties against specific plant pathogens have long been recognized in cultivated crops (Baker 1991; Schippers et al. 1995). This was recognized with continuous potato irrigated culture in Washington State in soils that developed Streptomyces scab antagonistic characteristics, which were transmissible to non-scab suppressive soils (Menzies 1959). Antagonist soils to several Rhizoctonia solani isolates were also identified in Hawaii (Kobayashi and Ko 1985) and in California (Baker et al. 1967), compared to non-suppressive Australian soils, a mechanism that was greatly nullified by soil steam treatments. The disease suppressive nature found in some soils is ultimately caused by, among other factors (Adams et al 1968a; Schippers et al. 1995), microbial rhizosphere activity, responsible for the "biological buffering" capacity of the soil, a valuable trait that can be modified by the crop sequence and by cultural management practices (Huber 1981; Schippers et al. 1995). Cultivated soils such as in alfalfa (O'Rourke and Millar, 1966), maize (Hornby and Ullstrup 1967), and lettuce (Bell et al. 1998b), normally contain native antagonistic organism populations that provide different levels of biological control against important soil-borne diseases (Baker 1991). A survey of a field continuously cropped with maize, soybean, wheat, and other crops showed a range of 11,000 to 22,000 fungi/g soil (6-19% of all fungal colonies found) which were antagonistic to Fusarium, with Penicillia, Aspergilli, Myrothecium, and Cephalosporium representing the most important antagonists (Williams and Kaufman 1962). The ratio of beneficial fungi species varied with the cultivated crop.

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Another soil survey found that both antagonistic and general nonantagonistic fungal and bacterial organisms provided some antifungistatic effects of soil-borne diseases such as Gliocladium and Pythium (Griffin 1962). In another study, rhizosphere bean soil sampling isolated 90 fungi genera. Important biocontrol specimens collected included Penicillum, Aspergillus, bacteria, and an actinomycete, altogether providing some level of control against fusarium and rhizoctonia bean root rot (Huber et al. 1966). Similar microbial population diversity (about 400 microbial isolates), a varied number of antagonists, plus specific beneficial fusarium isolates effective for suppression of fusarium wilt, were identified in watermelon (Larkin et al. 1996). Thus, rhizosphere biological control is a primary disease control mechanism in agricultural settings, illustrated by native fluorescent Pseudomonas and by Trichoderma (Taylor 1990). For example, the Pseudomonas fluorescens strain CHAO controls tomato Fusarium crown and root rot, and other pathogens (Duffy and Defago 1997). Pythium damping-off in lettuce was controlled by Trichoderma, and was also partially suppressed by Gliocladium and Enterobacter, plus the bacterial and fungal agents showed compatibility (Lynch et al. 1991). Also, in Nigeria, Bacillus subtilis soil-inhabiting isolates effectively protected Amaranth us hybridus roots from infection by the Choanephora cucurbitarum shoot disease (Ikediugwu et al. 1994), which indicates the need for local research for the isolation of effective biological controls. Several predacious fungi, e.g. Arthrobotrys, Cystopage, Dactylaria, Dactylella, Trichothecium, and Verticillium spp. attack nematode pests (Swarup 1990), indicating the possibility of enhancing nematode biological control with rhizobial beneficial fungal populations. In India, Bacillus, Glomus, and Pasteuria penetrans were also identified as nematode pest antagonists (Swarup 1990). Some bacterial isolates multiply in upper plant parts, one of which reduced anthracnose levels in cucumber (Leben 1964). Soils with native arthropod pest antagonists also exist. For example, the fungus Beauveria bassiana caused extensive sweetpotato weevil suppression in a soil of northern Taiwan (Su et al. 1988). Thus, with a better understanding of soil microbial biocontrol dynamics, cultural practices, such as the use of organic matter amendments, cover crops, conservation tillage, and crop rotations, could be implemented to enhance the soil biological buffering capacity (Huber 1981; Baker 1991; Sanginga et al. 1992).

Pest Management and Soil Organic Matter Levels. A basic tenet for organic farming is the goal to increase soil organic matter levels. The use of organic matter-based soil amendments was standard practice in many

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ancient agricultural systems, and is commonly used in small-scale farms of the tropics (Bridge 1996). High organic matter levels may not only increase soil quality and provide nutrients for crop growth, but may also increase rhizosphere microbial diversity and activity (Sanginga et al. 1992)-a fauna that, as discussed earlier, may become antagonistic to plant pathogen growth, and which may also increase activity of N-fixing bacteria such as Azospirillum, Azotobacter, Bacillus, Enterobacter, Klebsiella, and PseudOlnonas (van Bruggen 1995; Bridge 1996). Management practices that may promote rhizosphere microbial activity include continuous vegetative cover, soil plant residue applications, vegetational diversity over time and space, minimal soil disturbance, and the minimal application of toxic chemicals (Sanginga et al. 1992; van Bruggen 1995). Early research showed that the addition of complex carbohydrates such as maltose, dextran, starch, and cellulose, or crop residues such as coffee grounds (Adams et al. 1968b) or oats, which increased the soil c: N ratio, effectively suppressed several soil-borne diseases such as Fusarium, Rhizoctonia, Thielaviopsis, and Verticillium (Snyder et al. 1959; Green and Papavizas 1968; Papavizas et al. 1968; Papavizas and Adams 1969). Factors involved included a high C:N ratio, and the consequent promotion of a greater rhizobial microbial activity, which likely exhausted the limited supply of soil nutrients, which in turn reduced the population of soil-borne pathogenic microbes.

Organic Amendments for Nematodes Pest Management. A wide-range of amendments, such as oat straw (Johnson 1962) and N-containing (2-7%) amendments (Rodriguez-Kabana 1986), have also been identified for nematode pest control (Bridge 1996). An obstacle to effective nematode biocontrol is that many soils contain fungistatic factors fatal to conidia of nematophagus fungi. However, fresh alfalfa cuttings, rotten wood shavings, and chicken and steer manure amendments were able to overcome the soil fungistatic properties that killed these nematophagus fungi (Arthrobotrys, and Dactylella) , resulting in effective nematode control following the amendment treatments (Mankau 1962). Other crop residues that are effective for soil nematode population suppression include marigold, mustard and sunflower for root-knot nematode, M. incognita control (Akhtar and Alam 1992), as well as Argemone mexicana, Calotropis procera, Croton bonplandianum, Datura metel, and Solanum xanthocarpum, for both M. incognita and stunt nematode, Tylenchorhynchus brassicae, control in eggplant (Alam 1986). The application of organic amendments in nematode-infested soils presumably promotes the activity of nematode microbial antagonists

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(Cooke 1968; Bridge 1996). These include nematophagous fungi, endoparasitic fungal parasites, AM mycorrhizae, and predaceous organisms such as enchytraeids, collembola, and other nematodes (Bridge 1996). The potential for enhanced nematode biological control using agents that exist naturally in agricultural· soils seems promising. For example, over 30 nematode parasitic fungal species have been identified (Cooke 1968). However, inconsistent results have been obtained in the past in suppressing soil nematode populations through increased biological control with the use of organic amendment applications alone.

Composts. Compost applications, in addition to their benefits as soil nutrient amendments, also reportedly may be effective for soil-borne pathogen management. For example, disease suppression with the use of composts was reported in ornamentals infected with Pythium spp. (Schueler and Vogtmann 1990). Several compost treatments helped to reduce incidence of Pseudomonas bacterial wilt in tomato (Chellemi et al. 1992). Variables correlated with disease reduction levels, according to a stepwise regression procedure, included soil pH and organic matter content. Earthworm-based composts also effectively suppressed Phytophthora and Fusarium in tomato, and Plasmodiophora and Heterodera in cabbage (Szczech et al. 1993). In potted experiments, composts, representing 30% of the growing media, also reduced the severity of Mycosphaerella foot rot on pea seedlings (Schuler et al. 1993). 2. Physical Controls

Rotations. Continuous monocultures often result in incremental yield declines (Bullock 1992; Juo et al. 1995; Ryszkowski et al. 1998). This is due to soil quality decline; to disease-complexes as observed in bean (Snyder et al. 1959); or to pest outbreaks, as in the case of the maize rootworm complex that occurred with increased frequency in the U.S. after maize monocultures replaced the traditional maize rotations (Pimentel 1991). Rotations have long been practiced for the management of important nematode pests. For instance, seven-year rotations and fallow were followed in the Andes in pre-colonial times for management of the potato cyst nematode (Bridge 1996). However, shorter-duration rotations can be effective for management of nematode populations. One-year rotations with non-host cover crops such as cowpea and maize significantly reduced cyst nematode populations in soybean. One-year rotations were equally as effective as two- to four-year rotations (Ross 1962). Soybeansorghum rotations were also effective to reduce root knot nematode and cyst nematode levels on soybean (Rodriguez-Kabana et al. 1990). A five-

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year maize, one-year soybean rotation was effective in reducing incidence of brown stem rot, Cephalosporium gregatum, in soybean (Dunleavy and Weber 1967). However, other soil-borne pathogens, such as Aphanomyces euteiches root rot of pea, are more resilient. In eight-year rotation studies, increasing peas in the rotations from 1 to 3 times, resulted in proportional disease increased levels, and the disease propagules remained viable even in plots not grown with peas during the eight year cycle (Temp and Hagedorn 1967), indicating the need for additional complementary control measures. The greater microbial population number and diversity found under rotational programs than under monocultures (Williams and Schmitthenner 1962; Ryszkowski et al. 1998) may partially explain the greater levels ofbiocontrol and respective lower pathogen levels found in rotation programs than under continuous monocultures (van Bruggen 1995). The selection of crop rotation schemes also has an effect on the reduc'tion of the weed-seed pool in the rhizosphere. In Nicaragua, a cornsorghum rotation resulted in the lowest weed infestations (101 weed plants/m 2 ), compared to herbicide treatments (227/m 2 ), and to a cornsoybean rotation (330/m 2 ) (Eiszner et al. 1996).

No-til1. Little is yet known concerning the effects of no-till practices on disease development. Several environmental factors in a no-till system may contribute toward reduced pathogen growth, while others, such as increased pathogen survival in plant debris, may predispose the host plant to pathogen attack (Rothrock 1992; Sumner et al. 1995). In bell pepper, a no-till system with wheat stubble reduced Phytophthora propagule dispersal between plants, resulting in less overall disease pressure compared to bare-ground treatments (Ristaino et al. 1997). Direct disease suppression promoted by the wheat stubble in the no-till system could have been another, albeit untested, mechanism that prevented disease dispersal in this study. A more diverse microflora fauna, promoted by a high organic matter content, has generally been found in no-till than in conventional systems, plus crop residues from some plants, such as wheat, have also been found to release toxins suppressive of some crop pathogens (Ristaino et al. 1997). The practice of no-till farming may result in increased (Levine 1993), similar (Blumberg et al. 1997), or decreased (Zehnder and Linduska 1987) arthropod pest pressure compared to conventional tillage systems. Even though tillage may reduce pest numbers, under no-till systems alternative practices may be followed to manage problem pests, such as by controlling alternative weedy hosts around field borders to prevent oviposition and subsequent pest outbreaks (Levine 1993).

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No-till practices, by providing a more diverse environment, and a surface mulch, may also lead to an increased diversity of the arthropod faunal population, including beneficials, and consequently to fewer pest outbreaks (House and Stinner 1983; House and Brust 1989; Pimentel et al. 1989; Clark et al. 1993). This was observed with respect to spider numbers and species diversity after introduction of legume species into an unsprayed minimum-till system, as compared to plots receiving insecticide applications (Mangan and Byers 1989). In Nigeria, seven consecutive maize plantings under no-till resulted in less root lesion nematode levels than in cultivated treatments, presumably due to the greater activity of microbial antagonists found under the no-till system (Bridge 1996). No-till is conducive toward growth suppression of some weeds (House and Brust 1989), but perennials may become problematic, as observed in subhumid and semi-arid areas of Zimbabwe (Vogel 1994). In temperate areas, available herbicides are sometimes effective for weed management under no-till culture, as observed in asparagus (Boydston 1995). However, alternative weed management programs may be necessary in tropical areas where herbicides are not available. Also, several other aspects of the production system, such as poor stands due to the greater soil residue levels, may result in a lack of yield effects for no-till systems compared to standard cultivation practices, as observed in trials with shifting cultivation in the Amazon (Mt. Pleasant et al. 1992). All aspects of the production system, including rotation schemes, planting density (Mt. Pleasant et al. 1992), and sowing techniques thus need to be adapted for the effective implementation of these alternative production systems. Organic Mulches. Organic mulching techniques may provide complementary pest suppression in addition to contributing toward soil conservation and fertility. Soil-borne disease management, through the use of organic mulches, by preventing fruit contact with infected soil or splashing of fruit by infected soil from raindrops, was recently demonstrated in strawberry (Ellis et al. 1998). Organic mulches may also be effective for ground-living arthropod pest management. This was shown with the lower Colorado potato beetle larvae and reduced defoliation levels found in potato in response to straw and leaf mulches by Stoner (1993). Plausible reasons for the reduced beetle activity in potato are increased natural enemy activity under the mulch, and/or physical barriers to beetle movement under the mulch as compared to bare-ground cultivation (Stoner 1993). Organic mulches are widely used in vegetable agroecosystems for weed growth suppression. In okra, application of 5 t/ha of wheat straw

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plus one hand-weeding, conducted 45 days after planting, was as effective as a herbicide plus one hand-weeding for effective weed control and to obtain 8.9 t/ha yields from about 7 harvests (Singh et al. 1991). In addition, in some instances, the organic mulch promoted a greater bacterial, actinomycete, and soil fungal activity than the non-mulched treatments (Singh et al. 1991).

Biofumigant Species. Plant residues are known to release volatiles upon soil incorporation that have differential effects on soil microorganisms, including microbial respiratory enhancement (Owens et al. 1969) that may result in the preferential increase of particular microbial fungal and bacterial species. Some of these volatiles may be useful to break microbial dormancy periods to disrupt normal disease life cycles, or perhaps to stimulate activity of pathogen antagonistic microflora. Important volatiles with differential effects on soil microbial populations include acetaldehyde, isobutyraldehyde, isovaleraldehyde, 2-methylbutanal, valeraldehyde, methanol, and ethanol (Owens et al. 1969). Incorporated Brassica crop residues have been effective in reducing crop pests, including arthropods, nematodes, weeds, virus incidence, and soil-borne diseases (Boydston and Hang 1995; Brown and Morra 1997; Rosa et al. 1997). Incorporated cabbage residues controlled Aphanomyces root rot in pea (Papavizas 1966). Also Brassica isothiocyanate volatiles suppressed several pathogens, including Gaeumannomyces take-all of wheat (Angus et al. 1994) and gummy stem blight in combination with solarization in watermelon (Keinath 1996). Brassica plant residues also suppressed nematode pests, which was attributed to the glucosinolate toxic byproducts released by the crop residues upon decomposition (Halbrendt and Jing 1994). A range of nematode toxicity exists among Brassica species, and nematode species also show differential response to the biofumigation treatments (Halbrendt and Jing 1994; Rosa et al. 1997). In Washington State, 'Jupiter' rapeseed, B. napus, was the most effective Brassica cultivar, among the 12 tested, for reduction of root-knot nematode, M. chitwood races 1 and 2, levels in potato (Mojtahedi et al. 1991). Brassica species, used as cover crops and incorporated prior to planting, also show promise for weed control, as shown with white mustard, B. hirta, and with rapeseed, B. napus, for pea production (AI-Khatib et al. 1997). B. napus was used in a similar manner for potato production, resulting in less weeds and increased yields, compared to the use of a Sudan grass cover crop or fallow treatments (Boydston and Hang 1995). However, effective application ofbiofumigant techniques in commercial production settings is rare.

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Allelopathy. Plant allelopathy is a potential tool to control pests in the agroecosystem. However, allelopathic effects should be considered carefully to prevent any deleterious growth effects on cash crops grown in polycultures or in rotational programs. For example, sweetpotato residues decreased the shoot growth of the subsequent 'Jewel' and 'Centennial' sweetpotato crop by 20 to 74%. Sweetpotato residues also reduced N-fixing activity plus the top growth of subsequent cowpea plantings by 79-91 % (Walker and Jenkins 1986). Other crops with reported allelopathic effect include taro (Pardales et al. 1992) and asparagus (Weston 1996). Decomposing plant residues of crops such as barley, cowpea, and soybean release phytotoxins (Toussoun et al. 1968) that could be used for weed, pathogen, or nematode control as part of an integrated pest management (IPM) program. Toxins found in these residues include benzoic acid and phenylacetic acid (Toussoun et al. 1968). In California, toxins from barley residues were released and became active 7-10 days after field incorporation. Incorporated plant residues, such as those of alfalfa, also released volatiles that had differential inhibitory/stimulatory effects on saprophytic soil fungi at both low and high concentrations (Gilbert et al. 1969), indicating the potential of managing soil-borne pathogens through the use/incorporation of plant residues in no-till or cover crop production systems. Soil fungi show differential responses to the previous crop and/or crop residue species incorporation, responding primarily to soil nutrient availability, but also to particular residue characteristics (Williams and Schmitthenner 1960). Cultivated plants also contain or release toxins with pathogenic characteristics. For example, potato tubers produce anti-fungal compounds in response to Phytophthora attack (Tomiyama et al. 1968), as did carrot with respect to Thielaviopsis (Hampton 1962). Also, pea root exudates inhibited growth of Botrytis, Pythium, and Rhizoctonia (Schenk et al. 1991). Antibacterial substances are also produced by cultivated plants, as observed in cauliflower seed for Xanthomonas suppression (Malekzadeh 1966). Plant allelopathy can be incorporated in cropping systems for management of weed populations through the establishment of rotational cover crops, application of crop residues, in polycultures, or in living mulches (Rice 1984; Weston 1996). Allelopathic plants that may be used as cover crops to "smother" weed growth include buckwheat, black mustard (B. nigra), clovers (Trifolium and Meli10tus spp.), oats, rye, sorghum-Sudan grass hybrids, and wheat (Weston 1996). Factors that may affect the allelopathic potential of particular plants include cultivar, soil fertility status, soil microbial activity, tillage system, and other

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related environmental factors such as soil temperature and moisture (Weston 1996). It is also critical that the allelopathic plant be easy to manage, to prevent it from becoming a weed itself.

Solarization. Soil solarization, the practice of sterilizing the soil surface utilizing heat from sunlight and clear plastics, offers the potential for management of soil-borne diseases, arthropods, nematodes, and weeds. Solarization treatments were effective for the control of important vegetable crops diseases such as Cricon emella, Didymella, Fusarium, Paratrichodorus, Phytophthora, Pseudomonas, Pythium, Rhizoctonia, Sclerotinia, Sclerotium, Thielaviopsis, and Verticillium (Martyn and Hartz 1986; Chellemi et al. 1994; Keinath 1995; Lodha 1995; Chellemi et al. 1997); nematodes such as Meloidogyne javanica, M. incognita, M. hapla, Helicotylenchus, and Heterodera (Bridge 1996); and weeds such as Amaranth us spp., Chenopodium album, Cyperus rotundus, Ipomoea spp., Malva parviflora, Pennisetum glaucum, Portulaca oleracea, Solanum nigrum, and Sorghum halapense (Stapleton and DeVay 1986; Lodha 1995). In Florida, tomato yields were increased by 26% by a solarization treatment during a fall trial but not during the spring (Overman and Jones 1986). Solarization effectively reduced the incidence of verticillium, but root knot nematodes, M. incognita, were only controlled in the fall trials. Verticillium levels in tomato also were reduced through early- or full-season solarization in California (Morgan et al. 1991). In northern Florida, soil solarization in tomato resulted in less Phytophthora, Pseudomonas, and Fusarium to a depth of 25, 15, and 5 em, respectively, at depths in which soil temperatures were increased by 9,8, and 5°C, respectively, above those temperatures found in bare soils (Chellemi et al. 1994). The Florida trials indicated the differential pathogen sensitivity to solarization treatments. Elsewhere, solarization, which increased soil temperatures to 60 and 50°C at 2 and 10 em soildepths, respectively, reduced losses from Fusarium in watermelon, plus increased populations of beneficial saprophytic Fusarium spp., resulting in enhanced crop growth, as compared to non-solarized plots (Martyn and Hartz 1986). A combination of solarization plus incorporation of organic amendments, such as isothiocyanate-releasing Brassica species, may provide additional disease control, as observed in watermelon following a cabbage amendment-solarization treatment in South Carolina (Keinath 1996). Further research is needed to improve solarization techniques for management of specific pathogens, but complementary controls will likely be needed for cost-effective pest managenlent and to maximize yields (Keinath 1995). However, the research and validation trials conducted to date (Chellemi et al. 1997) indicate the feasibility,

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from an economic and production standpoint, for the incorporation of solarization treatments as part of an IPM program for vegetables.

Reflective Mulches. Reflective mulches may be effective for pest management in some instances. In South Carolina, three-year studies found that aluminum reflective mulches were effective in tomato to reduce pest losses from aphids, the tomato pinworm, and the tomato fruitworm (Schalk and Robbins 1987). Silver reflective mulches were also effective in reducing aphid numbers and in delaying incidence of aphidtransmitted viruses in zucchini, as compared to bare-ground or treatments with different colored mulches (Brown et al. 1993). In Indonesia, similar outcomes were observed in chili pepper grown with white or silver mulches, which resulted in fewer thrips, aphids, and in a delay in the onset of viral attack (Vos et al. 1995). Floating Covers. Light floating (laying over the top of the canopy) row covers (10 g/m 2 ) offer potential as a barrier for insect infestations in vegetables (Antill and Davies 1990). In Florida, spunbounded polyethylene floating row covers effectively excluded aphids, whiteflies, the Diphania pickleworm, and the Diphania melon worm on zucchini, resulting in greater marketable yields than untreated plants (Webb 1991; Webb and Linda 1992). In Mexico, floating row covers also effectively excluded pests and delayed the incidence of viral attack in muskmelon (Orozco-Santos et al. 1995). 3. Other Alternative Controls

Alternative Products. Alternative products with low toxicity and low potential for environmental pollution are available for pest control. For instance, bicarbonates and bicarbonates/spray oil combinations offer control against Alternaria, Botrytis, cucurbit anthracnose (Colletotrichum orbiculare), gummy stem blight (Didymella bryoniae), leaf spot (Ulocladium cucurbitae) , Sclerotium, and powdery mildew (Sph aerath eca fuliginea) (Ziv and Zitter 1992; Palmer et al. 1997). Potassium silicate sprays controlled Pythium and F. aphanidermatum (Cherif and Belanger 1992; Cherif et al. 1994), Fusarium (Miyake and Takahashi 1983), powdery mildew (Sph aeratheca fuliginea), and Erysiphe cichoracearum (Menzies et al. 1992) on cucurbits. However, precautions should be taken to prevent soil acidification from continuous Si applications (Miyake and Takahashi 1983). Similarly, phosphate and potassium salt applications induced systemic resistance in cucumber against powdery mildew infection (Reuveni et al. 1993, 1996). Alternative products

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such as 0.5 and 1% KH Z P0 4 , 5% NaHCO s , 0.75% JMS Stylet Oil, neem extract, wine vinegar, and acetic acid were also effective, in comparative studies with registered fungicides, for powdery mildew, Sphaerotheca, control (Pasini et al. 1997). Chitin and chitosan soil amendments also appear promising for management of plant diseases such as Fusarium in celery (Bell et al. 1998a).

Botanicals. Numerous materials from botanical origin offer pesticidal activity for management of important pests. In China, Yang and Tang (1988) listed 267 plant species with pesticidal activity. Plants from over 150 families have antimicrobial activity (Powell and Ko 1986). Also, plants from over 55 families contain nematicidal properties (Bridge 1996). Many of these species are locally available in tropical areas. For example, in the Philippines, a Dioscorea hispida extract showed rodent toxicity (Ocampo and Villa 1991) and, in India, garlic extracts showed high molluscicidal activity (Singh and Singh 1993). In a screen test of 57 plant species from 32 families, Powell and Ko (1986) found six species that were strongly inhibitory of soil-borne Phytophthora. Neembased products are popular for the management of several pests, such as aphids (Lowery et al. 1993), but the specific neem product needs to be well characterized, as control efficiency is affected by crop, pest species, and weather conditions. Jasmonic acid, which is a widely available naturally occurring plant product, also shows promise both for fungal disease management and for inducing resistance to arthropod pest attack (Avdiushko et al. 1997). Plant Disease Resistance and Cross-protection. Other alternative pathogen control mechanisms include development of disease-resistant cultivars, cross-protection, and disease-forecasting models (Taylor 1990). Advancements in the understanding of the mechanism of plant disease resistance, including defense response mechanisms and systemic acquired resistance (Hammerschmidt and Smith-Becker 1997) in response to viral (Seskar et al. 1998), bacterial (Smith-Becker et al. 1998), fungal (Meera et al. 1994; Larkin et al. 1996), and arthropod (Seo et al. 1997; Agrawal 1998) attack should unravel alternative avenues for inclusion in pest management programs (Delaney 1997; Stanley 1998). Similar possibilities are being explored through "cross-protection" techniques by inducing resistance to important pests through inoculation with mild pathogens (Lecoq et al. 1991; Reuveni et al. 1993), or with beneficial fungal isolates (Meera et al. 1994; Larkin et al. 1996). For example, in cotton, plants first attacked by spider mites showed induced tolerance toward verticillium infestation and, conversely, plants exposed first with verticillium showed

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slower mite developmental rates than uninoculated plants (Karban et al. 1987). Cross-protection for viral control, used effectively for Zucchini Yellows Mosaic Virus (ZYMV) control in zucchini (Lecoq et al. 1991; Rezende and Pacheco 1998) is another novel area for viral pest management. This approach was effective in some commercial cucurbit production areas such as in Hawaii 0. Cho and R. Shimabuku, unpublished data). E. Integrated Pest Management Programs

Little is known in the tropics concerning a host of pest-environment and pest-crop interactions, information which is critical for the design of efficient pest management programs (McSorley 1996). For example, there is little information about crop losses from nematodes (Swarup 1990), which precludes the establishment of economic injury levels or action thresholds-information that may be useful to plan rotations, to select cover crop species, or to determine whether any treatments are necessary. A comprehensive review of locally available information for specific crops is necessary to identify areas where preliminary IPM control strategies can be implemented, and also to identify critical areas where research is needed for further design and implementation of regional (area-wide) pest control programs. The extensive background biological research required for the development of concerted IPM programs was shown in Hawaii by the team approach used to develop management techniques for control of the tomato spotted wilt virus in tomato, pepper, lettuce, and other crops (Cho et al. 1989). In addition, economic data showing the potential for increased profits in production systems following IPM, as shown for celery in California (Trumble et al. 1997), will be useful for the implementation and dissemination ofIPM programs in the tropics. With a few exceptions (Rodriguez and Trumble 1992; Valenzuela and DeFrank 1995), IPM programs are non-existent throughout most of the tropics, and, where adopted, management information has often been borrowed from research conducted in developed countries. IPM programs for vegetables in the U.S. are also a recent phenomenon, with implementation programs established only within the past two to three decades (Pohronezny et al. 1978). However, efforts are underway to develop the baseline data necessary for implementation of IPM programs in several tropical areas. Advancements in the implementation of IPM programs may be reached by focusing on target pests that are amenable to standard IPM programs (Genung et al. 1978). Other priority and secondary pests are

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managed with conventional methods, until new data and experience reveals alternative control options. Knowledge of pest population dynamics, dispersion habits, natural enemies, and pest response to habitat manipulation is important in the design of sampling methodologies, control treatments, and timing of control measures. 1. Sampling and Monitoring. In Burkina Faso, simple sampling methodologies and economic thresholds were defined to time insecticide applications for the management of the Helicovera tomato fruitworm (Bouchard et al. 1992). Other rapid sampling methodologies developed in warm areas for important pests can often be borrowed, as a starting point for the design of IPM programs in tropical regions. Examples include the presence-absence sampling technique developed in Florida for Liriomyza leafminers (Schuster and Beck 1992); the presenceabsence technique developed in Australia for two spotted spider mite sampling in strawberry (Hepworth and MacFarlane 1992); the action threshold of one pepper weevil, Anthonomus eugenii Cano, per 400 terminal buds determined for intensive bell pepper production in Florida (Riley et al. 1992); the use of colored sticky traps for monitoring adult pepper weevils in pepper, also in Florida (Riley and Schuster 1994); aphid sampling techniques developed for cole crops in California (Weber et al. 1991); or the use of food baits for pre-sampling of wireworms in potato (Jansson et al. 1989). Similarly, pheromone-based controls or monitoring programs were developed in the U.S. for the management of the tomato pinworm (Steenwyk et al. 1983), beet armyworm, (McNally 1984), and of the sweetpotato weevil, Cylas formicarius (Mason and Jansson 1991). Attractant volatiles were also used in monitoring traps for thrips (Teulon et al. 1993). 2. Timing of Control Practices. The timing of pest control practices is an important tool available to the farmer as part of an IPM program. However, knowledge of the pest population dynamics is required for the farmer to make these decisions. For example, the importance of early spring plantings to reduce whitefly numbers in cotton was shown by Brazzle et al. (1997) in California. In North Carolina, early plantings also resulted in reduced Heliothis corn earworm damage in soybean (Terry et al. 1987). In some instances, the delay of pest control practices is warranted. For example, some vegetables can withstand considerable defoliation before yields are affected, showing substantial tolerance to pest attack before pesticide applications are necessary. In staked tomato, yields from 3-4 harvests were decreased only after 60% manual weekly defoliation of the

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lower canopy and not with the lower defoliation rates (Jones 1979). This trait was used for establishment of pesticide action thresholds. Thus, no pesticide treatments are required for leafminer control on tomatoes in Florida unless lower canopy defoliation exceeds 30% (Keularts et al. 1985), especially prior to the bloom growth state. After bloom, no controls are required unless lower canopy defoliation is exceeded by 50%. In addition to timing, the method of pesticide application can also be modified to achieve better pest management. For example, in the Netherlands, pesticide-treated leek seed was used for effective control of the onion thrips, Thrips tabaci (Ester et al. 1997). 3. Multi-tactic Approaches. Overall, the use of several management tactics within an IPM framework is likely to offer better results in the long run, and across several environmental settings, than the use of any one tactic alone (Lewis et al. 1997). For example, to manage a complex of arthropod pests in sweetpotato, the percent of control offered by different available techniques included pest resistance (64% control), the use of beneficial nematodes (21 %), and pesticides (31 %) (Schalk et al. 1993). Similarly, for pepper weevil control in the Pacific and Caribbean basins, potential multi-tactic approaches include destruction of volunteer weeds such as nightshade at least one month prior to planting, deep-disking of pepper plants after harvest, using pest-free transplants, use of tolerant cultivars, conservation of natural enemies, use of pesticide action thresholds, and the use ofbiorational pesticides (Schuster et al. 1996). Multi-tactic controls for nematode management in the tropics are multifaceted. They include cultivar resistance, prevention of inoculation and spread in the field, biological control, vegetational diversity, maintenance of traditional multiple cultivar systems, application of organic amendments, biological control, alternative tillage, soil solarization, and the use of other nonchemical, cultural, and physical controls (Bridge 1996; McSorley 1996). Weeds are perhaps the major challenge for small-scale vegetable crops production in the tropics, where they consume from 40-60% of the labor requirements for crop production (Eiszner et al. 1996). Promising weed control strategies that can be utilized within an IPM framework include intensive year-round production systems, typical in many Asian countries; the use of efficient and timely manual- and mechanicalweeding tools; preventive practices to minimize initial weedy soil seedpool levels, such as the use of transplants, flooding, precise water and fertilizer placement, and use of organic mulches; improving the timing of cultural management practices; the use of "smother" crops in the

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form of competitive cultivars, cover crops, or living mulches; modified triangular or square planting arrangements to maximize light interception of the cash crop; crop rotations; biological control, especially with fungi; as well as improved herbicide application technologies (William 1979; Putnam 1990; Elliot and Moody 1992). William (1979) pointed out the potential to manage nematodes, arthropods, and pathogens through the manipulation of weed populations in the agroecosystem (see Section C.4). Clearly, crop-weed-pest interaction dynamics need to be understood for the design of efficient cropping systems that will result in increased crop yields, improved nutrient cycling, and reduced pest levels.

V. CONCLUSIONS AND FUTURE PROSPECTS As this review indicates, agroecological techniques exist to develop and implement biologically-based cultural management programs, and habitat manipulation schemes that promote internal system nutrient cycling, improved soil quality, pest suppression, lower production risks, seasonal labor distribution, sustained yields, and regional food security. Likewise, over the past few decades, significant advances were made in virtually all cultural aspects for the commercial production of vegetables. Furthermore, research, experimental design, and statistical techniques were advanced for the design, evaluation, and validation of innovative area-wide cropping systems. In temperate regions, significant advances have been made in the area of vegetable stand establishment. Promising techniques include mechanization, seed treatments, biological control inoculants, and nutrient conditioning of seedlings prior to transplanting. Upon modification, many of these techniques are applicable to tropical settings. Research is also needed to develop alternatives to continued monocropping. The sustainability of monocultures may improve by increasing productivity and efficiency of resource utilization, as well as by extending the crop growing cycle to reduce the number of times the fields are tilled annually. Research with monocultures is needed in practically all aspects of the production process to evaluate new techniques developed in other regions or to modify local cultural practices. Relatively little research has been conducted in the area of vegetable production under polycultures. Work to date with agronomic crops indicates that advancements in the productivity of vegetable polycultures can be obtained by selecting adapted cultivars and by better understanding the underlying agroecological mechanisms that affect productivity. Thus,

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considerable research is necessary to improve the design and productivity of vegetable-based polycultures. Several nutrient management techniques were presented that may increase yields in tropical agroecosystems. These include the application of organic amendments, the use of cover crops, organic mulches, conservation tillage, and the improvement of resource utilization in polycultures through other habitat manipulation schemes. However, much adaptive research is required to implement these techniques with vegetables. Habitat manipulation strategies to minimize pest pressure in the tropics include the use of polycultures, cover crops, rotations, trap crops, and the establishment of insectary patches. Other alternatives available for pest management include build-up of soil organic matter to promote rhizosphere microbial activity, solarization, reflective mulches, the use of alternative products such as botanicals, and the implementation of integrated pest management (IPM) programs. However, extensive knowledge gaps still exist which prevent the implementation of these techniques for the production of vegetable crop in the tropics. The goal of future work will thus be to incorporate the rich indigenous knowledge that exists in the tropics with the latest agroecological techniques and applicable modern methods of crop production. Improved stand establishment, irrigation, nutrient management, biotechnology, pest management, postharvest management, and technology transfer techniques are necessary to reach sustained yield goals for the production of vegetables in the tropics. A trend exists throughout the tropics to increase the land area and yearround production of monocultures, resulting in a loss of germplasm resources, reduction of diversified production systems, and in a corresponding loss of woodlands, hedgerows, and other wild vegetation. Possible repercussions from the widespread biotic simplification of regional landscapes include an increase in the frequency of pest outbreaks; increased community dependence on external energy-intensive inputs such as food, pesticides, subsidized fertilizers (Prinz and Rauch 1987), and fossil energy (Pimentel et al. 1983); environmental deterioration in the more fragile/simplified agroecosystems; a greater scarcity of needed resources such as fuel-wood and fresh water; and continued impoverishment in rural areas that have for long experienced chronic economic instability. A greater understanding of tropical agroecosystems and the application of appropriate production techniques should help to improve crop productivity. However, contributions toward agricultural sustainability would have a greater impact if social equity was also improved in regions that are characterized by the marked uneven distribution of capital goods.

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5 Lettuce Seed Germination * Daniel J. Cantliffe, Yu Sung, and Warley M. Nascimento University of Florida, IFAS Horticultural Sciences Department Gainesville, Florida 32611-0690 1. Introduction II. Seed Structure III. Germination IV. Environmental Factors Affecting Germination A. Light B. Temperature V. Restriction of Lettuce Seed Germination at High Temperature A. Embryonic Growth B. Seed Integuments C. The Endosperm VI. Increasing Thermotolerance in Lettuce Seed A. Genotype B. Temperature and Light During Seed Maturation C. Hormonal Affects on Germination D. Seed Priming VII. Changes in the Embryo and Endosperm During Germination A. Embryonic Growth Potential B. Endosperm Weakening C. Endo-~-mannanase D. Other Enzymes Associated with Endosperm Weakening and Seed Germination VIII. Summary and Conclusion Literature Cited

I. INTRODUCTION Lettuce (Lactuca sativa L.) is an important salad crop produced yearround in the United States from seed, actually a one-seeded dry fruit (achene). Lettuce can be directly sown in the field or transplanted to *Florida Agricultural Experiment Station Journal Series No. R-06537. Horticultural Reviews, Volume 24, Edited by Jules Janick ISBN 0-471-33374-3 © 2000 John Wiley & Sons, Inc. 229

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optimize stands. If lettuce seeds are sown when soil or greenhouse temperatures are above 30°C, the maximum germination temperature range for many genotypes is exceeded (Gray 1975). As a result, germination can be erratic or completely inhibited, and crop or transplant production is thereby limited. Thus, lettuce seed germination is strongly temperature dependent. As temperature rises even 2 or 3°C above the optimum (usually 15-22°C), lettuce germination can sharply decline from nearly 100% to near 0% (Reynolds and Thompson 1971), a phenomenon known as thermoinhibition. This inhibition of germination upon imbibition at a supraoptimal temperature is not permanent. If the temperature returns to an appropriate degree for germination, the seeds are able to resume germination. An expanded period of imbibition during supraoptimal temperatures may induce a secondary dormancy called thermodormancy (Khan 1980/81). In this case, the seeds become dormant and will not germinate even if they are returned to favorable temperatures for germination. Thermoinhibition is a more transient condition than thermodormancy, and releasing thermoinhibition in lettuce seed can prevent the induction of thermodormancy. The ability of a lettuce seed to germinate at high temperature is termed thermotolerance. The problems of thermoinhibition and thermodormancy in lettuce seed have been the subject of interest to numerous researchers. Inhibition of germination of lettuce at high temperature has been attributed to several factors. These factors include: (1) reduction of seed covering permeability to oxygen and carbon dioxide (Borthwick and Robbins 1928); (2) physical barriers within the seed to water uptake (Speer 1974); (3) accumulation of metabolic products in the endosperm or embryo (Borthwick and Robbins 1928); (4) inhibitory effects of abscisic acid (McWha 1976); (5) mechanical restraint of the seed coverings (Ikuma and Thimann 1963b); (6) inhibition of the secretion of cell-waIl-weakening enzymes (Ikuma and Thimann 1963b; Dutta et al. 1997); (7) deficiency of the growth potential of the embryo (Nabors and Lang 1971a); and (8) nonfunction of phytochrome (Scheibe and Lang 1969). The physiology of germination of lettuce seed is complex. The effects of light and temperature on lettuce germination have been studied for many decades. Seed dormancy may be induced under various unfavorable environmental conditions, such as seed imbibition in darkness, high salinity, or at high temperature (Toole et al. 1956; Ikuma and Thimann 1964). Relief of dormancy can be obtained by a number of methods, such as exposure to red light (Scheibe and Lang 1965), application of plant growth regulators or chemicals (Keys et al. 1975; Dunlap and Morgan 1977a; Saini et al. 1986), and seed priming (Cantliffe et al. 1981).

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Researchers have described the mechanical, physiological, and biochemical mechanisms in germination of seed, but to date a review devoted to lettuce seed germination has not been published. An expanded research report entitled Lettuce Seed and Its Germination was published in 1928 by Borthwick and Robbins, in which the effects of temperature on lettuce seed germination were addressed. In the present review, the importance of the lettuce endosperm to germination, especially at high temperature, is described. Improving thermotolerance through genotype, seed maturation environment, priming, and hormone application are reviewed. Evidence for a role of endo-~-mannanaseand other enzymes associated with endosperm weakening and seed germination are described. Finally, specific involvement of ethylene during seed germination is addressed.

II. SEED STRUCTURE Borthwick and Robbins (1928) described the structure of the seed coverings that surround the embryo of the mature achene of lettuce: 1.

2.

3.

The pericarp, or fruit coat, is the outer surface of the seed and is a nonliving, ribbed structure consisting of thick-walled, lignified cells. The integument, or seed coat, is composed of the remnants of the outer epidermis and some parenchymatous cells and forms a nonliving layer between an inner, suberized, semipermeable membrane and an outer, thick-walled epidermis. The endosperm, a living tissue (8% of seed dry weight), adheres to the inner epidermis of the integument. Most of the endosperm is two cells thick except at the radicle end, which is three or more cells thick. The cell wall of the endosperm is thick and has numerous column-like protuberances.

The major storage components of the endosperm cell wall are mannopolysaccharides (carbohydrates, 3.4% of seed dry weight). The cytoplasm of the endosperm cells contain abundant protein bodies (0.23% of seed dry weight) and lipid storage material (4.3% of seed dry weight) (Balmer et al. 1978; Leung et al. 1979). The endosperm acts as a restriction or barrier for embryo growth and provides nutrients for growing seedlings following germination (Abeles 1986). The cotyledons are the major storage tissue (60-70% of seed dry weight). Most stored reserves in cotyledons are lipid (27% of seed dry weight) and protein (2.7% of seed dry weight), with a small amount of

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phytate, soluble sugars, and very little starch. The reserves in the cotyledons do not mobilize until stored reserves in the endosperm have been degraded (Halmer et al. 1978; Leung et al. 1979). III. GERMINATION

Germination is defined as the protrusion of the radicle through the surrounding seed coverings. The major events that occur in germination of lettuce seed include water imbibition, enzyme activation, storage tissue breakdown, initiation of embryo growth, rupture of the seed coverings, and establishment of the seedlings (Copeland and McDonald 1985). The processes of germination occur close to the tip of the embryonic axis (Ikuma and Thimann 1964). As lettuce seeds imbibe water, some of the most significant changes in the embryo occur in the plastids (Srivastava and Paulson 1968). After a short period of soaking, well-defined and fairly large plastids appear in all tissues. Plastids undergo a great deal of change in the hypocotyl but not much change in size or appearance at the root tip. Reserve proteins and lipids fill the dry embryo cells. Protein and lipid bodies are gradually depleted and eventually disappear after imbibition is complete. The order of events in the mobilization of stored reserves in the cotyledons, axis, and endosperm of germinated lettuce seeds has been established. The endosperm is the initial source of food reserves (proteins, lipids, and carbohydrates) for the growing embryo. Stored reserves (lipids, proteins, and phytate) in the cotyledons and axis mobilize after degradation of the endosperm (Leung et al. 1979). The degradation of endosperm requires a number of enzymes, which can be present as preformed enzymes or be synthesized de novo within the endosperm cells. All of the cytological prerequisites for enzyme synthesis are contained in the endosperm cells. For example, protein bodies can store enzymes or proteins that provide amino acids or small peptides for the synthesis of new enzymes. Lipid-containing spherosomes can connect to the endoplasmic reticulum and sometimes bear ribosomes. In addition, free ribosomes and mitochondria can be found in ground cytoplasm (Jones 1974). Under favorable circumstances for germination, radicle emergence and seedling establishment are the results of cell expansion and cell division of the growing embryo (Toole et al. 1956). The expansion of radicle cells precedes mitosis by many hours during lettuce seed germination. It is cell expansion that results in radicle protrusion; cell division plays little or

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no role in the germination process (Haber and Luippold 1960a). Srivastava and Paulson (1968) showed that, after 12 h of imbibition, the first detectable cellular changes during germination of 'Grand Rapids' occurred in epidermal and cortical cells about 0.5 mm behind the root apex. These cells were activated and expanded, then the radicle bent and penetrated the seed coverings. IV. ENVIRONMENTAL FACTORS AFFECTING GERMINATION A. Light Germination of some lettuce genotypes, such as 'Grand Rapids', is controlled by light. Photosensitive lettuce seeds having a functional phytochrome system are affected in their germination responses following red or far-red light irradiation (Borthwick et al. 1952). When 'Grand Rapids' seeds were imbibed at 18°e in darkness, there was a maximum germination percentage which could not be further increased by red light. At about 32°e, the seeds would not germinate in darkness and this could not be improved by red light. With these temperature limits, the maximum effect of red light on germination was reached at approximately 26°e (Evenari et al. 1953). Phytochrome is a photoreversible, photomorphogenetic pigment that exists in two forms: Pr, the red light-absorbing form, and Pfr, the far-red light-absorbing form. The Pfr form of phytochrome in hydrated tissue is unstable and can either undergo dark reversion to Pr or destruction, a process that represents a loss of photoreversibility (Toole et al. 1956; Butler et al. 1963). Red light increases lettuce seed germination as seed hydration increases from 8 to 15%; it reaches a maximum at moisture contents above 18%. Seeds have no response to red light at moisture contents below 8% (Hsiao and Vidaver 1971; Vertucci et al. 1987). Seeds that have imbibed water and have been irradiated with red light can be redried without affecting subsequent viability. The redried seeds will germinate in darkness and can be inhibited by a short exposure to far-red light after imbibition (Vidaver and Hsiao 1972). This suggests that phytochrome in mature lettuce seeds is primarily in the Pr form, and photoconversion to Pfr cannot happen in the dehydrated state (Kendrick and Russell 1975). The photoreceptive site in phytochrome-mediated lettuce seed germination corresponds to the tip of the hypocotyl (Ikuma and Thimann 1959). This site is at the upper half of the embryonic axis and never forms root hairs. At the beginning of the imbibition period, red light-induced

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growth occurred simultaneously in the hypocotyl and the radicle. Therefore, the light signal is probably transferred from the hypocotyl to the root (Inoue and Nagashima 1991). The transformation between Pr and Pfr is affected by temperature. Low temperature prevents the transformation of a physiologically active Pfr form to an inactive Pr form (Scheibe and Lang 1965). Evenari et al. (1953) proposed that there may be two limit points that can control phytochrome transformation. One mechanism ensures that germination of seed is not affected by light and operates up to the temperature limit below which germination cannot be decreased by light alone. This is the low temperature response, where light is not needed for germination. The other mechanism is light-sensitive, responding to the effects of red as well as far-red light and operates up to the temperature limit above which germination cannot be increased by light alone. This is the high temperature response, where germination is extremely light-sensitive and for which germination can cease above the high temperature limit. While photosensitive seeds sown over a suitable temperature range can germinate in darkness, at supraoptimal temperature a short exposure to red light after a period of imbibition is required to induce germination. Fielding et al. (1992) showed that there was a close link between phytochrome action and the upper temperature limit for seed germination. Increasing pfr levels in seed caused a successive increase in the upper temperature limit for germination. Kristie and Fielding (1994) indicated that the Pfr level required to induce 50% germination of lettuce seeds after a single light plus temperature treatment increased from about 11 % at 15° and 20°C to 86% at 30.5°C. Following a single light plus temperature treatment, seed germination at high temperature was limited solely by the availability ofPfr. The precise degree of upper temperature limit for seed germination was influenced by reversion of the pfr form to the Pr form. The block to germination at temperatures above the upper limit resided within the steps governing escape from photoreversibility rather than the later steps in the transduction chain between Pr and Pfr. Carpita and Nabors (1976) showed that phytochrome may be one of the molecules that turns over during thermoinhibition. The marked increase in the time needed to germinate after red light induction after return to 20°C was traced to a longer time needed before FR-reversal. The latter results indicated that the extra time needed may be necessary to synthesize new phytochrome. Germination of light-sensitive lettuce seeds can be stimulated or inhibited by applying plant growth regulators (Kahn 1960; Ikuma and Thimann 1963a; Khan 1968). Irradiation leads to the production of gibberellins (GAs) in lettuce seed. The GAs are involved in phytochrome-mediated

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growth responses in the axis (Kahn et al. 1957). Both GA and light induce changes in water potential of the embryonic axis of the seed, although gibberellin-promoted seed germination cannot be reversed by a period of far-red irradiation. The seed's response to GA is probably achieved differently from its response to irradiation (Kahn et al. 1957; Ikuma and Thimann 1960; Scheibe and Lang 1965). Abscisic acid (ABA) is a potent inhibitor of seed germination, including lettuce seed germination. In non-dormant seed, the amount of ABA falls sharply during imbibition (McWha and Hillman 1974). The inhibition of seed germination correlates with the amount of endogenous or exogenously-applied ABA (McWha 1976). Endogenous ABA may be reduced by leaching as well as by metabolic degradation (McWha and Hillman 1974; Dulson et al. 1988). Increase or decrease of ABA content in lettuce seed has not been associated with light or the action of phytocrome. Cytokinins can overcome inhibition of seed germination by ABA (Bewley and Fountain 1972). Cytokinins have minimal effect on germination in darkness, but promote germination in light (Miller 1958). Khan and Tolbert (1965) reported that light was essential for cytokinin reversal of ABA inhibition in light-requiring 'Grand Rapids' seeds but not in lightinsensitive 'Paris White' seeds. Germination of light-requiring seeds is modulated by GA and requires the presence of cytokinins when seeds germinate under stressful conditions. Cytokinins act on cotyledon expansion, whereas GA enhances axis elongation (Ikuma and Thimann 1963a). Similar to the role for ABA in lettuce seed germination, neither light nor phytochrome action have been linked to cytokinin presence or action. B. Temperature The optimum temperature for lettuce seed germination varies with genotype. Although the optimum temperature for germination of most lettuce genotypes is between 15° and 22°C, many genotypes will germinate well at temperatures ranging from 5° to 25°C. At temperatures above 25°C, germination rate generally decreases; above 27°C, both the rate and the percentage of germination are drastically reduced for most genotypes (Borthwick and Robbins 1928). The upper temperature limit for most lettuce seed germination is between 28° and 32°C (Damania 1986). When lettuce seeds are imbibed at high temperature, germination is inhibited. When lettuce seeds are kept at high temperature for an extended period of time, the induction of thermodormancy occurs (Khan 1980/1981). Lettuce seeds will not germinate once they are thermo dormant until that dormancy is overcome.

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Two stages of seed germination of 'Grand Rapids' lettuce were particularly sensitive to high temperature (Gray 1977). The first stage occurred during the first four hours of imbibition; the second stage was between the beginning of mitosis and the onset of radicle emergence. The restrictions on germination by high temperature were different between the two phases. In the first phase, germination of seeds was strictly inhibited, but in the second phase, high temperature had a relatively minimal effect on germination. Takeba and Matsubara (1976) observed that seeds that were imbibed for a period of time at low temperature could germinate in high temperature conditions. Moreover, the longer the seeds imbibed at 20°C, the higher their germination percentage was after they were transferred to high temperature. These results also suggested that a thermo-labile factor controlled the process of germination. When temperatures were above 30°C, the factor was inactivated, but it could be reformed or reactivated at 20°C. Thus, seeds imbibed at low temperature for a certain period of time might germinate when later exposed to high temperature (Takeba and Matsubara 1976), suggesting that thermoinhibition and thermodormancy can be bypassed prior to radicle protrusion. As previously discussed, different factors have been attributed to explain the failure of lettuce seeds to germinate at high temperature. These include the impermeability of seed coverings to gases (Borthwick and Robbins 1928) and water (Speer 1974), nonfunction of phytochrome (Scheibe and Lang 1969), inhibitory effects of ABA (McWha 1976), deficiency of the growth potential of the embryo (Nabors and Lang 1971a), inhibition of the secretion of cell-wall enzymes (Ikuma and Thimann 1963b), and mechanical restraints of the seed coverings (Ikuma and Thimann 1963b). More recently, considerable evidence suggests that the lettuce endosperm layer can directly restrict radicle protrusion, especially at high temperature (Nascimento et al. 1998a,b,c). V. RESTRICTION OF LETTUCE SEED GERMINATION AT HIGH TEMPERATURE Generally at temperatures above 25°C, germination of lettuce seed is blocked because of thermoinhibition. The nature of lettuce seed dormancy at high temperature, how to overcome it, and the role of the outer seed coverings or embryo in restricting lettuce seed emergence at high temperature has long been debated. One theory is that the endosperm layer restricts radicle emergence. The endosperm cells may secrete cell-waIl-degrading enzyme(s) to weaken the endosperm tissue,

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therefore allowing radicle emergence (Ikuma and Thimann 1963b). Another theory suggested that the emerging radicle cannot develop enough thrust to overcome the mechanical resistance of the endosperm without first weakening the endosperm cell wall (Nabors and Lang 1971a,b). Pavlista and Haber (1970) reported that germination of lettuce seed needed the mechanical force of the growing embryo pushing against the endosperm, along with the enzymatic weakening of the endosperm, in order to germinate, especially at higher temperatures. A. Embryonic Growth The growth of the radicle and germination of seed at high temperature are not identical phenomena. Foard and Haber (1966) found that localized expansion and mitosis occurred in thermodormant lettuce seeds, but did not contribute to the overall expansion of the embryo. From cytological studies of the lettuce embryo, Haber and Luippold (1960a,b) observed that the occurrence of mitosis and cell elongation could be regulated physically and chemically. For example, treatment with mannitol alone or a combination of kinetin and high temperature (37°C) caused mitosis prior to actual germination. However, at 37°C, when thiourea was present, high temperature caused radicle protrusion to precede cell division. Factors that prevent or promote germination are associated with the control of embryonic growth in lettuce seed. As temperature is raised to 30°C, germination is inhibited. Germination of lettuce seed can also be prevented at nonthermoinhibited temperature by placing them in an osmoticum such as mannitol (Kahn 1960). However, if the endosperm is removed or punctured, the embryo itself can germinate at high temperature or in osmotic restraint (Borthwick and Robbins 1928; Scheibe and Lang 1965). These results suggest that there are factors acting to prevent the ability of the embryo to develop sufficient force to penetrate the endosperm barrier. Takeba and Matsubara (1979) measured the embryonic growth potential of 'New York' lettuce seeds and demonstrated that intact seeds could not germinate at 35°C because the growth potential was not enough to overcome the restraining force of the seed coat. At high temperature, metabolic activity that normally increases during germination might be restricted, therefore reducing the accumulation of osmotic constituents. For example, as temperature increased from 15° to 35°C, small fat bodies did not disappear in 'New York' lettuce seeds (Takeba and Matsubara 1977). Moreover, the amount of amino acid accumulation was reduced, possibly related to the activity of glytamine synthetase (GS).

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Takeba (l983a,b) attempted to demonstrate that the activity of GS in 'New York' lettuce seeds was correlated to germination behavior at high temperature. High GS activity was detected in dry seeds, but this activity decreased rapidly during imbibition at 35°C, although the amount of GS protein did not change. The amount of ammonia increased abruptly during the early stages of imbibition at 35°C, suggesting blockage of ammonia assimilation at high temperature. The GS activity decreased to a low level during the first 12 h of imbibition at 35°C, but increased again during subsequent continued imbibition at low temperature (l5°C) before breaking thermo dormancy. Because the activity of GS in the embryonic axis was higher than that in the cotyledons, thermodormancy of 'New York' lettuce seeds was thought to be caused by the inactivation of GS during imbibition at high temperature. Red light increased the growth potential of lettuce seed at 25° and 30°C but not at 35°C. At 25° and 30°C, the osmotic potential in red lighttreated seeds decreased due to free amino acids. This decrease was sufficient to account for the changes in growth potential and enabled the embryo to overcome the resistance offered by the endosperm. At 35°C, the growth potential of seeds was considered to be less than the restraining force of seed coats; this low growth potential was thought to be a contributing factor for thermodormancy (Scheibe and Lang 1965; Scheibe and Lang 1969; Takeba 1980b,c). B. Seed Integuments Seeds develop from fertilized ovules and can be comprised of three genetically different components: the embryo, the endosperm, and the seed coat. Some species contain perisperm as storage tissue instead of endosperm, and some species may contain both tissues. Generally, these tissues enclose the embryo. All of these seed-enclosing structures may act as physical barriers interfering with the growth and emergence of the embryo. In many species, the seed coat, developed from the integument of the ovule, is the main protective barrier between the embryo and the external environment. The role of the seed coverings in seed germination was investigated in other species, including muskmelon (Welbaum et al. 1995), pepper (Watkins and Cantliffe 1983), and tomato (Groot and Karssen 1987). In lettuce, we speculate that the control of germination, especially at high temperature, appears to be exerted within the layers surrounding the embryo of which there are three: the pericarp, integument, and endosperm (Borthwick and Robbins 1928). The outermost coat is the longitudinally ribbed, non-living pericarp, of maternal origin (Drew and

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Brocklehurst 1984). Between the endosperm and the pericarp is the integument, a composite but delicate layer with semi-permeable properties. The lettuce endosperm has been thought to provide a mechanical resistance against radicle expansion, since seed germination was increased by puncturing or removing the endosperm (Borthwick and Robbins 1928; Evenari and Neumann 1952; Ikuma and Thimann 1963b; Prusinski and Khan 1990). We concur with this hypothesis. For imbibed seeds at high temperature, the greater delay and slower attainment of maximum germination suggests that metabolism inside the seed has been damaged or essential metabolites for germination have been reduced to low levels (Gray 1977). For instance, respiration in lettuce seed is inhibited at supraoptimal temperature because the endosperm and integumentary membrane restrict the free diffusion of oxygen inward and carbon dioxide outward. As temperature increases, oxygen requirements rapidly increase, but the seed coat gradually may become a barrier to the rapid exchange of gases so that the respiratory intensity is significantly decreased (Borthwick and Robbins 1928). Researchers have proposed that the inhibitory effect of ABA on lettuce seed germination at high temperature is due to its role in inhibiting the synthesis of cell wall-weakening enzymes in the endosperm (Braun and Khan 1975; Halmer and Bewley 1979). Dulson et al. (1988) found that the amount of ABA that was leached from isolated endosperm correlated with increased mannanase production in isolated lettuce endosperm. McWha (1976) demonstrated the effect of temperature on ABA content in 'Great Lakes' seeds during imbibition. When the seeds were maintained at temperatures at which germination was delayed or prevented, the ABA level fell more slowly or not at all. However, Braun and Khan (1975) reported that the ABA content in germinating seeds of 'Grand Rapids' diminished more rapidly at 25°C than at 35°C, but there was no relationship between ABA content and percentage of germination after imbibition for 24 h. It is unknown what amounts of endogenous ABA are present specifically at active sites or are necessary to effect control of germination in lettuce at any temperature. Dutta et al. (1994) used autohydrolysis to analyze the composition of cell walls and found that the autolytic activity of the isolated wall was related to germination conditions. In 'Pacific' lettuce seeds imbibed at 25°C, the rate of autolysis from the endosperm wall increased markedly in the period just prior to radicle emergence. The rate of autolysis at 32°C was reduced about 25% compared to that at 25°C. Seeds treated with ABA at 25°C did not germinate. The rate of autolysis from the endosperm cell walls of seeds treated with ABA was significantly lower than that of seeds imbibed without ABA. Therefore, the capacity for lettuce seed

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germination at high temperature may again be related to the activity of endosperm-cell-wall-degrading enzymes prior to radicle emergence. The seed coverings act as a physical barrier in the restriction of lettuce seed germination at high temperature. The force required to puncture the different seed tissues was measured with an Instron Universal Testing Machine in order to identify which part of the seed coverings played a major role in controlling germination (Tao and Khan 1979; Drew and Brocklehurst 1984; Wurr et al. 1987). In 'Grand Rapids', the major barrier to embryo growth was found to be the endosperm layer, which contributed 60% of the total resistance to puncture of the intact seed (Tao and Khan 1979), but, in 'Cobham Green', this value was only 40% (Drew and Brocklehurst 1984). Wurr et al. (1987) as well as Drew and Brocklehurst (1990) measured the forces required to penetrate seed layers of different thermosensitive cultivars and observed that the strength of the pericarp appeared to playa more important role than did the endosperm in germination. There were significant positive correlations between seed weight and the force required to penetrate the whole seed, but only one test cultivar had significant correlation between germination at high temperature and seed penetration forces. The results of the puncture test were not consistent, possibly because cultivar differences or variations of the positions of penetration of the seeds. Data on penetration force would be most meaningful if taken at the micropylar end (point where the radicle protrudes through the seed coverings). Sung et al. (1999) did in fact measure the force to penetrate the coverings at the micropylar end in thermosensitive and thermotolerant lettuce genotypes. All thermotolerant genotypes had lower endosperm resistance than the thermosensitive types. All genotypes were primed in order to enhance germination at 36°C. Seed priming decreased endosperm resistance among the thermosensitive genotypes. Thus, the association between endosperm weakening and the ability to germinate at 36°C was established. C. The Endosperm A number of different features might account for the resistance to germination imposed by the endosperm. The thick cell walls and dense cytoplasm of endosperm cells contribute to the strength of the endosperm layer (Ikuma and Thimann 1963b; Tao and Khan 1979). Balmer et al. (1976) reported that these thick cells act as a barrier to seed germination, especially under high imbibition temperatures. The chemical composition of the cell walls may also affect the rigidity of the endosperm. It is known that galactomannans in the endosperm cell walls of

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legume seeds result in a hard seed (Zamski 1995). Also, the cell wall column-like projections that mainly extend from the external cell wall to the internal wall (Borthwick and Robbins 1928) add rigidity to the tissue (Psaras 1984). The endosperm, possibly with the attached integumentary membrane, can exert a mechanical resistance to embryo expansion (Pavlista and Baber 1970). The lettuce endosperm, unlike that of several other species, is a living tissue, and comprises about 8% of the seed dry weight. Most of the lettuce endosperm consists of a distinct layer of two cells, except for the micropylar region, where there are three or more cells (Borthwick and Robbins 1928). The endosperm cell walls are comparatively thick. The part of the cell wall next to the integument is consistently thicker (25-30 /lm) than the inner walls (6-10 /lm) of the endosperm (Jones 1974). Unlike most plant cell walls, the contours of the wall of the lettuce endosperm are highly irregular, and numerous wall protuberances project into the cytoplasm (Jones 1974). The dense cytoplasm contains organelles of protein and lipid storage (0.23 and 4.3% of seed dry weight, respectively) (Jones 1974; Balmer et al. 1978; Leung et al. 1979). The lettuce endosperm cell wall comprises two-thirds of the total seed cell wall polysaccharide material (Balmer et al. 1975). It is composed largely of mannose-containing polysaccharides (58-74%) (Balmer et al. 1975; Dutta et al. 1994), probably (l,4)-~-mannans (Bewley et al. 1983). Some galactose (approximately 10%) is also present, suggesting the presence of galactomannans (Bewley et al. 1983). This hemicellulose consists of a linear backbone of ~-l,4-linked mannose units with branches of single ~-1,6-linked galactose residues (Ouellette and Bewley 1986). Other sugars, such as rhamose, fucose, arabinose, xylose, glucose, and uronic acids are also present in lettuce seed endosperm cell walls (Balmer et al. 1975; Dutta et al. 1994). The micropylar cell walls of lettuce endosperm is compositionally different from the lateral region. Cell walls from the micropylar region have a significantly higher proportion of arabinose and glucose (although mannose is still the predominant sugar) compared to walls prepared from the lateral region, which consist mostly ofmannans (Dutta et al. 1994). Lettuce endosperm is the initial source of food reserves for the growing embryo. Mobilization of the endosperm requires a number of enzymes that may be stored or synthesized de novo within the endosperm cells. The cells of lettuce endosperm contain all of the cytological apparatus for enzyme synthesis (Jones 1974). The galactomannan-rich polysaccharides are important food reserves utilized by the growing embryo after germination, but before the mobilization of the major reserves stored in the cotyledons (Balmer et al. 1978; Leung et al. 1979). Most of

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the carbohydrate for the growing lettuce embryo comes from the degradation of cell wall polysaccharides (Park and Chen 1974) that are subsequently converted to sucrose and transported to the embryo, primarily into the cotyledons (Park and Chen 1974; Halmer et al. 1978). Galactomannans are also found as a major component of the endosperm tissue of a number of species from Leguminosae (Reid and Meyer 1972; McCleary and Matheson 1975; Leung et al. 1981; Ganter et al. 1995; Buckeridge and Dietrich 1996), Palmae (Samonte et al. 1989; Daud and Jarvis 1992), Anonacea, Rubiaceae (Giorgini and Comoli 1996), Araceae (Nishinari et al. 1992), and Convolvulaceae. In most of these species, galactomannans are hydrolyzed in the post-germinative period to their monosaccharide constituents (mannose and galactose), which are then used by the growing embryo. Aside from the endosperm's importance as a food reserve, the structural complexity of the endosperm cell walls in lettuce is correlated with the role this tissue plays in restricting early embryo growth.

VI. INCREASING THERMOTOLERANCE IN LETTUCE SEED A. Genotype Thompson et al. (1979) observed significant differences at the upper limit of temperature tolerance among 23 cultivars. The response of seed germination in 22 cultivars of crisphead, cos, and butterhead types of lettuce was evaluated from 5° to 33°C (Gray 1975). The optimum temperature for germination in all cultivars was between 15° and 22°C, and there was a noticeable upper temperature limit for germination that ranged from 26° to 33°C. Generally, seeds of the crisphead type germinated well at 30°C, a temperature which inhibited germination in all the butterhead types. Damania (1986) examined 62 genotypes of lettuce germplasm obtained from different countries and reported that the upper temperature limits for germination for most genotypes ranged from 29° to 30°C. Sung et al. (1998a) were able to separate thermotolerant and thermosensitive genotypes from 21 genetic lettuce lines, including cos, butterhead, and crisphead types. A Spanish bibb lettuce 'Maturo' has been used as parental stock to import the thermotolerant character to cultivars such as 'Tall Guzmaine' and 'Floricos 83' (Guzman and Zitler 1983; Guzman 1986; Guzman et al. 1992). A wild accession, PI 251245 germinated 100% at temperatures above 30°C (Bradford 1985). Nascimento et al. (1998a) recently reported that seeds of thermotolerant 'PI251245' and 'Everglades' produced more endo-~-mannanaseat 35°C before radicle protrusion than

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did thermosensitive 'Dark Green Boston'. The enzyme is linked to endosperm wall degradation at the micropylar end of the lettuce seed. These results indicate the possibility of improving the ability of seeds to germinate at high temperature by breeding. Thompson et al. (1979) reported that the surface characteristics of black and white lettuce achenes were different. In black achenes, the longitudinal ridges were more protuberant, more highly sculpted, had more transverse rows, and had better developed trichomes than the white ones. Their studies of 23 lettuce cultivars revealed no significant correlation between the achene color and the germination response, although Damania (1986) reported that black-coated seeds tended to have lower temperature limits for germination than their white seeded counterparts. Such contradictory results fail to make clear whether or not the structural differences in seed coats are related to seed germination performance at high temperature. B. Temperature and Light During Seed Maturation The geographic region where lettuce seeds are grown significantly affects the performance of seeds over a range of germination. Germination performance of 80 samples of seed representative of 12 lettuce cultivars from nine growing areas indicated that the region where the seeds were grown significantly affected the ability of the seeds to germinate at high temperature (Harrington and Thompson 1952); seeds collected from hot climatic zones had higher temperature tolerance limits for seed germination. The quality of lettuce seeds harvested from mother plants may vary, even when the mother plants are grown in the same area and in the same cropping season. The lettuce inflorescence has a cymose cluster of flower heads, with the oldest flowers at the terminal end of the main axis (Jones 1927). Flowers open for no more than 2 h. Pollination and fertilization occur in less than 6 h, and seeds are mature 12 days after pollination. Throughout the flowering period, which lasts about 2 months, the rate of flowering and seed maturation is regulated by temperature (Jones 1927). Mother plants are thus exposed to varying air temperatures during the period of flowering. The temperatures during seed production affect seed quality; therefore, the quality of all the seeds produced through the season can vary (Damania 1986). Different environmental factors affect seed quality in various ways. The chemical composition of a seed can be influenced by its position on the plant, the environment under which the parent plant is grown, and the management of cultural practices. Temperature during seed maturation

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and development affects the seed's chemical composition, including oil quality and protein concentration, both of which have been investigated in food and fiber crops (Fenner 1992). The fatty acid composition of a number of seed oils has been reported to vary with the temperature under which the seeds have developed, with lower temperatures favoring increased unsaturated oils (Miquel and Browse 1995). In sunflower, the di-unsaturated linoleic acid (18:2) and the mono-unsaturated oleic acid (18: 1) had equal proportions when seeds matured at high temperature (about 28°C). This ratio, however, was 6:1 when seeds matured at low temperature (around 12°C) (Harris et al. 1980). Temperature also affects the protein concentration in seeds of some species. Higher protein concentration at high temperatures may simply be the result of a reduction in carbohydrate accumulation or oil content (Leffel 1988; Correll et al. 1994). In lettuce seed, major stored reserves are lipids and proteins. How high temperature is associated with a change of oil or protein composition or both and the relationship between this change and thermotolerance of seeds is still unknown. Gray et al. (1988) studied the effect of temperature on lettuce seed development, yield, germination, and seedling vigor. Seeds of 'Saladin' ('Salinas') produced at 30 /20°C germinated better at 30°C than those matured at 25°/15°C or 20 /10°e. Seed maturation temperature influenced the number of mature florets per plant, seed per floret, and mean seed weight. At 20 /10°C and 25°/15°C seeds matured slowly but, compared to seed matured at 30 0 /20°C, seed dry weight and seed size were increased to 50% and 15%, respectively. The number of seeds and seed yield per plant increased with an increase in temperature from 20 /10°C to 25°/15°C but then declined with a further increase to 30 0 /20°C. Root length of seedlings was not affected by the seed maturation temperature. Steiner and Opoku-Boateng (1991) investigated the effects of variation in ambient air temperature on 'Salinas' lettuce seed production. Mother plants were exposed to a wide range of temperatures during their flowering period in Fresno, California. Minimum temperatures during the day ranged from 11° to 22°C and the maximum ranged from 30° to 40°C. Seed size, weight, and yield were reduced, but germination percentage of those seeds increased with increasing minimum and maximum temperatures. Therefore, high temperature during seed development enabled the seed to subsequently germinate better at high temperature, but yield, in terms of total seed weight and/or total number of seeds, was not optimal, and seed size was reduced (Gray et al. 1988; Drew and Brocklehurst 1990). 0

0

0

0

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Continuous light during seed maturation increased germination in comparison to seeds that matured under 8 h of light per day (Thompson et al. 1979). Temperature and photoperiod conditions to which the ripening seed was exposed affected the subsequent germination behavior of the seed (Koller 196Z). When 'Grand Rapids' seeds maturated under high temperature (30 /Z3°C), high-temperature tolerance (Z6°C) increased. In addition, comparing the effect of diurnal (Z3°/17°C) and constant (Z3° or 17°C) thermoperiods on seed germination at high temperature suggested that seed maturation under constant temperature may induce greater germination at high temperature than seed maturation under varying temperatures. The level of temperature tolerance of the seeds was influenced by postharvest maturity of the achenes as well as environmental conditions including temperature, light, aeration, and moisture tension under which the crop was grown and harvested (Thompson et al. 1979). Seeds produced at 30 0 /Z0°C germinated more readily at 30°C than those produced at lower temperatures (Gray et al. 1988). Sung et al. (1998a) reported that lettuce seeds imbibed above Z7°C and that were matured at ZOO/10° or Z5°/15°C exhibited a lower percent germination than seeds that matured at 30°/ZOo or 35°/Z5°C. Seeds of 'Dark Green Boston' and 'Everglades' that matured at 30°/ZO°C exhibited improved thermotolerance over those that matured at the other three temperatures. Seeds of 'Valmaine' produced at ZOO/10°C exhibited 40% germination at 30°C, but seeds that matured at higher temperatures exhibited over 95 % germination. The upper temperature limit for germination of lettuce seed could thus be modified by manipulating the temperature during seed production. Nascimento et al. (1998a) observed more endo-~-mannanase activity before radicle protrusion in 'Dark Green Boston' seeds produced under 30 0 /Z0°C compared with those produced under ZOo/10°C. A relationship between the increase in enzyme activity before radicle protrusion and germination at high temperature was established for normally thermosensitive 'Dark Green Boston'. 0

C. Hormonal Effects on Germination

Induction of dormancy in lettuce seeds can be bypassed by GA application or red light; however, seeds still require other hormones during germination for alleviation of high temperature dormancy (Khan 1980/81). Cytokinins, ethylene, and GA, alone or in combination, have been involved in alleviating the effect of high temperature (Sharples 1973; Keys et al. 1975; Rao et al. 1975; Brmill and Khan 1976; Dunlap and Morgan

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1977a; Hegarty and Ross 1979). Braun and Khan (1976) indicated that thermodormancy in 'Mesa 659' and 'Grand Rapids' light-sensitive seeds could be overcome by adding a complex of GA, ethylene, and kinetin. Along with certain combinations of these hormones, GA was able to release thermodormancy in 'Grand Rapids' seeds at 32°C. However, the action of GA was not as effective in relieving thermodormancy of 'Mesa 659' seed at 32°C. At 35°C, the most effective relief for thermodormancy of these two cultivars was a combination ofGA plus kinetin and ethylene. In addition, CO 2 has been reported to enhance the stimulating effect of growth regulators in relieving thermodormancy (Negm et al. 1972; Keys et al. 1975; Saini et al. 1986). The effects of ethylene or cytokinins during early imbibition follow different pathways. Smith et al. (1968) dipped dry lettuce seeds in a kinetin solution for 3 min. Kinetin appeared to act at a very early stage of germination to relieve thermodormancy. Imbibing the seeds for various periods of time did not change the response to the dip treatment. In the early imbibition phase, the presence of ethylene was not required to release thermodormancy. Fu and Yang (1983) indicated that lettuce seeds did not respond to exogenous ethylene during the first 12 h of imbibition. The effect of ethylene on releasing dormancy was exerted during the imbibitional phase immediately before the emergence of the radicle, and the maximum effect of ethylene was exerted between 24 and 36 h of imbibition. These results suggest that ethylene and cytokinins have different action sites in overcoming thermodormancy. Both ethylene and kinetin can increase the growth of the embryonic hypocotyl (Takeba and Matsubara 1979; Abeles 1986). Kinetin is a more effective promoter than ethylene for reversing induced dormancy (Dunlap and Morgan 1977a). Kinetin acts to stimulate cotyledon expansion or hypocotyl longitudinal expansion or both (Ikuma and Thimann 1963a; Abeles 1986). Ethylene acts to control germination by promoting radial expansion of the hypocotyl. However, the longitudinal expansion of the hypocotyl is more effective in initiating germination than the radial expansion of the hypocotyl (Abeles 1986). Kinetin also strongly inhibits the growth of roots. Therefore, kinetin induces atypical germination, with cotyledon protrusion, a stunted radicle, or both. Once a wide opening is made at the cotyledon end of the endosperm coat, the embryo can slip through this opening. This presumably means that the hypocotyl can be a source of pressure to drive the radicle through the restraining endosperm. In this case, root growth does not appear to be required for germination since it is normally initiated after the radicle penetrates the endosperm (lkuma and Thimann 1963a; Braun and Khan 1976; Dunlap and Morgan 1977a).

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Small et al. (1993) reported that oxygen plus kinetin almost completely alleviated thermoinhibition in 'Grand Rapids' seeds. The results suggested that oxygen plus kinetin either caused seed to bypass an ethylene requirement for germination or increased the sensitivity of the seed to ethylene. In air at 38°C, seeds exhibited a high level of ethanolic fermentation. This was probably because of lower oxygen availability resulting from a decrease in oxygen solubility at this temperature. However, the seeds respired aerobically in a treatment of oxygen plus kinetin at 38°C. This treatment also increased the level of ATP in the seed which was able to satisfy the requirement for energy to drive germination processes at 38°C. Ethylene can stimulate germination and overcome dormancy of many seeds (Esashi 1991; Abeles et al. 1992). The inhibitory effect of high temperature on lettuce seed germination, for example, is overcome by exogenous ethylene (Abeles and Lonski 1969; Burdett 1972a; Negm et al. 1972; Keys et al. 1975; Rao et al. 1975; Dunlap and Morgan 1977a; Fu and Yang 1983; Abeles 1986; Saini et al. 1986; Khan and Prusinski 1989; Saini et al. 1989; Huang and Khan 1992). Despite the accepted involvement of ethylene in seed germination, the mechanistic details for its action have been poorly understood. High temperatures (35° to 40°C) inhibit ethylene production in a number of plant tissues (Yu et al. 1980). For instance, the negative effect of high temperature on chick-pea seed germination was due to low ethylene production (Gallardo et al. 1991). These authors also reported that treating the seeds with ethylene could alleviate the inhibitory effects of supraoptimal temperatures. In experiments where lettuce seeds were previously heated at 97°C for 8 or 16 h, seed germination as well as ethylene production were inhibited (Stewart and Freebairn 1969). Thus, the heat-treatment apparently inhibited ethylene production necessary for germination. In lettuce, ethylene synthesis or sensitivity to ethylene was decreased at high temperature during seed imbibition (Burdett 1972a,b; Dunlap and Morgan 1977a; Abeles 1986; Khan and Huang 1988). The high temperature appeared to inhibit the conversion of 1-aminocyclopropane-1carboxilic acid (ACC) to ethylene (Khan and Prusinski 1989). Gallardo et al. (1991) observed that high temperatures decreased the levels of free ACC. The requirement for endogenous ethylene production by lettuce seeds may be minimal for germination in a temperature range from 20° to 25°C; however, when lettuce seeds were imbibed directly at 35°C, ACC synthesis was not detectable (Huang and Khan 1992). The conversion of ACC to ethylene decreased as imbibition temperature of lettuce seed increased from 25° to 35°C (Prusinski and Khan 1990). In another

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species, apple, Yu et al. (1980) observed that an increase in temperature to 35°C caused an accumulation of endogenous ACC and ethylene production was greatly reduced. These authors suggested that conversion of ACC to ethylene was inhibited at high temperatures. The conversion of ACC to ethylene was more sensitive to high temperature inactivation than was ACC synthesis. Thus, the conversion of ACC to ethylene is the primary site of high temperature inactivation. Although high temperatures may inhibit ethylene production and consequently raise the threshold concentration of ethylene needed for lettuce seed germination, is the thermoinhibition caused by a reduction in the ability of seeds to produce ethylene? Early work by Abeles and Lonski (1969) reported that ethylene alone did not overcome thermodormancy in lettuce seeds. Ethylene action was limited to the early steps in germination, since treatment of dormant seeds with ethylene had no effect on germination (Abeles and Lonski 1969). Also, the reduction in lettuce germination with increasing temperature during imbibition was not due to a decrease in ethylene synthetic capacity of the seeds, since an increase in the rate of ethylene production was not a prerequisite of germination at supraoptimal temperatures (Burdett 1972a). Therefore, the mechanism of thermoinhibition in lettuce does not appear to be totally the result of reduced ethylene synthesis (Small et al. 1993). Ethylene had some effect on softening of the endosperm tissue; however, this effect was not correlated with the effect of ethylene as a germination promoter, and Abeles (1986) suggested that the action of ethylene in lettuce seed germination was the promotion of cell expansion in the embryonic hypocotyl. Dutta and Bradford (1994) also suggested that ethylene acts primarily on the embryo rather than on tissues enveloping it. They reported that ACC (via conversion to ethylene) extended the high temperature limit for lettuce seed germination by acting in the embryo to maintain a water potential sufficiently low to promote the initiation of growth at higher temperatures. It is evident that ethylene influences biochemical processes in seeds. Ketring (1977) suggested some possibilities for the mechanism of ethylene during seed germination: (1) interaction with growth regulators (e.g., ABA) at a basic level of metabolism, (2) a combination of growth promoters may be required to maximize a given physiological response, (3) a given physiological response is not specific for a single growth promoter, and (4) enzyme synthesis and secretion. Abscisic acid reduced ethylene production by dormant, imbibed peanut seeds and inhibited ethylene production and germination of after-ripened peanut seeds (Ketring and Morgan 1972). ABA also inhibited both ethylene production and germination of chick-pea seeds (Gallardo et al. 1992)

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and after-ripened apple embryos (Kepezynski et al. 1977). Exogenous ethylene reversed the inhibitory effects of ABA on dormant seeds (Ketring and Morgan 1972). The release of dormancy in lettuce seeds by ethylene, however, was not a result of removal of ABA-like compounds (Rao et al. 1975). Ethylene may interact with light or GA to promote germination at high temperature. For example, ethylene promoted dark germination only in the presence of GA (Dunlap and Morgan 1977a). Gibberellin slightly stimulated ethylene production in peanut seeds (Ketring and Morgan 1970). The action of GA in lettuce seeds could be through promotion of ethylene synthesis, or ethylene could stimulate germination by a separate mechanism (Stewart and Freebairn 1969). Burdett and Vidaver (1971) found that both ethylene and GA were necessary to stimulate germination of lettuce seeds at high temperature. However, GA did not promote lettuce germination by stimulating ethylene synthesis (Burdett and Vidaver 1971). In addition, the reversal of thermo dormancy in lettuce by ethylene occurred only when seeds were incubated in the light (Dunlap and Morgan 1977b). Heat treatment at 30°C of Spergula arvensis prevented ethylenepromoted germination in the dark, but the inhibition was reversed by red light (Olatoye and Hall 1972). The inability of lettuce seeds to germinate at supraoptimal temperatures was due neither to a rapid loss of far redabsorbing phytochrome nor to inadequate ethylene synthesis (Burdett 1972b). Moreover, Abeles and Lonski (1969) reported that the ability of ethylene to initiate a small increase in germination of lettuce seeds was apparently not through phytochrome control of ethylene production. It would appear that red light does not promote germination of lettuce seeds by influencing their ethylene production. Ethylene evolution from irradiated lettuce seeds began to increase 2 h prior to radicle protrusion, whereas the dark-incubated (nongerminating) seeds produced a low, constant amount of ethylene (Saini et al. 1989). Thus, endogenous ethylene was essential for the light-induced relief of thermoinhibition of lettuce seed germination. Under high osmotic conditions, the promotive effect of ethylene in lettuce seed germination was under the control of phytochrome (Negm and Smith 1978). Saini et al. (1986) reported that endogenous ethylene synthesis and action are essential for the alleviation of thermoinhibition of lettuce seeds by combinations of GA 3 , kinetin, and CO 2 , Negm et al. (1972) reported that CO 2 is required for ethylene action in overcoming thermodormancy in lettuce seeds, but ethylene does not enhance respiration. Cytokinins stimulate ethylene production by some seeds (Khan and Huang 1988). The relief of salt stress and thermoinhibition of lettuce

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seed germination by kinetin was accompanied by an enhancement in the pregermination ethylene production. When cytokinins and ethylene are used together, the stress of high temperature is alleviated in a synergistic fashion (Rao et al. 1975; Braun and Khan 1976). Part of the mechanism of thermoinhibition could be the failure of ATP content to reach a level sufficient to satisfy the requirements for germination at 38°C (Small et al. 1993). Ethephon used to break dormancy of Indian rice grass seeds resulted in increased ATP levels in the seeds after 24 h of germination (Tao et al. 1974). Ethephon also enhanced polyribosome formation by lettuce seeds during germination in light (Rao et al. 1975). A similar increase in the synthesis of ribosomes by ethephon and ethylene has been observed in other species (Marei and Romani 1971). Ethylene has also been reported to stimulate the synthesis of some enzymes (Cervantes et al. 1994; Hasegawa et al. 1995). Separation of cells due to the activation of cell wall-dismantling enzymes, such as endo-~­ 1,4-glucanases, was recently reported as an ethylene effect (Casadoro et al. 1998). Other cell wall-degrading enzymes show ethylene dependency, such as endopolygalacturonase, some isoforms of a-galactosidase, ~-arabinosidase, and galactanase (Pech et al. 1998). In some fruits, the climacteric ethylene rise was accompanied by an increase in a-galactosidase and ~-mannosidase activities (Moya et al. 1998). Thus, it is reasonable to assume that ethylene might overcome the inhibitory effect of high temperature on lettuce seed germination by activating cell wall enzymes responsible for endosperm digestion. The exact timing of ethylene production during seed germination must be determined to clarify this question. Under normal conditions, ethylene production by seeds begins immediately after the onset of imbibition and increases with time; however, the pattern of ethylene production by seeds during germination differs among species. For example, Takayanagi and Harrington (1971) found only one peak of ethylene production during germination of rape seeds, coinciding with the emergence and elongation of the radicle, cotyledon expansion, and splitting of the seed coat. In oat seeds, ethylene production was observed prior to radicle protrusion and gradually increased (Meheriuck and Spencer 1964). In lettuce, a major surge in ethylene evolution was observed at the time of visible radicle protrusion (Saini et al. 1986). A peak of ethylene production was also correlated with radicle protrusion (Fu and Yang 1983). According to Small et al. (1993), however, the major increase in ethylene evolution occurred after lettuce radicle protrusion. Thus, it appears that this question is still inconclusive.

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The embryo is the major site of ethylene production (Ketring and Morgan 1969; Esashi and Katoh 1975). Ethylene concentrations effective at stimulating seed germination of dormant seeds are in the range of 0.1-200 Jll L-1 depending on the species (Corbineau and Come 1995). For lettuce, 10 Jll L-1 of ethylene was reported as being close to optimal for promoting seed germination (Burdett and Vidaver 1971). The differential capacity of various cultivars to produce ethylene during stress generally corresponded with their inability to germinate at high temperature (Prusinski and Khan 1990). These authors reported that the genotypic variability in seed coat characteristics might influence ethylene production and performance of seeds under stressful conditions. The seed coat may reduce the performance of the growing embryo in two ways under stressful conditions: first, it may serve as a mechanical barrier and, second, it may create a hypoxic environment unfavorable for the conversion of ACC to ethylene (Prusinski and Khan 1990). Aminoethoxyvinilglycine (AVG) is an inhibitor of ethylene synthesis but has little influence on lettuce seed germination. For example, germination of lettuce seeds at 35°C (Khan and Prusinski 1989) or at 25°C (Huang and Khan 1992) was not inhibited by AVG even though the chemical inhibited ethylene production. These results suggest that the seeds may have a very low ethylene requirement, fulfilled by the residual ethylene synthesis occurring even in the presence of AVG (Saini et al. 1986). However, AVG and cobalt ions reduced germination in lettuce and the effect was overcome by the addition of ethylene (Abeles 1986). Also, inhibitors of ethylene action, such as silver and 2,5-norbornadiene, reduce germination and their effect can be reversed by added ethylene. Ethylene probably plays a role in lettuce seed germination at high temperature. Nascimento et al. (1998c) enhanced seed germination of thermosensitive 'Dark Green Boston' at 35°C by adding ACC during imbibition. Endo-~-mannanase activity was also increased, whereas AVG decreased enzyme activity and germination. They suggested that ethylene overcomes the inhibitory effect of high temperature in thermosensitive lettuce seeds by weakening of the endosperm due to increased endo-~-mannanaseactivity. D. Seed Priming

Priming consists of imbibing seeds in an osmotic solution for a specific period of time at a certain temperature. The osmoticum is usually a solution of inorganic salts or polyethylene glycol (PEG) in water. The concentration of osmoticum must be adjusted to a level high enough to inhibit radicle protrusion, thereby permitting prolonged metabolic

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reactions during the lag phase of water uptake (Heydecker and Gibbins 1978; Karssen et al. 1989). Successful priming depends on treatment duration, temperature, aeration, and the water potential of the priming solution; in addition, species, cultivar, seed quality, dehydration after priming, and seed storage conditions also affect the results (Heydecker and Gibbins 1978; Guedes 1979; Parera and Cantliffe 1994). Seed priming can shorten the period from sowing to seedling establishment and reduce the risk of seeds being exposed to adverse environmental and biotic factors in the field during this critical period (Khan et al. 1978). Bradford (1986) as well as Parera and Cantliffe (1994) detailed methods for successful osmotic priming in various species, including lettuce. In several cultivars of lettuce, a variety of osmotica successfully released thermo dormancy. For example, seeds of 'Valmaine' lettuce, which are sensitive to high temperature during germination, had significantly more germination at 30°C after seeds were primed in -5.1 bar K3P0 4 + PEG (Cantliffe et al. 1981). Thermodormancy in seeds of 'Mesa 659' was overcome by priming for 2 weeks in a solution of -8.4 bar PEG at 16°C (Khan et al. 1980/1981). Seeds of 'Minetto' were primed in an aerated solution of 1 % K 3P0 4 at 15°C for 20 h to bypass thermodormancy (Guedes and Cantliffe 1980). These results indicated that thermodormancy in lettuce can be successfully overcome by priming. Priming initiates several physiological and biochemical changes in lettuce seeds. For instance, it shortens the time before the onset of RNA and protein synthesis, increases the amount of RNA and protein synthesis, increases the activity of many enzymes, and accelerates metabolic rate (Mayer 1977; Khan et al. 1978). In lettuce seed, protein synthesis is enhanced during or following priming (Khan et al. 1978). Cycloheximide, an inhibitor of protein synthesis, inhibits the rate of protein synthesis and early germination in primed and nonprimed seeds of lettuce (Khan et al. 1980/1981). Khan et al. (1980/1981) suggested that improvement of seed germination by seed priming may be influenced by an enhancement in activities of protein synthesis. Cantliffe et al. (1984) suggested that the osmotic potential of the lettuce radicle should be increased so that cell elongation could occur, since this process was inhibited by high temperature. In primed lettuce seeds, increased accumulation of soluble amino nitrogen compounds and other hydrolytic products in the radicle tips could be a mechanism for overcoming thermodormancy. Georghiou et al. (1983) and Psaras et al. (1981) reported that, in the germinating seed, the endosperm cells opposing the radicle were highly vacuolated and storage materials were mobilized prior to radicle protrusion. However, the endosperm cells at

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the lateral and cotyledonary end remained unchanged. This was verified by Sung et al. (1999), wherein primed thermosensitive lettuce seeds germinated at 35°C and a similar "clearing" of endosperm cells specific to the micropylar end was observed in the primed seeds before radicle growth was initiated. Khan (1980/1981) observed that the rate, quality, and quantity of enzyme synthesis were influenced by priming. Karssen et al. (1989) has suggested that osmotic priming facilitated quick and uniform germination in tomato by stimulating cell wall extensibility in the radicle and weakening the endosperm cell walls. The embryo had expanded growth prior to the emergence of the radicle and the endosperm tissue enclosing the embryo restricted further hydration until weakening of its cell walls occurred so as to permit radicle emergence. Priming of tomato seeds may lead to more rapid germination by modifying these mechanisms. For example, a rapid expansion of embryo from primed tomato seeds was attributed to changing the extensibility in the radicle cell wall during priming (Groot et al. 1988). The 50% reduction in germination time was a function of the reduction of the mechanical resistance of the endosperm tissue in primed seed. In lettuce seeds, Karssen et al. (1989) noted that a comparison of cultivars indicated that the deeper the dormancy, the larger the effect of the enclosing structures on restraining radicle protrusion. Guedes et al. (1981) viewed morphological changes in 'Minetto' lettuce seeds during priming with a scanning electron microscope and observed that the outer layer of endosperm cells were gradually loosened after 9 h of priming. This loosening may be symptomatic of wall weakening and possibly is one of the mechanisms during seed priming involved in enhancing seed germination at high temperature. Sung et al. (1998a,b) were able to demonstrate a decrease in endosperm resistance force in lettuce genotypes that were primed. Puncture tests were conducted to measure the force required to penetrate the endosperm at the micropylar tip of five lettuce genotypes. Puncture force declined as imbibition time increased in primed and nonprimed seed of all genotypes except thermosensitive 'Dark Green Boston'. The force required for penetration of the endosperm in nonprimed seeds of 'Dark Green Boston' increased with time and that for primed seeds was reduced. In thermosensitive 'Valmaine', penetration force remained similar in both primed and nonprimed seeds. In three thermotolerant genotypes, penetration force decreased in primed and nonprimed seeds during imbibition. Priming lowered the initial force necessary to penetrate the endosperm. Nascimento et al. (1998a,b) found that during priming of a thermosensitive lettuce genotype endo-B-mannanase activity was induced after 24 h. After drying and immediately upon reimbibition, endo-B-

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mannanase levels were high, leading to rapid germination at 35°C. Thus, a connection between priming, thermotolerance and endo-~-mannanase activity has finally been established.

VII. CHANGES IN THE EMBRYO AND ENDOSPERM DURING GERMINATION A. Embryonic Growth Potential Growth potential of the embryo is measured by the osmotic potential that limits seed germination to 50% (Scheibe and Lang 1965). A negative water potential in embryonic cells is essential for seed germination and is the principal driving force for cell expansion. Cells change osmotic potential (via solute production) or pressure potential (via increased wall loosening) to change their water potential (Ray et al. 1972). Solute accumulation prior to radicle growth can lower the osmotic potential and generate sufficient turgor pressure to allow the embryo to penetrate the endosperm barrier. The ability of the embryo to absorb water from its environment and to initiate growth is dependent on the osmotic potential of its cells (Nabors and Lang 1971a,b; Takeba 1980a). The time taken for a seed to initiate radicle growth is inversely proportional to the difference between the embryonic water potential during imbibition and its threshold water potential which prevents radicle growth: the greater the differences, the sooner radicle growth will begin after imbibition (Bradford 1990). As mentioned, germination of lettuce seed can be controlled by light, temperature, hormones, priming, or a combination of these. All can influence the growth potential of the embryonic axes. Researchers have suggested that phytochrome control of germination in positively photoblastic lettuce seed is mediated through increasing the growth potential of the embryo, measured by the osmotic potential required to achieve 50% seed germination (Scheibe and Lang 1965; Nabors and Lang 1971b). Nabors and Lang (1971b) measured the growth potential of the embryo using mannitol and polyethylene glycol as osmotica and determined that the force needed for the radicle to penetrate the seed coat was at an osmotic potential of 0.16 to 0.38 M mannitol. In osmotica, light-treated embryos of positively photoblastic seeds developed a water potential 0.3 M lower than that of dark-treated embryos, which was sufficient for seed germination. The phytochrome-mediated growth increase in the embryonic axes is an integrated function of the cells: increased wall loosening is coupled with rising osmotic constituents (Carpita et al. 1979a).

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Increasing the growth potential in the embryo can at least partially overcome the resistance offered by the endosperm (Nabors and Lang 1971b). Carpita et al. (1979b) indicated that the growth of embryonic axes and the degradation of stored reserves were not different in red- and far-red-treated seeds. However, axes of red-treated seeds increased their secretion of H+ and their uptake of K+ and Na+ more than those of far-red-treated seeds. A possible explanation for this increased secretion was that a phytochrome-stimulated proton pump initiated water potential changes that allowed the embryos to relieve the mechanical restraint of the seed coverings. Takeba (1980a) suggested that changes in the growth potential of the embryonic axes could be best explained by changes in the amount of an osmotic substance in the axes. An accumulation of glutamate (Glu) and glutamine (GIn) in germinating seeds indicated that Glu and GIn possibly acted as osmotic substances for germination of 'New York' lettuce seeds. Takeba (1980b) reported that accumulation of Glu and GIn took place only in the tips of growing axes during the first 24 h of imbibition at 18°C. The amount of Glu and GIn accumulated was enough to account for increasing the growth potential of the embryonic axes. Since an increase in the osmotic potential of the embryonic axes is a necessary step for growth, it would be important to determine precisely whether an increase in substances in the growing axes is the cause or the result of growth. Glutamine is formed from Glu and ammonia by the action of glutamine synthetase (GS) in plant tissues. The activity of GS is not only affected by temperature but also has been demonstrated to be mediated by phytochrome (Takeba 1980d). Both red light and GA can increase the level of GS in seeds before the initiation of axis elongation. This increase is completely suppressed by cycloheximide (Takeba 1983b, 1984). Sakamoto et al. (1990) demonstrated that red light increased the translatable mRNA for GS in lettuce seeds. It is assumed that GS is synthesized de novo. Radicle protrusion in lettuce results from cell elongation rather than cell division (Haber and Luippold 1960a; Bewley and Black 1994). The different sequences of the inceptions of cell division and cell expansion are dependent on temperature, indicating different mechanisms for germination at different temperatures (Haber and Luippold 1960a). For example, at high temperature, mitoses may occur before cell expansion. A negative water potential in the cells of the embryo is essential for seed germination and is the principal force for cell expansion. The ability of the embryo to absorb water and to initiate growth is dependent on the osmotic potential of its cells (Nabors and Lang 1971a,b; Takeba 1980b).

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The differences in water potential in the embryonic axis from the accumulation of osmotically active compounds (Carpita et al. 1979; Takeba 1980b) was considered sufficient for radicle protrusion and embryonic axis elongation (Nabors and Lang 1971a,b; Bewley and Halmer 1980/81). Thus, increasing the growth potential in the embryo, in fact, might be a way to overcome the mechanical resistance of endosperm. Three possible causes for initiation of radicle growth have been suggested: (1) solute accumulation (which lowers the osmotic potential in the radicle cells and increases embryo turgor), (2) cell wall loosening (which increases extensibility) of the radicle, or (3) weakening of the tissues surrounding the radicle tip, thus allowing it to elongate (Bewley and Black 1994; Bradford 1995). In lettuce, two different mechanisms for overcoming the mechanical restraint of the endosperm at high temperature have been postulated: (1) the mechanical force of the growing embryo, which exerts pressure against the endosperm envelope, and (2) weakening of the endosperm tissue. It is possible that both mechanisms operate together in allowing lettuce seed to germinate. B. Endosperm Weakening

Takeba and Matsubara (1979) suggested that at high temperature, lettuce seeds were unable to germinate because the embryonic growth potential was insufficient to overcome the restraining force of the seed coat. However, thermoinhibition of lettuce seed germination is not due to a failure of the embryo to absorb water or develop sufficient turgor (Bradford 1990; Weges et al. 1991). In a study using chlorine treatments during lettuce seed imbibition, Pavlista and Haber (1970) observed considerable embryo elongation within the confines of the endosperm without protrusion of the radicle. Thus, other mechanisms might be involved in radicle protrusion at high temperature. Structural and morphological changes occurring in the endosperm prior to lettuce seed germination (Psaras et al. 1981; Georghiou et al. 1983) suggest another hypothesis for radicle protrusion. Georghiou et al. (1983) observed structural changes opposite the radicle end in lettuce seeds germinating under red light. Modification of the cytoplasm of endosperm cells in the micropylar region was observed before radicle protrusion, and these changes were a prerequisite for the completion of lettuce seed germination (Georghiou et al. 1983). The absence of structural changes in the endosperm cells at high temperature could provide the basis for the induction of secondary dormancy (Georghiou et al. 1983). Takeba and Matsubara (1977) found that small fat bodies disappeared from lettuce seeds during early stages of imbibition at 20°C, but

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not at 35°C. Phytochrome destruction and/or reversion to an inactive form (Toole 1973), or the inactivation of a thermolabile factor (Takeba and Matsubara 1976), could explain the absence of structural changes in endosperm cells at high temperatures. In addition, Pavlista and Valdovinos (1978) observed disruptions in lettuce endosperm before germination occurred. In an extensive study, Sung et al. (1999) verified that endosperm cells in the micropylar region were structurally altered during germination at high temperature. Changes included separation of the endosperm layer from the integument, depletion of protein bodies, formation of empty vacuoles, cytoplasm condensation, and rupture of the endosperm cell walls with subsequent embryo growth toward this opening. Sung et al. (1999) concluded that weakening of the endosperm layer was a required prerequisite to radicle protrusion. Weakening of the endosperm has also been reported in other species (Watkins and Cantliffe 1983; Karssen et al. 1989; Dahal and Bradford 1990; Groot and Karssen 1992). In the last few years, evidence has emerged showing that some seeds germinate via enzymic degradation of the endosperm. Weakening of the endosperm in the micropylar region prior to radicle protrusion was also observed in anatomical studies of pepper (Watkins et al. 1985) and Datura ferox (Sanchez et al. 1990). Evidence that weakening of the endosperm could be a prerequisite to radicle protrusion was provided for tomato seeds (Groot et al. 1988; Ni and Bradford 1993; Leviatov et al. 1995; Nomaguchi et al. 1995). In these species, endosperm weakening was suggested to be mediated by endo-~-mannanase.The increase in endo-~-mannanaseactivity was linearly correlated with decreasing resistance of endosperm to penetration (Hilhorst and Karssen 1992). However, endo-~-mannanaseactivity in the endosperm cap of tomato seeds was not sufficient to permit seeds to complete germination (Toorop et al. 1996). Production of hydrolyases within the endosperm and secretion into the cell walls, causing weakening and allowing the radicle to protrude, has been proposed for other species (Black 1996; Bewley 1997a). In lettuce, Ikuma and Thimann (1963b) proposed that the action of an enzyme produced by the embryo enables the radicle tip to penetrate through the restricting tissues. Possible chemical weakening of the endosperm near the radicle end was also suggested by Pavlista and Haber (1970). Other enzymes, including cellulase, pectinase, and pentosanase, were effective at promoting germination of dormant seeds of lettuce (Ikuma and Thimann 1963b). Increased activity of carboxymethylesterase prior to endosperm degradation has also been reported (Pavlista and Valdovinos 1975). However, Bewley (1997b) has claimed that lettuce seeds do not produce cellulase. Lettuce endosperm cell

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walls are composed largely of galactomannans (Halmer et al. 1975). Thus, endo-~-mannanaseis probably the enzyme most likely involved in the cell wall degradation at the micropylar end, leading to endosperm weakening and subsequent radicle protrusion.

c.

Endo-fl-mannanase

The time of appearance of endo-~-mannanase(EC 3.2.1.78) activity during germination has been largely debated. In some studies, mannanase activity detected in dry seeds or after the first hours of imbibition might have been due to activation of preexisting enzyme in lettuce (Dutta et al. 1994) or the retention of enzyme produced during seed development (Hilhorst and Downie 1995). Thus, some researchers have speculated that growth conditions during tomato seed development might affect endo-~-mannanase levels. Imbibed seeds of fenugreek and carob did not exhibit endo-~-mannanaseactivity until after completion of germination (Reid et al. 1977; Spyropoulos and Reid 1988; Kontos and Spyropoulos 1995). In lettuce, early studies detected mannan hydrolysis only as a post-germinative event (e.g., after radicle protrusion) (Halmer et al. 1975, 1976; Bewley and Halmer 1980/81). For example, mannanase activity increased about 100-fold in all regions of the endosperm during 15 h following germination (Halmer et al. 1978). Recently, Dutta et al. (1997) reported that a cell-waIl-bound endo-~-mannanase was expressed in lettuce seed endosperm prior to radicle protrusion and was regulated by the same conditions that govern seed germination. These authors suggested that this enzyme is likely to be involved in the weakening of the endosperm cell walls. Endosperm cell walls exhibit endo-hydrolase activity (Dutta et al. 1994). These endo-hydrolases are highly substrate specific (Huber and Nevins 1977). Endo-~-mannanase is targeted toward the mannan-rich endosperm cell walls (Halmer et al. 1975, 1976). Experiments have shown that endo-~-mannanase is synthesized de novo (Bewley and Halmer 1980/81; Halmer 1989). Estimates ofMr by chromatography and electrophoresis were 46,000; endo-~-mannanase was a major protein secreted by lettuce endosperm (RaImer 1989). Two-dimensional PAGE indicated that mannanase exists as three isoforms, with pIs between 4.75 and 4.9. Maximum enzyme activity was achieved at pH 5.0 (Downie et al. 1994; Dutta et al. 1997). However, low enzyme activity represented by the cell wall has been reported in lettuce (Dutta et al. 1994, 1997). This low activity may be due to the limited number and specific nature of the bonds susceptible to catalysis during autolysis within lettuce endosperm (Dutta et al. 1997). Also, endo-~-mannanaseis tightly bound to the endosperm

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cell walls. More structural details of matrix polysaccharides and their linkages between the polymers of endosperm cell walls need to be known. Lettuce genotypes differ in endo-~-mannanase abundance and isozyme complements (Dirk et al. 1995). No isozymes could be detected when the lettuce embryo and endosperm were separated (Dirk et al. 1995). Similar results were found in tomato seeds, where fewer isoforms were produced when the embryo and endosperm were dissected prior to completion of germination and incubated separately (Voight and Bewley 1996). The differences in the pI values of the isozymes in the seeds and plant parts of several crops suggests that endo-~-mannanase is variable in its amino acid composition, or in the glycosylation patterns, or both (Dirk et al. 1995). A cDNA encoding a (l,4)-~-mannanase from endosperm of germinated tomato seeds has been recently isolated and characterized (Bewley et al. 1997). Also, in tomato, multiple isozymes of endo-~-mannanasewere reported in germinating seeds (Dirk et al. 1995; Nonogaki et al. 1995; Toorop et al. 1996; Voigt and Bewley 1996). In this species, the enzyme isoforms produced in the micropylar and lateral regions were different (Nonogaki and Morohashi 1996; Voigt and Bewley 1996). It is possible that only specific isozyme(s) are regulated in a quantitative manner with germination rates (Dahal et al. 1997). Multiple forms of endo-~-mannanasewere also isolated from the endosperm of different legume seeds (McCleary and Matheson 1975). For instance, several isozymes of mannanase were detected in the endosperms of fenugreek and carob (Kontos et al. 1996). Cell walls isolated from lettuce endosperm are capable of autohydrolytic activity, indicating the presence of cell-waIl-bound hydrolases (Dutta et al. 1994). The endosperm is the site ofmannanase production, although it is controlled by the embryo (Balmer and Bewley 1979). In fenugreek, Spyropoulos and Reid (1985) and Malek and Bewley (1991) also reported the involvement of the embryonic axis in the regulation of endosperm mobilization. Mannanase production is subject to inhibition. The endosperm may contain some inhibitor(s) that needs to be removed in order to have enzyme synthesis. Some studies have demonstrated that the volume of buffer used during incubation of isolated endosperms affects the production of endo-~-mannanase. For example, isolated endosperms incubated in large volume (1 mL) produced substantial quantities of mannanase, whereas those incubated in a small volume (20 ~L) did not (Dulson et al. 1988; Dulson and Bewley 1989). Dulson et al. (1988) concluded that endogenous ABA regulated mannanase production in isolated lettuce endosperms. In another study,

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treatment with ABA suppressed both lettuce germination and endo-~­ mannanase activity (Dutta et al. 1997). ABA has also been reported to inhibit both germination and endo-~-mannanase activity in seeds of tomato (Hilhorst and Downie 1995; Nomaguchi et al. 1995; Nonogaki and Morohashi 1996; Voigt and Bewley 1996) and fenugreek (Malek and Bewley 1991). Endo-~-mannanaseactivity inhibition by ABA in tomato seeds was dependent on tissue localization. ABA was incapable of inhibiting endo-~-mannanase activity in the endosperm cap, but did inhibit activity in the lateral endosperm (Toorop et al. 1996). However, Dahal et al. (1997) reported that ABA did not act by inhibiting the induction of mannanase activity in tomato seeds and concluded that ABA can prevent endosperm weakening in the absence of the embryo. Endo-~-mannanaseactivity is also regulated by environmental factors. Red light-treated seeds of lettuce produced high amounts of endo-~­ mannanase but only after radicle protrusion (Bewley and Halmer 1980/81). Conversely, seeds imbibed in the dark produced little mannanase and did not germinate (Halmer and Bewley 1979). Endo-~­ mannanase was strongly promoted in the micropylar region of Datura seed endosperm under red light (Sanchez and Miguel 1997). Imbibition of lettuce (Dutta et al. 1997) and tomato (Leviatov et al. 1995) seeds, respectively, at high or at low temperature, decreased enzyme activity. Incubation of seeds of 'Pacific' lettuce at high temperatures resulted in total prevention of germination and almost complete suppression of endo-~-mannanase(Dutta et al. 1997). Temperature might reduce enzyme synthesis inhibiting some factors involved in the regulation of endo-~-mannanase production by the endosperm on those species. Development of mannanase activity in tomato seeds was temperature-dependent (Dahal et al. 1997). Moreover, the optimum temperature for enzyme activity was around 35-40°C (Groot et al. 1988). Leviatov et al. (1995) verified that the expression of increasing endo~-mannanase activity in the micropylar region of the endosperm of tomato at low temperature was characteristic of cold-tolerant germinating lines. In this study, there was a positive relationship between germination ability at low temperatures and endo-~-mannanaseactivity in the six progeny lines. In a different study, Downie et al. (1997) reported increased endo-~-mannanaseactivity in white spruce seeds during dormancy alleviation by chilling. Gibberellins also affect endo-~-mannanase synthesis. Ikuma and Thimann (1 963a) suggested that germination of lettuce seeds promoted by gibberellin was due to a stimulation or activation of hydrolytic enzymes. Treatment with GA was accompanied by significant enhancement of

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endo-B-mannanase activity in lettuce seeds (RaImer and Bewley 1979) and Datura (Sanchez and Miguel 1997). In the later study, hydrolase activity and germination were blocked by paclobutrazol, an inhibitor of GA synthesis. Using GA-deficient gib-1 mutant tomato, Groot et al. (1988) concluded that the weakening of endosperm prior to radicle protrusion was mediated by a GA-induced enzymatic degradation of the mannan-rich cell walls. In lettuce, alleviation of thermoinhibition with GA was accompanied by significant enhancement of mannanase activity (Dutta et al. 1997). It has been well documented that seed priming, an osmotic treatment, circumvents thermodormancy of lettuce seeds and allows germination at higher temperatures (Guedes and Cantliffe 1980; Cantliffe et al. 1981; Khan 1980/81; Wurr and Fellows 1984; Valdes et al. 1985). Increased activity of endo-B-mannanase (Karssen et al. 1989) and galactomannan hydrolyzing enzyme (Nonogaki et al. 1992) was observed during seed priming of tomato and lettuce (Nascimento et al. 1998b). Whether endo-B-mannanase is in itself sufficient for germination to be completed has been an open question. In tomato, endo-B-mannanase can increase in the absence of radicle protrusion, or vice-versa (Bewley 1997a). In addition, the amount of endo-B-mannanase necessary to ensure endosperm weakening has not been determined. Thus, although endosperm weakening is likely to be essential for seeds to complete germination, how this is achieved remains unknown. In lettuce seeds, Dutta et al. (1994) verified during autolysis a substantial release of arabinose and galactose, in addition to mannose. Thus, endo-B-mannanase may not be rate limiting and additional wall-hydrolyzing enzymes other than endoB-mannanase may be involved in the lettuce endosperm weakening. Recent work reported by Nascimento et al. (1998a,b,c) has more closely associated endo-B-mannanase with the ability of lettuce seeds to germinate at 35°C. In their studies, thermotolerant genotypes produced more enzyme than thermosensitive genotypes. The enzyme could be detected before radicle protrusion and was produced in quantity during priming of a thermosensitive lettuce genotype, 'Dark Green Boston'. Further, lettuce seeds that were matured under high temperature germinated better and produced more endo-B-mannanase at 35°C than seeds matured under lower temperature. Finally, an association was established between the ability of ethylene to overcome the inhibitory effect of high temperature in thermosensitive seeds and weakening of the endosperm due to increased endo-B-mannanase activity. Previously, Sung et al. (1998a,b and 1999) closely established the connection between endosperm weakening and the ability of these same genotypes to germinate at 35°C.

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D. Other Enzymes Associated with Endosperm Weakening and Seed Germination Galactomannans consists of a backbone of ~-1 ,4-linked mannose subunits with ~-1,6-galactose side chains. This polymer requires three different enzymes for its complete mobilization: endo-~-mannanase, a-galactosidase (EC 3.2.1.22)' and -~-mannanosidase(~-mannosidaseor exo-~-mannanase,EC 3.2.1.25) (Bewley et al. 1983; Ouellette and Bewley 1986). These enzymes may originate in different regions of the seed, and they all behave differently during germination. The ~-1 ,4-linked mannan backbone is hydrolyzed via the action of endo-~-mannanase; and the ~-1,6-galactose side chains are released by a-galactosidase. ~­ mannosidase further catalyzes the hydrolysis of the oligomannans produced by endo-~-mannanase.An exo-polysaccharase, a-galactosidase, and endo-~-mannanaseact cooperatively to effect the hydrolysis of the lettuce endosperm cell walls (Leung and Bewley 1983). ~-mannosidase is another enzyme that occurs during lettuce seed germination but is present exclusively within the cotyledons (Bewley et al. 1983). The likely substrates for this enzyme are the products of endosperm cell wall mobilization, mannobiose and mannotriose, which diffuse to the cotyledons (Bewley and Balmer 1980/81; Ouellette and Bewley 1986). The synthesis of ~-mannosidase could be induced by its substrate, or by some factor(s) released from the degrading endosperm (Bewley and Balmer 1980/81). Glycosidases are not believed to be responsible for in situ weakening of wall structure (Dutta et al. 1997). The galactomannan polysaccharides from different species have different proportions of Dgalactose and D-mannose, but always consist of a ~ (1-4) mannan backbone with single D-galactose branches linked ~ (1-6), (Smith and Montgomery 1959). The D-galactose content can vary from 10 to 50% according to the species (McCleary and Matheson 1975). ~-mannanase has limited ability to hydrolyze galactomannans with high galactose contents (McCleary and Matheson 1975). Seeds containing these galactomannans had very active a-galactosidases. In dry carob seeds there is substantial activity of a-galactosidase (Kontos and Spyropoulos 1995), while in Senna occidentalis (Edwards et al. 1992), a-galactosidase is present during seed development. Thus, it has been suggested that in developing seeds the action of this enzyme reduces the high galactose/mannose ratio of the newly synthesized galactomannan (Kontos and Spyropoulos 1996). Other cell wall enzymes with putative functions in seed germination have also been reported. In muskmelon, besides endo-~-mannanase, endo-1 ,4-~-glucanase activity was observed about 5 h before radicle protrusion (Welbaum and Wang 1997). ~-1,3-

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glucanase was reported in tobacco seeds, increasing in the micropylar region of the endosperm before radicle protrusion (Leubner-Metzger et al. 1996). Expression of a putative arabinosidase mRNA has also been detected in tomato seeds (Dahal et al. 1997). All these studies show evidence for enzymatic mechanisms for weakening the tissues constraining the embryo, allowing the radicle to protrude from the seed. Making inferences from those studies, it is reasonable to match the cell wall composition of the restraining tissue, in this case, lettuce endosperm, with the probable major enzyme involved, in this case, endo-~-mannanase. VIII. SUMMARY AND CONCLUSIONS

The lettuce seed is unique in that its germination appears to be controlled by so many factors. At temperatures close to ±5.0°C optimum, germination is less affected by environmental factors, hormones, or priming. The loosening and breakdown of the endosperm, expansion and growth of the embryonic axis, and the growth of the radicle all occur in an orderly fashion. However, as temperature begins to increase above the optimum, germination becomes less orderly and fails to proceed if too high a temperature is perceived by the seed. The causes of thermoinhibition and thermodormancy have been studied for many decades. Hundreds of publications have been dedicated to reasons why lettuce seed will not germinate and to conditions or treatments that will allow it to germinate. In this review we have made a case for specific treatments that completely bypass dormancy in lettuce, namely, maturing seeds at high temperature, use of thermotolerant genotypes, seed priming, and the use of certain hormones such as ethylene. We have also tried to make a case for how these factors might control germination, i.e. by controlling the production of endo-~-mannanaseat the micropylar end of the seed before radicle expansion. Recently, we have published data that clearly shows more endo-~­ mannanase activity in primed lettuce seeds than non-primed seeds (Nascimento et al. 1998a,b) and more endo-~-mannanaseactivity in seeds matured at 30°120°C than at 20°/10° day: night temperature (Nascimento et al. 1998a). Increased endo-~-mannanaseactivity correlated with the ability of the seed to germinate at high temperature. Also, endo-~­ mannanase activity could be clearly detected before longitudinal radicle expansion at the micropylar end of the seed. Further providing ACC during germination at 35°C increased endo-~-mannanaseactivity and germination (Nascimento et al. 1998c). Thermotolerant genotypes produced more endo-~-mannasethan thermosensitive genotypes.

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The literature provides a clear view of the ability of the endosperm to restrict germination at high temperature. Factors that control or contribute to the ability of a lettuce seed to germinate at high temperature are most likely the result of control of the endo-~-mannanasesystem needed for cell wall loosening at the micropylar end. This may also be a contributing factor causing a restriction of embryo growth potential. Ethylene may play an important role in controlling this process. The exact role for ethylene in germination is unknown, but ethylene evolution, addition, and/or the addition of ACC have been associated with increased endo-~-mannanaseactivity and the ability of lettuce seed to germinate at high temperature. This is certainly an intriguing topic for further investigation. LITERATURE CITED Abeles, F. B. 1986. Role of ethylene in Lactuca sativa cv 'Grand Rapids' seed germination. Plant Physiol. 81:780-787. Abeles, F. B., and J. Lonski. 1969. Stimulation of lettuce seed germination by ethylene. Plant Physiol. 44:277-280. Abeles, F. B., P. W. Morgan, and M. E. Saltveit, Jr. 1992. Ethylene in plant biology. 2nd ed. Academic Press, San Diego, CA. Ashraf, M., and C. M. Bray. 1993. DNA synthesis in osmoprimed leek (Allium porrum L.) seeds and evidence for repair and replication. Seed Sci. Res. 3:15-23. Bewley, J. D. 1997a. Breaking down the walls: A role for endo-~-mannanase in release from seed dormancy? Trends Plant Sci. 2:464-469. Bewley, J. D. 1997b. Seed germination and dormancy. Plant Cell 9:1055-1066. Bewley, J. D., and M. Black. 1994. Seeds: Physiology of development and germination. 2nd ed. Plenum Press, New York. Bewley, J. D., R. A. Burton, Y. Morohashi, and Y. Fincher. 1997. Molecular cloning of a cDNA encoding a (1-4)-~-mannan endohydrolase from the seeds of germinated tomato (Lycopersicon esculentum). Planta 203:454--459. Bewley, J. D., and D. W. Fountain. 1972. A distinction between the actions of abscisic acid, gibberellic acid, and cytokinins in light-sensitive lettuce seed. Planta 102:368-371. Bewley, J. D., and P. Halmer. 1980/81. Embryo-endosperm interactions in the hydrolysis of lettuce seed reserves. Israel J. Bot. 29:118-132. Bewley, J. D., D. W. M. Leung, and F. B. Ouellette. 1983. The cooperative role of endo-~­ mannanase, ~-mannosidaseand a-galactosidase in the mobilization of endosperm cell wall hemicelluloses of germinated lettuce seed. In: C. Nozzolillo, P. J. Lea, and F. A. Loewus (eds.), Rec. Adv. Phytochemistry, 17. Plenum Press, New York. Black, M. 1996. Liberating the radicle: A case for softening-up. Seed Sci. Res. 6:39-42. Borthwick, H. A., S. B. Hendericks, M. W. Parker, E. H. Toole, and V. K. Toole. 1952. A reversible photoreaction controlling seed germination. Proc. Nat. Acad. Sci. (USA) 38:662-666. Borthwick, H. A., and W. W. Robbins. 1928. Lettuce seed and its germination. Hilgardia 3:275-304.

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6 Viroid Dwarfing for High Density Citrus Plantings Ronald f. Hutton NSW Agriculture Yanco Agricultural Institute Yanco. NSW 2703 Australia Patricia Broadbent NSW Agriculture Elizabeth Macarthur Agricultural Institute Menangle. NSW 2568 Australia Kenneth B. Bevington NSW Agriculture Agricultural Research and Advisory Station Dareton. NSW 2717 Australia

I. Introduction A. Intensive Planting Strategies in Citrus B. Viroid Dwarfing II. Causal Agent A. Detection of Viroids B. Mode of Action of Viroids in Causing Dwarfing C. Risks in Using a Pathogen for Tree Size Control III. Use of Viroids for Tree Size Control A. Rootstock B. Scion C. Time of Inoculation IV. Vegetative Growth A. Tree Uniformity B. Tree Growth Rates and Canopy Size C. Shoot Growth Patterns

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V. Reproductive Growth A. Flowering B. Fruit Growth C. Yield D. Fruit Quality VI. Intensive Viroid-Dwarfed Plantings VII. Economic Considerations VIII. Management IX. Summary and Conclusions Literature Cited

I. INTRODUCTION

A. Intensive Planting Strategies in Citrus The potential for dwarfing of citrus using viroids to develop a predictable range of tree size control for use in more intensive citrus plantings was pioneered in Australia (Long et al. 1972). This review summarizes 30 years of Australian research on viroid dwarfing of citrus in high density plantings undertaken by NSW Agriculture at Gosford (33°S, 151°E) on the Central coast, at Yanco (34°S, 146°E) in the Murrumbidgee Irrigation Areas (MIA) and at Dareton (34.1°S, 142°E) in the Sunraysia region of New South Wales, Australia and relates this work to some more recent international studies in Israel, Southern Africa, and the USA. The adoption of more intensive planting strategies utilizing higher planting densities and smaller-sized trees is considered essential to ensure the continued viability of citrus production. Interest in higher density planting is being driven by the necessity to improve early returns on investment and to make more efficient use of resource inputs. Smaller trees are being sought to reduce harvesting costs. The development of suitable intensive planting systems would also facilitate the adoption of new cultivars which would enable the industry to become more responsive to changing market demands. Uniform tree size control is critical to the success of high density citrus plantings to prevent overcrowding and loss of orchard productivity. For any planting system to be practical, it must be possible to maintain trees within their allotted space in the orchard without adversely affecting production. 'Carrizo' and 'Troyer' citranges (Citrus sinensis (1.) Osbeck x Poncirus trifoliata (1.) Raf.) are well-adapted to the sandy soils of the Sunraysia citrus growing areas of the Murray Valley of Australia and are the predominant rootstocks used for new plantings in the region. P. trifoliata is the best suited rootstock for the heavier soils of the MIA and together with the citrange rootstocks, are the principal rootstocks

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used for citrus in the inland production areas. Trees grown on these rootstocks are high-yielding and produce good quality fruit. However, tree growth is generally quite vigorous and trees have the capacity to attain a large size at maturity, especially on citrange (Bevington and Cullis 1990). Research in California has shown that vigorous stock/scion combinations are not suited to high density plantings as the excessive amount of pruning required to maintain trees within their allotted space at close spacings is detrimental to fruit production (Boswell et al. 1970). Trees on 'Carrizo' and 'Troyer' citranges are commonly planted at low to medium densities (400-600 trees/hal in Australia. Although such orchards are highly productive at maturity, early yields are low and it takes a relatively long period of time for the orchard to reach peak production. Different means have been considered for achieving smaller trees for high density orchards, including the use of naturally stunted scions, pruning and hedging, root restriction, dwarfing rootstocks, angle planting, growth retarding chemicals, and viroid inoculation. Only a few of these have been found applicable for extensive use in citrus production (Mendel 1969; Castle 1978; Wheaton et al. 1978; Bitters et al. 1979; Cohen 1981; Hutton 1986; Roose 1986). Use of the dwarfing trifoliate rootstock 'Flying Dragon' is not practical in cool climates, since tree growth is too slow (Golomb 1988; Hutton 1991) and rate of canopy development is insufficient. Use of smaller trees grown in hedgerows at higher densities to increase productivity in citrus has been reported where vegetative vigor has been controlled with the use of hedging (Wheaton et al. 1978; Castle 1985). Recent reports from Israel indicate that restricted wetted zone drip irrigation may also be an effective means of reducing tree vigor in high density citrus plantings by limiting the size of the effective root zone (Golomb 1988). B. Viroid Dwarfing In the 1930s and 1940s, the Australian citrus industry was decimated by Phytophthora root rot affecting trees on sweet orange and rough lemon stocks (Fraser 1949). P. trifoliata was the only rootstock available that was resistant to Phytophthora citrophthora Sm. & Sm., but it had a reputation as an unreliable stock (Benton et al. 1950). Some cultivars on this stock were dwarfed and unthrifty, with obvious bark scaling of the stock, a symptom described in Australia as scaly butt (Fraser and Levitt 1959) and in California as exocortis (Fawcett and Klotz 1938). Other trees had varying degrees of dwarfing. In these trees, a depression in growth rate occurred at three or four years after planting and by eight to ten years

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the size differential between normal and dwarfed trees was pronounced (Fraser et al. 1961). In dwarfed trees, in the absence of butt scaling, the stock was broader than the scion trunk and often predominantly shouldered or benched at the bud union, in contrast to normal trees in which the stock was fluted and evenly expanding from the bud union to the crown roots. The bark of the stocks of dwarfed trees varied from smooth to thin flaky bark scales or persistent corky pustules arranged singly or grouped or in horizontal bands (Fraser and Broadbent 1979). A high proportion of dwarfed trees had an indented ring at the bud union, visible when the bark was removed. Both the dwarfing associated with exocortis and non-scaling dwarfing were shown to be transmissible by bud inoculation and perpetuated by direct propagation (Benton et al. 1950; Fraser et al. 1961). Propagations of 'Bellamy' nucellar navel (G. sinensis (L.) Osbeck) orange on P. trifoliata stock were inoculated as nursery trees with budwood from nine dwarfing, non-scaling budlines [sweet oranges (G. sinensis (L.) Osbeck)-'Washington' navel (3531, 3532, 3533, 3534, 3535, 3536) and 'Valencia' (3537) or 'Marsh' grapefruit (G. paradisi Macf.) (3538, 3539)]. Trees of each propagation were planted in 1955 at the Horticultural Research and Advisory Station at Gosford (Somersby section). Dwarfing was evident within 5 years. Today, 43 years later, the trees remain healthy, producing good crops of high-quality fruit. Inoculum from the selections with the best yield per tree size ratio was used for statistically designed dwarfing trials at the Yanco Agricultural Institute, planted in 1961,1963,1964, and 1986, and at the Horticultural Research and Advisory Station at Dareton in 1966 and 1987. In these trials, we have examined the efficiency of crop production in relation to tree size, dwarfing in relation to rootstocks and spacing, irrigation, and other management requirements. II. CAUSAL AGENT A. Detection of Viroids The range of bark scaling and dwarfing symptoms encountered on P. trifoliata in the field was attributed to strains of the exocortis pathogen (Fraser and Levitt 1959; Calavan and Weathers 1961; Weathers and Calavan 1961), which were distributed unevenly and independently in the tree and, therefore, transmitted unevenly to progeny trees (Fraser and Broadbent 1979). The development of'Etrog' citron (G. medica L.) as an indicator for exocortis (Calavan et al. 1964) enabled the classification of field sources as mild, moderate, and severe based on the degree of plant

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stunting, leaf epinasty, and corking of the midrib. Consequently, Broadbent et al. (1971) attributed graft transmissible dwarfing to mild exocortis, based on symptom expression in 'Etrog' citron. When the severe bark scaling isolates were transmitted to Gynura aurantiaca DC, a small infectious RNA molecule (citrus exocortis viroid, CEVd) was isolated (Semancik and Weathers 1972). Similar RNA molecules could not be detected in extracts of citron infected with mild strains or transmitted to Gynura aurantiaca (Kapur et al. 1974; Schwinghamer and Broadbent 1987; Broadbent et al. 1988). It was not until the development and application of the more sensitive sequential polyacrylamide gel electrophoresis (sPAGE) procedures for viroid detection (Rivera-Bustamante et al. 1986; Semancik 1986) that additional citrus viroids were detected (Duran-Vila et al. 1986; 1988a,b), causing less severe symptoms in 'Etrog' citron. The "strain" differences of exocortis could now be explained as the effects of these individual viroids. However, some confusion still remains with the continued reference to these viroids as belonging to the exocortis syndrome (Duran-Vila et al. 1986; 1988a,b), even though this diverse collection of independently transmissible RNA molecules (275-340 nucleotides) is distinctly different from the 371 nucleotide CEVd. Gillings et al. (1988, 1991) found that all graft-transmissible dwarfing isolates in Australia carry CVd-IIIa, sometimes in the presence of CVdIIa (Table 6.1). These designations were made on the basis of molecular size and relative mobility in sPAGE. Rakowski et al. (1994) subsequently found that the sequence of CVdIII in Australian dwarfing isolate 3538 is 99.3% identical to the type CVd-IIIb, differing only in the inclusion of a single A:U pair to the string of three A:U pairs in the left terminal region. Likewise, the CVd-III from a dwarfed 'Allen Valencia' orange (2045) has been sequenced (Stasys et al. 1995) and is identical to the type CVd-IIlb. Taylor et al. (1997) found that the sequences of CVd-III vary only slightly between Australian isolates. CVd-III from 3532 was identical to the CVd-IIlb type sequence of Rakowski et al. (1994), while isolates from 3538 and 3539 have a deletion at positions 3 and 4 and an insertion at position 295, which may cause a slight change at the very end of the hairpin structure. Isolate 3539 had further differences at positions 284 (A not G) and 287 (C not U) (Taylor et al. 1997). Thus, the Australian strains of CVd-III, associated with dwarfing, are most likely to be CVd-IIIb and not CVdIlIa, as originally suggested by Gillings et al. (1988). Whereas the viroid dwarfing agents used in Australia contain only one or two viroids (CVd-IIlb and sometimes CVd-IIa), the graft transmissible dwarfing agent # 225T used for experimental dwarfing of grapefruit

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

282

Table 6.1. Viroids associated with graft transmissible dwarfing in Australia (Gillings et al. 1991). Symptoms Poncirus trifoliata Viroid budline 033 3536 3531 3532 3534 3538 3539 Teralba

Rangpur lime

Scaling Y

Stunting

+

+ + + + + + + +

Cachexia x

Stunting + ? + ?

+

+ ?

Viroid IDZ

Etrog citron Leaf symptoms W S S M M M M M M

CEVd

IIa

+ +

+ + + + +

IIb

IIc

IIIb + + + +

+ +

+ + +

ZCitrus viroid consensus catalogue (Duran-Vila et al. 1988a,b), viroid-like RNAs were identified by relative mobility, molecular weight estimates, and symptoms on 'Etrog' citron. Y+

persistent, extensive scaling of the rootstock (typical exocortis);

= no symptoms.

trees indexed positive for cachexia on biological indicators (Broadbent and Dephoff 1992); - = no symptoms.

x+

wSeverity of leaf symptoms: S

= severe, M = mild, on 'Etrog' citron Arizona 861.

(G. paradisi Macf.) and Oroblanco (G. grandis (L.) Osbeck X G. paradisi Macf.) trees in Israel was found to contain at least five citrus viroids with estimated sizes of 284 (CVd-IV and a novel chimaeric viroid of CEVd and HSVd: Puchta et al. 1991), 295, 299 (a sequence variant of hop stunt: Puchta et al. 1989a,b), 318 (a novel chimaeric viroid of ASSVd and CEVd: Ashulin et al. 1991), and 371 nucleotides (CEVd) (Bar-Joseph 1993; Hadas and Bar-Joseph 1991). One dwarfing budline (3536) initially used in Australia, in the absence of bark scaling of P. trifoliata, contained citrus exocortis viroid (CEVd), while another (3539) indexed positive for cachexia (xyloporosis) on 'Orlando' tangelo (G. reticulata Blanco x C. paradisi (Schwinghamer and Broadbent 1987; Broadbent and Dephoff 1992). These two dwarfing isolates were subsequently dropped from later field trials. A critical factor in establishing a causal link between specific viroids and dwarfing is the use of defined inoculum. In Australia, gel-purified viroids have been inoculated into viroid-free scions (,Prior Lisbon' lemon (G.limon (L.) Burn.), 'Ellendale' tangor (G. sinensis (L.) Osbeck x G. reticulata Blanco) and 'Bellamy' navel orange on trifoliate orange rootstocks to determine dwarfing responses (Gillings et al. 1991). Trials are now 5-7 years old and are showing dwarfing effects due to single and mixed viroid inocula (Taylor et al. 1997).

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS

283

B. Mode of Action ofViroids in Causing Dwarfing The mode of action of viroids in causing dwarfing is unknown and has not been researched. However, there are several molecular mechanisms by which viroids could induce a dwarfing effect by interrupting or disabling normal tree functions. These include interfering with either mRNA splicing (Diener 1981; Gross et al. 1982), or pre rRNA processing (Solymosy and Kiss 1985; Jakab et al. 1986), competition for RNA polymerase II or RNA polymerase (Rackwitz et al. 1981; Palukaitis and Zaitlin 1987), and interrupting protein transport (Haas et al. 1988; Symons 1989). Meduski and Velten (1990) identified sequence similarity between the negative strand of potato spindle tuber viroid and a highly conserved region of large ribosomal RNAs, indicating the potential for specific interaction between the two. Viroids can also affect the peroxidaselIAA oxidase systems of some plants (Rodriguez and Flores 1987; Yaguchi and Takahashi 1985; Takahashi et al. 1992). C. Risks in Using a Pathogen for Tree Size Control Roistacher (1992) cited a number of reasons for his concern about the use of viroids for tree size control. These include the potential for mutation, mechanical transmissibility, heightened susceptibility of viroid dwarfed trees to frost (Garnsey 1983), potentially deleterious effects from the indiscriminate use of viroids by nurserymen, and the possibility that viroids might be causal agents of certain new diseases (Semancik et al. 1997). However, a disease relationship has not been established for all citrus viroids (Semancik et al. 1997) and these concerns are based on the assumption that all viroids are agents of disease. In Australia, dwarfing viroids have not been associated with disease and change in the pathogenicity of the dwarfing viroid CVd-IIIb has not occurred in more than 40 years of field trials. Hadas and Bar-Joseph (1991) recommended that only endemic viroids that are widely spread without causing damage and with a limited host range should be used. These precautions would rule out the use of CEVd (which causes bark scaling of P. trifoliata) and CVd-II group of viroids (because of their wide host range). No vectors are known. Decontamination of cutting implements to avoid mechanical transmission is essential if viroid inoculation is to be carried out in the nursery. In Australia, inoculation in the field during the first year after planting is recommended (Broadbent et al. 1986, 1992) to avoid the possibility of compromising high health status citrus material from a budwood scheme and the possibility of transmission to other hosts. Because of the close similarity in sequences ofCVd-IIa (associated with dwarfing)

284

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

and CVd-IIb, which is the causal agent of cachexia (Semancik et al. 1988, 1992; Levy and Hadidi, 1993; Taylor et al. 1997), budlines containing the CVd-II group of viroids are no longer used in Australia for dwarfing. While symptoms of gum pocket (Marais et al. 1996; Roistacher et al. 1993) on P. trifoliata have been associated with CVd-IIIb, no symptoms have been seen in Australia. While Garnsey (1982) found that CEVd infected trees were more frost sensitive than healthy trees, the reverse has been seen in Australia, where viroid dwarfed trees, having reduced summer and autumn flush, are less damaged by frost. III. USE OF VIROIDS FOR TREE SIZE CONTROL

The effects of citrus exocortis viroid (CEVd) and mild citrus viroids (CVd's) on field performance of a number of different stock-scion combinations have been reported over the past 30 years (Fraser et al. 1961; Long et al. 1972; Cohen 1968; Mendel 1969; Stannard et al. 1975; Bevington and Bacon 1977; Bacon 1980; Cohen et al. 1980; Cohen 1981, Hutton and Cullis 1981; Broadbent et al. 1986; Amir et al. 1988; Ashkenazi and Oren 1988; Golomb 1988; La Rosa et al. 1988; Nauer et al. 1988; BarJoseph 1993; Perez et al. 1992; Semancik et al. 1997). In all instances, reductions in scion trunk cross-sectional area, tree height, canopy volume and a commensurate reduction in tree yield were reported, but no differences were noted in fruit quality factors such as fruit size, color, juice percentage, percent citric acid or Brix (Benton et al. 1950; Cohen 1968; Hutton and CUllis 1981; Polizzi et al. 1991; Albanese et al. 1996; La Rosa et al. 1996). Research carried out by NSW Agriculture personnel over more than 40 years has shown that viroid dwarfing is an effective means of controlling tree size of 'Washington' navel and 'Valencia' orange trees on P. trifoliata and citrange (P. trifoliata x Citrus sinensis) rootstocks without affecting cropping efficiency or fruit quality (Bevington and Bacon 1977; Bacon 1980). Tree size can be reduced by 50% on P. trifoliata and by 20-50% on 'Carrizo' and 'Troyer' citrange, depending on the dwarfing budline used for inoculation. Yields of inoculated trees are reduced directly in proportion to the reduction in canopy surface area. The time lag observed before the effects on canopy size or yield become apparent, following inoculation with viroids, is one of the principal advantages of viroid inoculation for regulating tree size in citrus. Tree performance is essentially unaffected during the first 5 years after planting. During this interim period, viroid inoculated trees often show slightly enhanced yield efficiency. The delay following inoculation before canopy

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS

285

growth rates are reduced ensures rapid early development of a high canopy bearing volume per hectare to promote high early orchard productivity (Bevington et al. 1996). Results from the various investigations into viroid dwarfing in diverse geographic and climatic areas in Australia have demonstrated that a predictable response to viroid inoculation can also be expected under different growing conditions. Trials carried out in Israel (1965-1980) with a range of scions carrying numerous viroids when grafted on P. trifoliata and 'Troyer' citrange gave inconsistent results with respect to tree size control (Bar-Joseph 1993). But in all cases, trees planted at high density outyielded conventionally spaced trees on a planted area basis for a period of 10 years after planting. Factors that can affect the response to viroid inoculation include the composition of the viroid complex (Bar-Joseph 1993), the timing of inoculation, and the rootstock and scion used. A. Rootstock In a low density trial at Yanco, trees on P. trifoliata (Australian selection 22) gave the largest response to dwarfing isolates, with significant effects becoming apparent within 5 years of planting (Bacon 1980). Inoculating trees on 'Troyer' and 'Carrizo' citranges produced a response in 7 to 8 years (Fig. 6.1). Inoculations with non-scaling dwarfing isolates containing CVd-IIIb +/- CVd-IIa reduced tree size of Valencia orange on 'Troyer' or 'Carrizo' citrange by 23% compared to uninoculated trees, 11 years after planting (Bacon 1980). Scaling isolates containing CEVd reduced the size of trees on citrange by nearly 50% and they were considerably smaller than non-scaling CVd inoculated trees. In a second low density trial at Dareton, 'Bellamy' nucellar navel orange trees on a range of rootstocks were inoculated with either dwarfing selection 3532 (CVd-IIIb + CVd-IIa) or 3539 (CVd-IIIb + CVd-IIb) and compared to uninoculated trees. Inoculations with 3532 or 3539 reduced tree size on P. trifoliata, 'Troyer' and 'Carrizo' citranges and 'Rangpur' lime (G.limonia Osb.) and after 9 years, canopy surface area was reduced by 51 %,25% and 19% respectively relative to uninoculated trees (Fig. 6.2). Bevington and Bacon (1977) reported no effect of viroid inoculation on tree size on 'Cleopatra' mandarin(G. reticulata Blanco), sweet orange or rough lemon (G. jambhiri Lush.). Both dwarfing isolates produced similar results, and even though isolate 3539 produced symptoms of cachexia (gumming, pegging, and pitting) in 'Rangpur' lime stocks, tree vigor and productivity was unaffected. The selections of P. trifoliata used in all dwarfing trials in Australia are the small leaf and small flower type. In Israel there are differences

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

286 450

a

400

b

350

d

e

300

e=

250

~

c

0

~

E

2 ·0 ~

e

200

~

150 100 LSD (P=O.05)

50 ns

ns

0 1

2

ns

1 4

TTT T TT 5

10

Tree age ( years)

Fig. 6.1. Effect of inoculation and rootstock on trunk circumference of young 'Valencia' oranges at Yanco Agricultural Institute (Re-drawn from Bacon, 1980). Treatments were: viroid free = [AJ, dwarfing budline (CVd-IIIb + CVd-IIa) = [BJ, scaling budline (CEV + CVdIIIb + CVd-IIa) = [C]. a Citrange [A]; b = Citrange [B]; c = Poncirus trifoliata [A]; d = Citrange [B+C]; e = Poncirus trifoliata [C].

in the dwarfing response among trifoliate orange clones: 'Benecke', 'Local', 'Pomeroy', 'Rubidoux', and 'Jacobsen', which vary from mild to highly responsive (Ashkenazi and Oren 1988). In Israel, citrumelo does not respond to dwarfing inoculations (Golomb 1988). In field trials in Italy (Albanese et al. 1996), CVd-ilI had no effect on 'Moro' sweet orange and 'Clementine' mandarin (G. reticulata Blanco) trees on sour orange rootstock (G. aurantium 1.), but reduced trunk circumference and canopy volume of trees on trifoliate orange and 'Troyer' citrange rootstock by 30-35% at 10 years after planting. The success of the dwarfing inoculations will depend on how well suited the rootstock is to the soil conditions. Without ideal growing conditions and management, the success of viroid dwarfed high density plantings will be limited (Golomb 1988; Bar-Joseph 1993; Bevington 1997). However, one unexpected benefit of viroid infection has been increased tolerance to root rot (Rossetti et al. 1980).

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS

287

25 T;==================;--------~ 0 Uninoculated .3532 f213539j

I

20

1 e<:

f::e<: 15


'.I

oS:..

t;l ,.., Q..

10

0

e<: ==

U

5

0

Poncirus trifoliata

'Troyer' citrange

'Carrizo' citrange

'Rangpur lime'

Rootstock

Fig. 6.2. Effect of viroid dwarfing inoculations on mean canopy surface area of 9-yearold 'Bellamy' nucellar navel orange trees on Poncirus trifoJiata, 'Troyer' citrange, 'Carrizo' citrange, and 'Rangpur' lime rootstocks at Dareton. Treatments were: Uninoculated (no viroid); viroid isolate 3539 (CVd-IIIb + CVd-IIb); viroid isolate 3532 (CVd-IIIb + CVd-IIa). Note: There was no effect of viroid inoculation on 'Cleopatra' mandarin, 'Sweet orange', or 'Rough lemon'.

B. Scion

The response of the scion depends on its sensitivity to the individual viroids present in the dwarfing inoculum, e.g., dwarfing inoculations of 'Ellendale' tangor on 'Troyer' citrange stocks in Queensland, with dwarfing isolate 3539 (CVd-IIIb + CVd-IIb), were disastrous, because of the development of cachexia symptoms in the 'Ellendale' tangor scion. In trials using purified viroids (Taylor et al. 1997), CVd-IIb caused cachexia symptoms, while the closely related CVd-lla had no effect on tree health or tree height and CVd-IIIb reduced tree size. Other research into the effect of CVd inoculation of mandarin scions has recently been conducted by Yandilla Park Pty. Ltd., a large citrus producer located in South Australia, to evaluate the performance of 'Ellendale' tangor and 'Kara' mandarin on P. trifoliata and 'Carrizo' citrange rootstocks. In all cases, tree size was reduced by inoculation and earlier cropping was observed on some inoculated trees (A. Edwards, pers. commun.).

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

288

Most dwarfing trials in Australia have been for oranges ('Washington' navel and 'Valencia'). Although individual tree yields on sensitive stocks are reduced by inoculation due to the reduction in canopy surface area (Bacon 1980), production per hectare can be maintained or even increased because the smaller tree size allows higher planting densities (Long et al. 1972; Hutton 1991). There were no significant differences in juice content, acidity or the total solids-to-acid ratio between viroid inoculated orange trees on citrange rootstocks and uninoculated trees (Bacon 1980). However, 'Valencia' orange trees on P. trifoliata inoculated with CEVd budline 033 (CEVd+CVd-IIIb + CVd-IIa) produced fruit with significantly higher soluble solids (Bacon 1980). Nauer et al. (1988) also showed there were no significant differences in fruit quality of navel oranges on two rootstocks when infected with CEVd or various mixtures of citrus viroids when compared to viroid-free shoot tip grafted controls. Viroid dwarfing of 'Marsh' grapefruit has not been investigated to any extent in research trials in Australia. In one minor trial, isolates 3538 (CVd-IIIb) and 3539 (CVd-IIIb + CVd-IIb] reduced height and width of trees on P. trifoliata, with a corresponding reduction in yield. At 17 years, tree size reduction and a commensurate yield reduction were evident (Table 6.2) and no deterioration in fruit quality occurred as a result of the viroid inoculations. Increased yields of viroid-dwarfed (GTD#225T inoculated) 'Oroblanco' trees on trifoliate orange rootstock grown at high density in Israel have been reported by Bar-Joseph (1993). New growth of these trees was also considerably shortened after topping (30-60 cm) compared to the uninoculated trees (>100 cm). The possibility of reducing the vigor of lemons by viroid inoculation was investigated in a trial at Somersby (Broadbent et al. 1988). Both 033 (CEVd+CVd-IIIb + CVd-IIa) and viroid isolate 3538 (CVd-IIlb) significantly reduced tree height and width of 'Prior Lisbon' and 'Taylor Eureka' lemon trees on rough lemon rootstock, but not on P. trifoliata. The tree size reductions for 'Eureka' lemon on rough lemon were 28%

Table 6.2. Effect of viroid inoculation of 'Marsh' grapefruit on Poncirus trifoliata on tree size and yield. Inoculation Nil 3539 3538

Height x width (m)

Cumulative fruit no.

Total weight (kg)

3.8 x 4.1 2.8 x 3.4 2.6 x 3.4

5473 3242 3335

1090 717 725

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS

289

for CEVd and 15% for CVd-IIIb, and from 6 years, cropping efficiency was better than for uninoculated trees, which could compensate for the smaller tree size. There was also a 9% reduction in fruit weight, but there was no effect of inoculation on juice quality or summer cropping. A second trial using purified viroids to inoculate nursery trees of 'Prior Lisbon' lemon on P. trifoliata was planted at high density at Somersby in 1991. In this trial a combination of CEVd+CVd-IIIb had the greatest effect on trunk circumference and tree height (Taylor et al. 1997). 'Tahiti' limes inoculated with "mild exocortis" and planted at high density in Brazil were reduced in size. The infected trees outyielded nucellar budlines at normal spacing by 30-40% per hectare in the first four years after planting (Salibe 1988). C. Time of Inoculation Time of inoculation influences the extent of the dwarfing response. Trees inoculated in the nursery with a dwarfing budline (3532 CVdIIIb + CVd-IIa) were the most dwarfed (Fig. 6.3), whereas trees that

Time of inoculation after planting ---D.··Non-inoculated --.--Nursery - - . - Year 2 ~ Year 2 - 0 - - YearS

e

---II- Year - 0 - - Year

1 3

24.0

u

';' 20.0 u

1:1

f

~

16.0

·5

12.0

e = u

:::

=

~

8.0

~ ~

~ 4.0 0.0

+---j---+---j---+----+---t---j--j----f------/

o

2

3

4

S

6

7

8

9

10

Years after planting

Fig. 6.3. Effect of inoculation time with dwarfing viroid isolate 3532 on mean trunk circumference (em) of 'Washington' navel trees on Poncirus trijoliata (Re-drawn from Stannard et al. 1975).

290

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

were inoculated in the field one, two, three, or five years later were successively less dwarfed (Stannard et al. 1975). The dwarfing response for orange trees on P. trifoliata takes at least four years to become apparent (Stannard et al. 1975). This response time and the possibility of inoculating trees in an orchard situation have distinct benefits. Growth of trees inoculated soon after planting is as rapid as in uninoculated trees for the first four years, allowing trees to rapidly fill their allotted space and produce good yields per hectare. Accidental inoculation after this time will not have any effect on tree size. By contrast, growth of the trifoliate dwarfing rootstock, 'Flying Dragon' (P. trifoliata) , is slow from the time of planting (Fig. 6.4) and planting density would have to be significantly increased to compensate for the very slow canopy development observed in cool Mediterranean climates (Golomb 1988; Hutton 1995). Inoculation of trees in the orchard following planting avoids the possibility of contaminating other propagations in a nursery by transmission of the viroids on budding knives and pruning tools.

Fig. 6.4. Canopy development in 9-year-old 'Valencia' orange trees grown at Yanco Agricultural Institute. Treatment contrasts were: (A) uninoculated trees on Poncirus trifoliata (clone 22) at 664 trees/ha; (B) viroid (3532) dwarfed trees on Poncirus trifoliata (clone 22) at 1000 trees/ha; (C) uninoculated trees on 'Flying Dragon' (trifoliate orange) at 1000 trees/ha.

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS

291

IV. VEGETATIVE GROWTH A. Tree Uniformity A characteristic of the viroid dwarfing trials in Australia has been the uniformity of response to viroid inoculation. By contrast, a marked variation in tree size has occurred in grapefruit trees on trifoliate orange rootstock inoculated with GTD#225T in Israel (Hadas et al. 1989), due to a segregation or exclusion of one or more viroids from the complex. Variability in tree size does occur in some budlines in Australia, e.g. 'Thompson' grapefruit was introduced from California in the 1950s as a few budsticks. The first propagations on trifoliate orange resulted in trees with exocortis and some apparently healthy trees. Subsequent propagations from non-scaling trees resulted in dwarfed and normal-sized trees (Fraser and Broadbent 1979). This budline was subsequently shown to carry CEVd, CVd-IIa, and CVd-IIIa (Gillings et al. 1988). 'Allen Valencia' orange (2045) produces trees of variable size (Fraser and Broadbent 1979), but contains only CVd-IIIb (Gillings et al. 1988; Stasys et al. 1995). B. Tree Growth Rates and Canopy Size In 1986 and 1987, long-term field trials were established at Yanco in the MIA and at Dareton in the Sunraysia region of southern NSW to investigate the production potential and management requirements of high density citrus plantings grown under different irrigation management. In the trial at Yanco, reduced trunk growth of viroid inoculated trees on P. trifoliata first became apparent two years after planting, but did not become evident for a further three years in canopy size measurements. Growth rates were reduced and trunk cross sectional area (TCSA) increments were significantly lower (P
292

R. HUTTON, P. BROADBENT, AND K. BEVINGTON 14

n

12

-e

....

c=J Uninoculated; 668 treeslha E22222J 3532; 790 treeslha

A

3532; 1011 treeslha 033;1011treeslha E:=J 033; 1337 treeslha ~ ~

10

~

-=e

1.0

.S

8

6

F

C=

-<

U

00.

r-<

~

f=

2

r= r= r= r= f= r= f=

0

= r=

4

F

t= f=

f= 1= f= f=

f=

r=

E

r==

I--

7

6

14 - , - - - - -

12

~

--,

I~~..

Uninoculated; 666 trees/ha

~

3532; 2127 trees/ha

B

E22222J 3532; 1000 trees/ha

I

6

7

Tree age Fig. 6.5. Effect of planting density and viroid inoculation on annual trunk cross sectional area (TCSA) growth increment in 6- to 7-year-old 'Valencia' orange trees grown on (A) 'Carrizo' citrange at Dareton and (B) Poncirus trifoliata at Yanco Agricultural Institute. Viroid isolate 3532 = CVd-IIa + IIIb; viroid isolate 033 CEVd+CVd-IIIb + CVd-IIa.

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS

293

TCSA increments were recorded on 3532 and CEVd (033) inoculated trees in each year (Fig. 6.5). By the end of the eighth growing season, the TCSA of 3532 and 033 inoculated trees was 22% and 28% smaller than the TCSA ofuninoculated trees, while tree height had been reduced by 15% and 17%, respectively. There were no significant viroid inoculation by irrigation method interactions for trunk growth or canopy size. However, the response to inoculation was more evident with drip irrigated trees than with those irrigated by micro-sprinkler, because of their more uniform growth. Growth of both uninoculated and inoculated trees during the first 8 years after planting was unaffected by within row spacing. There was also no consistent effect of row spacing on tree growth within viroid inoculation treatments. The four-year delay before any growth rate reduction occurred following viroid inoculation, which occurred at both Yanco and Dareton, and the extent of tree size reduction observed is consistent with previ0us trial results on citrange rootstocks (Bevington and Bacon 1977; Bacon 1980; Hutton 1991). C. Shoot Growth Patterns Vegetative shoot growth patterns were studied in long-term field trials at Yanco and Dareton. At Yanco, less total vegetative shoot growth and significantly less total spring vegetative growth (P<0.05) was recorded for viroid inoculated trees. This observation is consistent with the smaller annual increment in TCSA and tree height recorded on these trees and explains the gross difference in tree size recorded for the two tree vigor levels. Examination of calculated mean shoot length, number of leaves per shoot, and mean leaf area per shoot for the spring growth flush revealed no significant difference between the growth flushes of viroid inoculated and uninoculated trees. The only difference was the number of spring shoots developed. The reduced number of spring flush shoots initiated on viroid inoculated trees resulted in less total shoot length being developed. The summer flush growth contribution to total seasonal vegetative shoot growth formed only 14% and 17% in viroid inoculated and uninoculated trees, respectively. Shoot growth in autumn contributed similar amounts of vegetative shoot growth to that recorded for the summer flush, but it was more pronounced on viroid inoculated trees than on uninoculated trees. However, the observed difference in autumn shoot growth did not have a significant impact on total seasonal vegetative shoot growth. The morphology of autumn shoot growth was different, with more shoots

294

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

of smaller internode length and fewer leaves developing on viroid trees than uninoculated trees whose autumn growth was sparse and appeared as rank water shoots in the top half of the tree canopy. Annual vegetative shoot growth was significantly less on viroid inoculated trees grown on 'Carrizo' citrange at Dareton. Although growth was reduced, timing and duration of growth flushes was not affected by viroid inoculation treatments. Also, annual variation in the intensity of vegetative growth followed the same trends in both uninoculated and inoculated trees. However, despite viroid inoculated trees on citrange rootstock being smaller than uninoculated controls, the greater vigor of this rootstock produced larger trees than viroid dwarfed trees of the same age grown on P. trifoliata at Yanco. Warmer winter temperatures at Dareton also resulted in greater spring flush development and this, combined with significant summer shoot growth evident in trees grown on citrange rootstock, resulted in more rapid canopy development and larger tree size in 'Valencia' oranges than that recorded at Yanco, where virtually no summer flush was evident. Effects of viroid inoculation on shoot growth at Dareton were predominantly expressed through a reduction in the amount of growth that occurred during the major summer growth flush. The main effect of inoculation was a reduction in the number of shoots initiating growth (Fig. 6.6). Total shoot production on inoculated trees during summer and autumn was reduced by 28 to 47%. Expressed on the basis of tree size, the intensity of shoot production (number of shoots/TCSA) was significantly (P
295

6. VIROID DWARFING FOR mGH DENSITY CITRUS PLANTINGS 45 .----;1

I

-l!l

40

- - urunOC,mm'd ----ts- Viroid 3532

35

___ Viroid033

I

30

0 0

.;;

.....0 25 L.

,.Q


20

Z

15

e::I

10 5 0

~

J:,

~ ~

iii

~

M

..0

..0

";<

";<

I1l

V)

I1l

00

N

Date

a

::;;s

:±

a

~ 00

N

..

0..

1

..

0..


N

Fig. 6.6. Effect of viroid inoculation on number of shoots initiating growth during summer and autumn growth flushes of 'Valencia' orange on 'Carrizo citrange' rootstock at Dareton. Viroid isolate 3532 = CVd-IIIb + CVd-IIa; viroid isolate 033 = CEVd+CVd-IIIb + CVd-IIa.

V. REPRODUCTIVE GROWTH A. Flowering The timing and intensity of flower production has been the same for all trees whether viroid dwarfed or uninoculated and only varied between seasons. The total amount of reproductive shoots (leafy and leafless inflorescences) was also the same per unit of canopy volume, and no significant difference between tree vigor levels has been observed on percentage fruit set. This observation explains why cropping efficiency of orange trees on P. trifoliata rootstock is not affected by viroid dwarfing and supports previously reported results showing that the number of fruit per cm 2 leaf area is unaffected by viroid dwarfing (Hutton 1986). B. Fruit Growth Seasonal growth patterns and trends in mean weekly growth rates have been identical for fruit grown on viroid inoculated and uninoculated

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

296

t

70 , . . . . - - - - - - - - - - - - - - - - - - - - - - , :u:mnOCUlmed;666 trees/ha

60 50

--Q.l Q.l

L..

.3532;1000trees/ha

~3532;2127 trees/ha

40

~

e....

~

.; 30 L.. ~

20

10

o 6

7 Tree age (years)

8

Fig. 6.7. Effect of viroid inoculation on yield per tree from 6 to 8 years after planting 'Valencia' orange trees on Poncirus trifoliata at Yanco Agricultural Institute. Viroid isolate 3532 = CVd-IIIb + VCd-IIa.

trees (Bevington et al. 1996). Reduction in tree size and vigor following viroid inoculation had no adverse effects on fruit development or mean fruit diameter at harvest. C. Yield Although lower ·trunk growth increments are recorded on inoculated trees 2 to 3 years after planting, effects on canopy size reduction were insufficient to affect yield until 4 years later. Significant effects of viroid inoculation on annual tree yield were evident in both the Yanco and Dareton trials 6 years after planting (Fig. 6.7). The reduction in yield of viroid inoculated trees was directly related to the reduction in canopy size, which was reduced in the same proportion as TCSA and tree height (Table 6.3), and yield efficiency was not reduced. Lower yields on inoculated trees are entirely due to a reduction in the number of fruit carried on smaller trees. Fruit size differences observed between years are purely a function of crop load. Average fruit size is unaffected by inoculation. The absence of effects of viroid inoculation

6. VlROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS

297

Table 6.3. Trunk cross-sectional area (TCSA) and height of uninoculated and viroid inoculated 'Valencia' orange trees at Yanco, Australia, nine years after planting.

Viroid inoculation Uninoculated 3532 (CVd-IIIa + CVd-IIa) 3532 (CVd-IIIa + CVd-IIa) SE

Row spacing (m)

TCSA (cm 2 )

Height (m)

5.6 4.0 1.5; 3.2

55.2 39.7 31.6 1.8

2.3 1.9 1.7 0.5

on reproductive growth explains why no adverse effects of inoculation on yield efficiency, expressed as crop weight per unit TCSA, have been recorded. Yield efficiency is also unaffected by closer tree spacing within rows (Hutton and Cullis 1981).

D. Fruit Quality In the 13 years of tree spacing research conducted between 1974 to 1987 at the Yanco Agricultural Institute on 'Late Valencia' oranges, no significant effect of tree density or the components of density (within-row or between-row spacing) could be found on fruit quality (Hutton and Cullis 1981; Hutton 1986). However, at very high densities fruit coloring was delayed and fruit size was marginally smaller, irrespective ofviroid status of the trees. VI. INTENSIVE VIROID-DWARFED PLANTINGS

Present planting trends favor the use of smaller, more closely spaced productive trees which develop higher yields per unit of ground area. Close planting and good tree size control are essential for the success of this practice (Castle 1978; Phillips 1978; Wheaton et al. 1978). The first examination of intensive viroid dwarfed plantings (Hutton and Cullis 1981; Hutton 1986) was undertaken in the MIA at Yanco, Australia in 1974 (Fig. 6.8) and the fundamental outcomes of this work have been supported by more recent work in Australia at both Dareton and Yanco (Bevington et al. 1996), Israel (Ashkenazi and Oren 1988; Bar-Joseph 1993), South Africa (Piner 1988; Rabe et al. 1992), Brazil (Roberto et al. 1992), and Italy (Intrigliolo et al. 1992; Tribulato et al. 1992). Substantial increases in productivity were achieved early in the life of these high density plantings relative to conventionally planted orchards

298

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

Fig. 6.8. Aerial photograph taken at Yanco Agricultural Institute. (A) Trials to evaluate the effects of dwarfing viroid inoculation on 2 stock/scion combinations at 2 planting densities. (B) Spacing trials for viroid dwarfed and uninoculated trees.

(Hutton and Cullis 1981; Hutton 1986; Cullis and Verbyla 1992) and this was shown to be purely a function of tree density for any given row spacing (Fig. 6.9). Higher cumulative yields recorded over the productive life of such plantings (Fig. 6.10) would generate greater profitability to citrus growers due to the demonstrated early productivity gains. Trees grown at increasing planting densities developed both a smaller trunk cross-sectional area (TCSA) and smaller canopies (Fig. 6.11). The same inverse relationship was observed for yield with tree size, where lower yields per tree were recorded for smaller trees grown at high density. Because of this, individual tree yield (kg) was less than for conventionally planted trees of similar age, but by increasing tree densities to generate more canopy per unit land area (Fig. 6.12), yield (tonnes/ha) was significantly increased due to the greatly increased fruit bearing volume developed soon after planting. The plant density-productivity relationship identified is simply the result of yield potential being related to rapid development of canopy bearing volume (Boswell et al. 1975; Wheaton et al. 1978; Castle 1985). There is a positive relationship between tree yield and canopy size (Fig. 6.13).

299

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS A

600 500 ';'

-=

~

400

'0 ~

.~

'0 Qi

= e

300

:;

= u 200 u

-<

100 0 4

3

5

6

7

8

9

10

11

Tree age (years)

600

B

T

i

l:~

500

--.-2M

,-... Cll

~

400

_3M

'0 ~

.~

'0

~

300

Cll

:;

e u= u

-<

200

100

0 3

4

5

6

7

8

9

10

11

Tree age (years)

Fig. 6.9. Effect of within row spacing on accumulated yield of 'Valencia' oranges on Poncirus trifoliata at row spacings of A 2 ill and B = 5 ill at Yanco Agricultural Institute.

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

300

600 ___ Uninoculated 222 tree/ha

500

- . - Viroid dwarf 864 tree/ha

';'

~ 400 :5! .S:!

~ 300

.i::

JS

e

::: U

200100

o "l'---~~+-~~+-~~-t-~~-t-~~+-----t--~t-~-I 5

9

7

11

13

15

17

19

21

Tree age (years)

Fig. 6.10. Effect of planting density and viroid dwarfing on cumulative yield in 'Valencia' oranges on Poncirus trifoliata rootstock at Yanco from 1964 to 1985. Viroid isolate was 3532 (CVd-IlIb + CVd-Ila).

900

•

800

y= -1016x + 849.2

~

R2

~

"" 700 ....~

•

5

~ C':l

~

600

C':l

'; 500 Q ==

--5

•

0.664

• • •

~ 400 ~ Q

"" .,:;c: ~

::: ==

""

Eo-<

300 200 100 0 0

0.1

0.2

0.3

0.4

0.5

0.6

Tree density (trees/m 2)

Fig. 6.11. Effect of planting density on trunk cross sectional area of ll-year-old viroid dwarfed (3532) 'Valencia' orange trees on Poncirus trifoliata at Yanco Agricultural Institute.

301

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS

Fig. 6.12. Aerial photograph of 7-year-old 'Valencia' orange trees on Poncirus trifoliata showing differences in canopy surface area (CSA) developed at higher tree densities in an irrigation management trial at Yanco Agricultural Institute. A, D, F: uninoculated trees at 664 trees/ha; C, E, G = viroid (3532) dwarfed at 2127 trees/ha, and B, D = viroid (3532) dwarfed at 1000 trees/ha.

600 y= 19.89x+2.42

500

2

R

'Z' ~

•

0.963

lo<

~ 400

e

:s .;!:l

»

'"0

300

~

~

"3 ::I

200

== c.l c.l

-<

1 I

100

•

o, 4

Cumulative yield (kg/tree)

-Linear (Cumulative yield (kg/tree))

8

12

16

20

24

CSA (m2 /tree)

Fig. 6.13. Relationship of canopy surface area (CSA) to accumulated yield (kg/tree) of viroid dwarfed 'Valencia' orange trees on Poncirus trifoIiata at Yanco Agricultural Institute. Viroid isolate was 3532 (CVd-IIIb + CVd-IIa).

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

302

The relationship between plant density and crop yield is an asymptotic one (Fig. 6.14) where, with increase in density, yield rapidly rises to near maximum levels and then levels out at an increasing rate at very high planting densities (Holliday 1960; NeIder 1966). In citrus, however, the very high levels of productivity recorded for uninoculated trees grown at high density (Fig. 6.14) could not be sustained without imposing tree size control (hedging), since cropping moved higher up the tree canopy as the trees grew taller with increasing tree age, and general orchard management operations (spraying and harvesting) became impossible. The use of viroid dwarfing to control vegetative vigor overcomes this problem, and high density CVd dwarfed plantings remain highly productive throughout their mature bearing life. Using this philosophy, high density orchards are designed to maximize the bearing volume, yields, and net returns over the life of the orchard from soon after planting. This is in contrast to conventional wider-spaced orchards that are designed to attain maximum bearing volume, yield, and net returns at maturity, with little consideration of the elapsed time and costs needed to reach mature bearing age. The current high density management trial at Dareton is the first trial in which the performance ofviroid inoculated trees on 'Carrizo' citrange

700 y1 = 150.31Ln(x) + 735.15

600 ~ ..c

~

"C ~

';;'

R 2 =0.6025

500 []

400

y2 = 159.14Ln(x) + 691.65

[]

R 2 = 0.7762

"C

~

S

:I

e:I tj tj

-<

300 200

t I

100 0

0

0.1

0.2

0.3

Density (trees/m

0.4 2

0.5

0.6

)

Fig. 6.14. Effect of planting density on accumulated yield of ll-year-old 'Valencia' orange trees on Poncirus trifoliata at Yanco Agricultural Institute. Viroid isolate was 3532 (CVd-IIIb + CVd-IIa).

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS

303

rootstock have been evaluated at close spacings. During the first 8 years after planting, yields per hectare have been much greater from viroid inoculated trees planted at high density than from uninoculated trees planted at medium density. The lack of competition on tree growth and yield to date highlights the importance of appropriate selection of tree spacings to match tree growth rates to delay the onset of adverse effects of overcrowding. This has allowed trees at each density to develop maximum yield potential. Present indications are that planting densities between 800-1,000 trees/ha would be satisfactory for viroid inoculated trees on citrange rootstocks in replant situations. VII. ECONOMIC CONSIDERATIONS

A number of assumptions must be made in constructing an economic model for the effect of tree spacing on profitability. Some costs vary with density and others are independent of spacing. Costs varying with density include trees, planting, material costs for fungicide and insecticide, and costs associated with equipment travel, since closer row spacings result in more kilometers traveled. Other costs are independent of tree density and are applied on a per hectare basis. Various investment options using 20-year cash flow budgets to determine the effect of planting density on profitability were analyzed for 'Valencia: oranges on P. trifoliata grown in the MIA. Cash flow analyses (Elton and Hutton, unpublished) were constructed for viroid dwarfed orange plantings grown at medium density (740 trees/hal, high density (1,000 trees/hal, and very high density (1,250 trees/hal compared to a standard density non-dwarf planting (556 trees/hal. For the standard density plantings, actual data from research trials at the Yanco Agricultural Institute were used. The remaining yield data were generated using the yield prediction model (Cullis and Verbyla 1992) developed from the spacing experiments for viroid dwarfed trees conducted between 1974 and 1986 at the same location. Yield profiles used in the analyses are summarized (see Fig. 6.15). Breakeven points were influenced by whether the nursery trees were purchased or propagated by the grower. For grower propagated trees, the very high and high density options generated a positive cash flow in year 4. For the medium density planting, the cash flow became positive in year 5, while a positive cash flow was achieved in year 6 for the standard density planting. The cumulative cash positions became positive in year 8 for the very high density planting. For the high and medium density options, the new development would break even in years 9 and 10,

304

R. HUTTON, P. BROADBENT, AND K. BEVINGTON 80 70 60

c;-

50

.Cl

~ 40

::E .~ ;;...

30 ---.20 10 -

uninOCUlated~"556treeS/h~

----A---

Viroid 3532; 740 trees/ha

-0-

Viroid3532; 1000/trees/ha

_

Viroid 3532; 1250 trees/ha

9

11

-------

0 3

5

7

13

15

17

19

Tree age (years)

Fig. 6.15. Predicted annual yield profiles from the model ofCullis and Verbyla, 1992 for viroid (3532) dwarfed 'Valencia' orange trees on Poncirus trifoliata at three densities, and actual yields recorded for standard double planted uninoculated 'Valencia' orange trees at Yanco Agricultural Institute.

respectively, whereas the standard density option would not break even until year 11. After that point, cumulative returns per hectare from mature orchard trees was high for all planting density options (Fig. 6.16). Where nursery trees were purchased, net positive cash flows were not achieved until much later. Peak debt was significantly higher and cumulative cash flow did not become positive until year 18 for the very high density option. Break even occurred in year 17 for the high and medium density options. However, the standard density planting did not break even until year 19. The internal rate of return (IRR) is a related measure of profitability. The IRR is that interest rate which just balances the present values of cash receipts and cash outlays. A decision rule is to accept projects in which the IRR is greater than or equal to the opportunity cost earning rate, i.e., the discount rate which you would use if you were calculating the net present value (NPV) of the project. The return on investments as measured by IRR (using a 5% discount rate) suggests that all higher density plantings using grower propagated trees would be profitable. However, the internal rates of return for similar developments using purchased

305

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS 80000 , . . . - - - - - - - - - - - - - - - - - - - - - ,

~

l

3532; 1250 trees/ha

i - 0 - 3532; 1000 trees/ha

60000

A

i

-...-3532; 740 trees/ha --.- Uninoculated; 556 trees/ha

40000 ~

0

10:

~

= i: = :;

20000

~ ~

u=

0

==

-20000

-40000

-60000

Year

80000 1

60000

3532; 1250 trees/ha - 0 - 3532;

B

1000 trees/ha

-...- 3532; 740 trees/ha 40000

--.- Uninoculated; 556 trees/ha

~

0

10:

~

= ~ :;=

20000

~ ~

0 20

== =

U

-20000

-40000

-60000

Year

Fig. 6.16. Cumulative cash flows over 20 years for 'Valencia' orange trees planted at four densities; (A) trees purchased from a nursery at ADD$ 8.50 and (B) grower propagated trees at ADD$ 2.29.

306

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

nursery trees suggest that these investments would not be profitable (Elton and Hutton, unpublished). Investment costs are higher for high density plantings, resulting in higher negative cash flows during the early years of establishment. However, due to high density plantings reaching maximum productivity at a very young age, higher cumulative net cash flows may be expected over the life of the orchard due to higher relative returns for fruit in the initial years. When considering NPV criteria, the high and very high density planting options rank the highest, and hence indicate that this is a most desirable investment alternative. These analyses have shown that profitability is directly related to planting density. The advantages of early productivity and a shortened time period to achieve positive cash flows are obvious, but this cultural practice also offers the opportunity to capitalize on rapidly developing a marketable quantity of new varieties or cultivars to meet specific market windows. The greater investment required to establish high density plantings is more than offset by early productivity gains and the potentially higher price premiums which might be realized by supplying quality fruit for specific markets. The economic factors favoring high density plantings of viroid dwarfed trees are the early income on investment and the containment of fixed costs by increasing production efficiency. The disadvantages are the higher costs for planting and increased need for optimum orchard management due to greater numbers of trees per hectare. However, fungicidal and insecticidal spray applications are more efficient.

VIII. MANAGEMENT The results of all studies in Australia have confirmed the advantages of using graft transmissible viroid dwarfing to control tree size in high density orange plantings on P. trifoliata and citrange rootstocks. Inoculation of trees during their first year in the field is recommended to avoid mechanical transmission ofviroids in the nursery. The time lag observed before effects on canopy size or yield become apparent, following viroid inoculation, is one of the principal advantages of using viroid inoculation for regulating tree size in citrus. During this interim period, viroid inoculated trees often showed enhanced yield efficiency. The delay in reduction of canopy growth rates following viroid inoculation allows normal canopy development to occur during the first few years after planting, which ensures rapid early development of a high canopy bearing volume per hectare to promote high early orchard productivity. Sustained produc-

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS

307

tivity is dependent on controlling excessive vegetative growth to eliminate adverse effects of crowding and on maintaining high cropping efficiencies. The best strategy for managing vegetative growth in bearing citrus trees is to promote spring flush development and to reduce summer/ autumn growth. Appropriate management strategies to achieve this will depend on the stage of development of the orchard. During the first 3 to 4 years after planting, maximum vegetative growth needs to be encouraged. Following this period, the balance between vegetative growth and cropping needs to be progressively adjusted until trees fill their allotted space in the orchard. Once trees attain their required size, only sufficient vegetative shoot production is required to sustain yields. The use of viroid inoculation for tree size control is well suited to this overall strategy. Observations on vegetative and reproductive growth patterns made after this initial establishment phase indicated that yield efficiency (yield/tree size) of viroid inoculated trees was not adversely affected by the reduction in tree vigor. Effects of viroid inoculation on reduced canopy growth were restricted to a reduction in the magnitude of vegetative growth occurring during summer and autumn, confirming the dominant role of these flushes in regulating canopy expansion. Inoculation had no effect on spring flush growth, flowering intensity, inflorescence type, fruit set, or fruit growth rates. Apparently, the reduction in tree vigor and reduced shoot growth during summer and autumn is insufficient to adversely affect flowering. Timing and duration ofvegetative flushes were not affected by viroid inoculation and annual variation in the intensity of vegetative growth flushes followed the same trends in both viroid inoculated and uninoculated trees. Vegetative growth impacts on yield through its influence on canopy development and tree size. It is also essential for generating flowering sites in the following spring. However, it has been suggested that only a single growth flush is required for maintenance of yield in citrus (Chapman 1986). The extent of vegetative growth and vigor can be modified by rootstock selection, viroid inoculation, crop load, and nutrition. The need to regulate summer/autumn vegetative growth of citrus growing in a cool Mediterranean environment arises more from the perspective of controlling tree size, rather than from consideration of effects on fruit development, as summer growth appears neither competitive with fruit growth nor does it enhance crop development. However, reducing the extent of summer/autumn growth places increased emphasis on promoting adequate spring vegetative growth for maintenance of yield. This can be assured through paying appropriate attention to the

308

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

irrigation and nutritional needs of the trees during the spring growth period of the growing cycle. The successful application of deficit irrigation strategies to manipulate vegetative growth depends on the phenological separation of the main periods of shoot and fruit growth (Chalmers 1988). With 'Valencia' oranges growing under climatic conditions of the Sunraysia and MIA, most summer and autumn vegetative growth occurs during the period of maximum fruit growth rate. Recent experimental results (Bevington et al. 1996) suggest that opportunities for controlling excessive vegetative growth in 'Valencia' oranges by applying periods of water stress (deficit irrigation) will be limited. The use of deficit irrigation to regulate vegetative growth during spring is not considered a practical option because of likely adverse effects on flowering and fruit set. Method of irrigation (microsprinkler or drip) had a major effect on growth and productivity of high density plantings (Bevington et al. 1996). This was related to differences in application interval, frequency of fertigation, and size of the wetted zone. Total water consumption and nutrient removal on a planted area basis was greater where trees were grown at high density (Hutton 1995). This was due to the development of greater root densities per unit ground area in close planted trees, which resulted in more efficient use of irrigation water and fertilizers. Water use efficiency (kg fruit per liter of water applied) of close planted trees was significantly increased, especially in the first three cropping years, despite using more water per unit ground area (Fig. 6.17). Tree density does not affect mineral composition of leaves of individual trees (Hutton et al. 1998), despite greater depletion of nutrients on an area basis in high density plantings. Similar observations on foliar elemental levels in high density plantings of other crops have been reported (Atkinson 1973; Cripps et al. 1975; Wertheim 1985). Drip irrigation in combination with continuous nitrogen fertigation further enhanced water use efficiency by promoting early tree growth and productivity of both uninoculated and viroid inoculated trees compared to conventional microsprinkler irrigation (Bevington et al. 1996). Enhanced early canopy development was achieved through stimulation of summer and autumn shoot growth and a consequent increase in fruit set and yield efficiency resulted. Drip irrigation has also resulted in considerable savings in water with substantial improvement in water use efficiency (WUE) relative to microsprinkler irrigation systems. Similar observations have been reported in high density plantings of apples, especially in the early years after planting (Atkinson 1978). Careful attention should be paid to maintaining adequate soil moisture

309

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS 900 __ Uninoculated; 666 trees/ha

800

----{r-

700 C.:;

.:::

ell

3532; 1000 trees/ha J..JJ'-'-, ..,1-,-,

trees/ha

600

~ "'" 500

~ "0 .~

Q.

c..

= "'" =

~

~

400 300 200 100 0 1989

1990

1991

1992

1993

1994

1995

Year

Fig. 6.17. Effect of planting density and viroid dwarfing on water use efficiency in the initial 6 crops harvested from 'Valencia' orange trees on Poncirus trifoliata at Yanco Agricultural Institute.

levels with use of higher frequency irrigation schedules in young high density citrus plantings (Phillips 1969). This is especially so where trees have relatively shallow root systems characteristic of some less vigorous rootstocks (Castle and Krezdorn 1977). Ideal stock/scion combinations for high density planting should exhibit moderate vigor and high early cropping efficiencies. In this regard, trees on P. trifoliata are better suited to high density planting than trees on citrange, although, in combination with viroid dwarfing, high density plantings on citrange are also highly productive. Appropriate selection of tree spacing will be dependent on tree growth rates and vigor. Matching tree growth rates to spacing is important to optimize productivity per hectare and maximize potential returns from high density plantings. A crop management model for high density orange plantings based on data collected in Australian studies (Bevington et al. 1996) shows linkages between key growth processes and management inputs (Fig. 6.18). Key components of the model are regulation of vegetative growth and canopy development, flowering intensity, fruit set, and crop load.

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

310

Vegetative Growth Flowering Intensity

Fruit Set

Crop Load

Fruit Size

Yield (kgltree)

Production Iha

Fig. 6.18.

A crop management model for high density viroid dwarf orange plantings.

6. VIROID DWARFING FOR HIGH DENSITY CITRUS PLANTINGS

311

IX. SUMMARY AND CONCLUSIONS Australian field trials have shown that inoculation of trees with viroid CVd-IIIb with and without CVd-IIa reduced the tree size of selected sweet orange and grapefruit scions on trifoliate orange and citrange rootstocks without causing deleterious effects to tree health or fruit quality. Tree size reduction is not apparent in field grown trees inoculated after 5 or more years of age. Furthermore, the lack of accidental dwarfing through mechanical transmission of CVd's to uninoculated trees in long term field trials has demonstrated that this technique of tree size control is horticulturally sound. Closely spaced viroid dwarfed trees have been shown to outyield conventional plantings on a planted area basis and fruit quality was not affected by either the presence of viroids or close planting. Tree performance (cropping efficiency) was also shown to be greater for viroid inoculated trees. The time lag observed before effects on canopy size or yield become apparent, following viroid inoculation, is one of the principal advantages of using viroid inoculation for regulating tree size in citrus. No effects on canopy development become apparent until 4 to 5 years after inoculation, while effects on yield are delayed for a further one to two years. During this interim period, viroid inoculated trees often show enhanced yield efficiency. The delay following inoculation before canopy growth rates are reduced allows normal canopy development to occur during the first few years after planting, which ensures rapid early development of a high canopy bearing volume per hectare to promote high early orchard productivity. Observations on vegetative and reproductive growth patterns made after this initial establishment phase indicated that yield efficiency (yield/tree size) ofviroid inoculated trees was not adversely affected by the reduction in tree vigor. Effects of viroid inoculation on reduced canopy growth were restricted to a reduction in the magnitude of vegetative growth occurring during summer and autumn, confirming the dominant role of these flushes in regulating canopy expansion. Inoculation had no effect on spring flush growth, flowering intensity, inflorescence type, fruit set, or fruit growth rates. Apparently, the reduction in tree vigor and reduced shoot growth during summer and autumn is not sufficient to adversely affect flowering. Timing and duration of vegetative flushes were not affected by viroid inoculation and annual variation in the intensity of vegetative growth flushes followed the same trends in both inoculated and uninoculated trees.

312

R. HUTTON, P. BROADBENT, AND K. BEVINGTON

Ideal stock/scion combinations for high density planting should exhibit moderate vigor and high early cropping efficiencies. In this regard, trees on P. trijoliata are better suited to high density planting than trees on citrange, although in combination with viroid dwarfing, high density plantings on citrange are also highly productive. Appropriate selection of tree spacing will be dependent on expected tree growth rates and vigor. Matching tree growth rates to spacing is important to optimize productivity per hectare and maximize potential returns from high density plantings. Based on tree growth rates and yields recorded in Australian trials, planting densities of 800-1,000 trees/ha would appear suitable for viroid inoculated trees on citrange rootstock whilst a density of 1,000 trees/ha is more suitable for trees on P. trijoliata rootstock. The financial viability of viroid dwarfed high density plantings is dependent on cost of trees, namely, whether trees are purchased from a nursery or propagated by the grower. Tree costs are greater in high density plantings and the peak debt is higher during the early years of orchard establishment. However, maximum productivity is reached at a very young age in high density orchards and the greater cumulative net returns achieved in the long term (15 to 20 years) suggest that planting citrus at high density is a desirable investment option, with profitability being directly related to planting density. Economic factors favoring high density plantings of viroid dwarfed trees are the early income on investment and the containment of fixed costs by increased production efficiency.

LITERATURE CITED Albanese, G., R. La Rosa, M. Tessitori, E. Fuggetta, and A. Catara. 1996. Long term effect ofCVd-III inoculation on citrange, trifoliate and sour orange rootstocks. p. 367-369. In: Proc. 13th IOCV Conf., Riverside, CA. Amir, A., S. Ashkenazi, A. Shaked, and M. Kahn. 1988. Exocartis viroid [CEV] dwarfed trees in Yisreel Valley, Israel. In: Proc. 6th Int. Citrus CongI. 2:913-915. Ashkenazi, S., and Y. Oren. 1988. The use of citrus exocartis virus (CEV) for tree size control in Israel: practical aspects. In: Proc. 6th Int. Soc. Citriculture 2:917-920. Ashulin, 1., O. Lachman, R. Hadas, and M. Bar-Joseph. 1991. Nucleotide sequence of a new viroid species, citrus bent leaf viroid (CBLVd) isolated from grapefruit in Israel: Nucleic Acids Res. 19:4767. Atkinson, D. 1973. Annu. Rep. East MaIling Research Sta. 1972. p. 58-59. Atkinson, D. 1978. Use of soil resources in high density planting systems. Acta Hort. 65:65-89. Bacon, P. E. 1980. Effects of dwarfing inoculations on the growth and productivity of Valencia oranges. J. Hart. Sci. 55:49-55.

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Bar-Joseph, M. 1993. Citrus viroids and citrus dwarfing in Israel. Acta Hort. 349:271-276. Benton, R. J., F. T. Bowman, L. Fraser, and R. G. Kebby. 1950. Scaly butt and stunting of citrus. Dept. Agr. N.S.W. Sci. Bul. 70, 20p. Bevington, K. B. 1992. Initial growth and yield of high density plantings of Valencia orange as influenced by microsprinkler and drip irrigation. Proc. Int. Soc. Citriculture 2:709-710. Bevington, K. B. 1997. High density planting strategies for oranges. 1997. National Citrus Field Day Handbook. Mildura. Nov. 1997. Bevington, K. B., and P. E. Bacon. 1977. Effects of rootstocks on the response of navel orange trees to dwarfing inoculations. Proc. Int. Soc. Citriculture 2:567-570. Bevington, K. B., and B. R. Cullis. 1990. Evaluation of rootstocks for Marsh and Davis grapefruit in the Murray region of New South Wales. Australian J. Expt. Agr. 30: 405-411. Bevington, K. B., R. J. Hutton, S. Papasidero and S. Gee. 1996. Development ofphenological based crop management model for high density citrus. Final Report to Horticultural Research and Development Corporation. CT216. ISBN 0731098226. Bitters, W. P., D. A. Cole, and C. D. McCarthy. 1979. Facts about dwarf citrus trees. Calif. Citrogr. 64:54-56. Boswell, S. B., L. N. Lewis, C. D. McCarty, and K. W. Hench. 1970. Tree spacing of Washington navel orange. J. Am. Soc. Hort. Sci. 106:307-312. Broadbent, P., and C. M. Dephoff. 1992. Virus indexing in the New South Wales citrus improvement scheme. Australian J. Expt. Agr. 32:493-502. Broadbent, P., J. B. Forsyth, K. B. Bevington, and R. J. Hutton. 1986. Citrus tree size control with dwarfing agents. Calif. Citrogr. 71:8-10. Broadbent, P., J. B. Forsyth, R. J. Hutton, and K. B. Bevington. 1992. Guidelines for the commercial use of graft-transmissible dwarfing in Australia: Potential benefits and risks. Proc. Int. Soc. Citriculture 3:697-701. Broadbent, P., L. R. Fraser, and J. K. Long. 1971. Exocortis virus in dwarfed citrus trees. Plant. Dis. Rptr. 55:998-999. Broadbent, P., M. R. Gillings, and B. 1. Gollnow. 1988. Graft-transmissible dwarfing in Australian citrus. p. 219-225. In: Proc. 10th IOCV Conf., Riverside, CA. Broadbent, P., P. Nicholls, and B. Freeman. 1988. Effect of graft transmissible dwarfing agents on lemons. p. 211-218. In: Proc 10th IOCV Conf., Riverside, CA. Calavan, E. c., E. F. Frolich, J. B. Carpenter, C. N. Roistacher, and D. W. Christiansen. 1964. Rapid indexing for exocortis of citrus. Phytopathology 54:1359-1362. Calavan, E. c., and L. G. Weathers. 1961. Evidence for strain differences and stunting with exocortis virus. p. 26-33. In: Proc. 2nd IOCV Conf., Univ. Florida, Gainesville. Castle, W. S. 1978. Controlling citrus tree size with rootstocks and viruses for higher density plantings. Proc. Florida State Hort. Soc. 91:46-50. Castle, W. S. 1985. Citrus rootstocks for tree-size control in Florida. Calif. Citrogr. 70(4):81-84,92. Castle, W. S., and W. H. Krezdorn. 1977. Soil water use and apparent root efficiencies of citrus trees on four rootstocks. J. Am. Soc. Hort. Sci. 102(4):403-406. Chalmers, D. J. 1988. Manipulation of plant growth by regulating plant water deficit and limiting the wetted zone. In: Proc. 4th Int. Micro-Irrigation Congress, Albury, Australia. Chapman, J. C. 1986. Growth management of mature citrus with applied nitrogen. Acta Hort. 175: 173-177. Cohen, M. 1968. Exocortis virus as a possible factor in producing dwarf citrus trees. Proc. Florida State Hart. Soc. 81:108-115. Cohen, M. 1981. Beneficial effects of viruses for horticultural plants. Hort. Rev. 3:394-411.

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Cohen, M., W. S. Castle, R L. Phillips, and D. Gonsalves. 1980. Effect of exocortis viroid on citrus tree size and yield in Florida. p. 195-200. In: Proc. 8th IOCV Conf., Riverside, CA. Cripps, J. E. L., F. Melville, and H. 1. Nicol. 1975. The relationship of Granny Smith apple tree growth and early cropping to planting density and rectangularity. J. Hort. Sci. 50: 291-299. Cullis, B. R, and A. P. Verbyla. 1992. Nonlinear regression modelling and time dependent covariates in repeated measures experiments. Australian J. Stat. 34:145-160. Diener, T. O. 1981. Are viroids escaped introns? Proc. Nat. Acad. Sci. (USA). 50145015. Duran-Vila, N., R Flores, and J. S. Semancik. 1986. Characterisation ofviroid-like RNAs associated with citrus exocortis syndrome. Virology 150:75-84. Duran-Vila, N., J. A. Pina, J. F. Ballester, J. Juarez, C. N. Roistacher, R Rivera-Bustamante, and J. S. Semancik. 1988a. The citrus exocortis disease: a complex of viroid RNAs. p. 152-164. In: Proc. 10th IOCV Conf., Riverside, CA. Duran-Vila, N., C. N. Roistacher, R Rivera-Bustamante, andJ. S. Semancik. 1988b. A definition of citrus viroid groups and their relationship to the exocortis disease. J. Gen. Virol. 69:3069-3080. Fawcett, H. S., and Klotz, L. J. 1938. Exocortis of trifoliate orange. Citrus Leaves 28:8. Fraser, L. R 1949. Gummosis disease of citrus in relation to its environment. Proc. Linn. Soc. NSW 74:5-18. Fraser, L. R, and P. Broadbent. 1979. Virus and related diseases of citrus in New South Wales. NSW Department of Agriculture. Surrey Beattey & Sons Pty. Ltd., Chipping Norton, Sydney. Fraser, L. R, and E. C. Levitt. 1959. Recent advances in the study of exocortis [scaly butt] in Australia. p. 129-133. In: J. M. Wallace (ed.), Citrus Virus Diseases. Univ. Calif. Div. Agr. Sci., Berkeley. Fraser, L. R, E. C. Levitt, and J. E. Cox. 1961. Relationship between exocortis and stunting of citrus varieties on Poncirus trifoliata rootstock. p. 34-39. In: Proc. 2nd IOCV Conf., Univ. Florida, Gainesville. Garnsey, S. M. 1983. Increased freeze damage associated with exocortis infection in navel oranges on Carrizo citrange rootstock. Proc. Florida Sta. Hort. Soc. 95:3-7. Gillings, M. R, P. Broadbent, and B. 1. Gollnow. 1991. Viroids in Australian citrus: Relationship to exocortis, cachexia and citrus dwarfing. Australian J. Plant Physiol. 18: 559-570. Gillings, M. R, P. Broadbent, B. 1. Gollnow and C. Lakeland. 1988. Viroids in Australian citrus. p. 881-895. In: Proc. 6th Int. Citrus CongI., Tel Aviv, Israel. Golomb, A. 1988. High density planting of intensive groves. A challenge and realization. p. 921-930. In: Proc. 6th Citrus CongI., Tel Aviv, Israel. Gross, H. J., G. Knipp, H. Domdey, M. Raba, P. Jank, C. Lossow, H. Alberty, and H. L. Sanger. 1982. Nucleotide sequence and secondary structure of CEV and CSV. Eur. J. Biochem. 121:249-257. Haas, B., A. Klanner, K. Ramm, and H. L. Sanger. 1988. The 7s Rna from tomato leaf tissue resembles a signal recognition particle RNA and exhibits a remarkable sequence complementarity to viroids. EMBO J. 7:4063-4074. Hadas, R, and M. Bar-Joseph. 1991. Variation in tree size and rootstock scaling of grapefruit trees inoculated with a complex of citrus viroids. p. 240-243. In: Proc. 11th IOCV Conf., Riverside, CA. Hadas, R, M. Bar-Joseph, and J. S. Semancik. 1989. Segregation of a viroid complex from

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a graft-transmissible dwarfing agent source for grapefruit trees. Ann. Appl. BioI. 115:515-520. Holliday, R 1960. Plant population and crop yield. Field Crop Abstr. 167: 247-254. Hutton, R J. 1986. The influence of tree size control and plant density on citrus productivity. Acta. Hort. 175:249-254. Hutton, R J. 1991. Principles of high density planting. ACGF Annu. General Meeting, Leeton, NSW. Hutton, R J. 1995. High density citrus management trials. 1995 National Citrus Field Day Handbook. Leeton. Nov. 1995. Hutton, R J., G. D. Batten, A. B. Blakeney, S. Papasidero, and T. Finlay. 1998. Evaluate NIRS for rapid determination of leaf nutrients in oranges for quality fruit production. Final Report to Horticultural Research and Development Corporation. CT433. Sydney, Australia. Hutton, R J., and B. R Cullis. 1981. Tree spacing effects on productivity of high density dwarf orange trees. Proc. Int. Soc. Citriculture 1:186-190. Intrigliolo, F., G. Reforgiato Recupero, and A. Giuffrida. 1992. Effect of planting density and rootstock on performance of Valencia orange. Proc. Int. Soc. Citriculture 2:705-708. Jakab, G., T. Kin, and F. Solymosy. 1986. Viroid pathogenicity and pre-rRNA processing: a model amenable to experimental testing. Biochem. Biophys. Acta 868:190-197. Kapur, S. P., L. G. Weathers, and E. C. Calavan. 1974. Studies on strains of exocortis virus in citron and Gynura aurantiaca. p. 101-109. In: Proc. 6th IOCV Conf., Univ. Florida, Gainesville. La Rosa, R, G. Albanese, M. Renis, and A. Catara. 1988. Viroids and viroid-like RNAs on citrus plants. Proc. Int. Soc. Citriculture 2:903-908. La Rosa, R, M. Tessitori, and E. Fuggetta. 1996. Observations on the effects ofviroid inoculation of grapefruit grafted on Carrizo citrange planted at high density. p. 363-366. In: Proc. 13th Conf. IOCV, Riverside, CA. Levy, L., and A. Hadidi. 1993. Direct nucleotide sequence ofPCR-amplified DNA's ofthe closely related citrus viroids ria and lib (Cachexia). p. 180-186. In: Proc. 12th IOCV Conf., Riverside, CA. Long, J. K., L. R Fraser, and J. E. Cox. 1972. Possible value of close-planted, virus-dwarfed orange trees. p. 262-267. In: Proc. 5th IOCV Conf., Univ. Florida, Gainesville. Marais, L. J., R F. Lee, J. H. J. Breytenbach, B. Q. Manicom, and S. P. van Vuuren. 1996. Association of a viroid with gum pocket disease of trifoliate orange. p. 236-244. In: Proc. 13th Conf. IOCV, Riverside, CA. Meduski, C. J., and J. Velten. 1990. PSTV sequence similarity to large rRNA. Plant Mol. BioI. 14:625-627. Mendel, K. 1969. New concepts in stionic relations in citrus. Proc. Int. Soc. Citriculture. 1:387-390. Nauer, E. M., C. N. Roistacher, E. C. Calavan, and T. L. Carson. 1988. The effect of citrus exocortis viroid [CEV] and related mild citrus viroids [CV] on field performance of Washington navel orange on two rootstocks. p. 204-210. In: Proc. 10th IOCV Conf., Riverside, CA. NeIder, J. A. 1966. Inverse polynomials, a useful group of multi-factor response functions. Biometrics 22:128-141. Palukaitis, P., and M. Zaitlin. 1987. The nature and biological significance oflinear PSTVd molecules. Virology 157:199-210. Perez, R, R Rodriguez, A. Gonzalez, N. del Valle, and N. Duran-Vila. 1992. Dwarf citrus trees for high density plantings. Proc. Int. Soc. Citriculture 2:711-713.

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Phillips, R 1. 1969. Dwarfing rootstocks for citrus. p. 401-406. In: Proc. 1st Int. Citrus Symp., Univ. California, Riverside. Phillips, R 1. 1978. Citrus tree spacing and size control. Proc. Int. Soc. Citriculture, 319-324. Phillips, R 1. 1980. Citrus tree spacing and size control. Proc. Int. Soc. Citriculture, Griffith, NSW, Australia. 1:319-324. Piner, G. F. 1988. Planting citrus at ultra high densities: A review of developments in Southern Africa with special reference to angle planting. Proc. Int. Soc. Citriculture 2:931-940. Polizzi, G., G. Albanese, A. Azzaro, M. Davino, and A. Carter. 1991. Field evaluation of dwarfing effect oftwo combinations of citrus viroids on different citrus species. p. 230233. In: Proc. 11th IOCV Conf., Riverside, CA. Puchta, H., K. Ramm, R Hadas, M. Bar-Joseph, R Luckinger, K. Friemuller, and H. 1. Sanger. 1989b. Nucleotide sequence of a hop stunt viroid (HSVd) isolated from grapefruit in Israel. Nucleic Acids Res. 17:1247. Puchta, H., K. Ramm, R Luckinger, R Hadas, M. Bar-Joseph, and H. 1. Sanger. 1991. Primary and secondary structure of citrus viroid IV (CVd-IV), a new chimaeric viroid present in dwarfed grapefruit in Israel. Nucleic Acids Res. 19:6640. Puchta, H., and H. 1. Sanger. 1989a. Sequence analysis of minute amounts ofviroid RNA using the polymerase chain reaction. Arch. Virol. 106:335-340. Rabe, E., N. Cook, G. Jacobs, and H. P. van der Walt. 1992. Current status of research on citrus tree size control in Southern Africa. Proc. Int. Soc. Citriculture 2:714-720. Rackwitz, H. R, W. Rohde, and H. 1. Sanger. 1981. DNA dependent RNA polymerase II of plant origin transcribes viroid RNA into full length copies. Nature 291:297-301. Rakowski, A., J. A. Szychowski, Z. S. Avena, and J. S. Semancik. 1994. Nucleotide sequence and structural features of the group III citrus viroids. J. Gen. Virol. 75:3581-3584. Rivera-Bustamante, R, R Gin, and J. S. Semancik. 1986. Enhanced resolution of circular and linear molecular forms of viroid and viroid-like RNA by electrophoresis in a discontinuous-pH system. Anal. Biochem. 156:91-95. Roberto, S. R, 1. C. Donadio, O. Sempionato, J. R M. Cabrita, and J. D. De Negri. 1992. Tree growth in high densities and costs for planting citrus in Brazil. Proc. Int. Soc. Citriculture 2:721-722. Rodriguez, J. 1., and F. Flores. 1987. Effects of citrus exocortis viroid infection on the peroxidaselIAA-oxidase system of Gynura aurantiaea and Lyeopersieon eseulentum. Biochem. Physiol. Pflanz. 182:449-457. Roistacher, C. N. 1992. Dwarfing of citrus by use of citrus viroids: Pros and cons. Proc. Int. Soc. Citriculture 3:791-796. Roistacher, C. N., J. A. Bash, and J. S. Semancik. 1993. Distinct disease symptoms in Poneims trifoliata induced by three citrus viroidsfrom three specific groups. p. 173-179. In: Proc. 12th IOCV Conf., Riverside, CA. Roose, M. (1986). The potential for dwarfing rootstocks for citrus. Calif. Citrog. 71:225-229. Rossetti, V., J. Pompeu, O. Rodriguez, M. H. Vechiato, M. 1. da Veiga, D. A. Oliveira, and J. T. Sobrinho. 1980. Reaction of exocortis-infected and healthy trees to experimental Phytophthora inoculations. p. 209-214. In: Proc. 8th IOCV Conf., Riverside, CA. Salibe, A. A. 1988. Exocortis mild strains to control Tahiti lime tree size. p. 423. (Abstract). Proc. 10th IOCV Conf., Riverside, CA. Schwinghamer, M. W., and P. Broadbent. 1987. Association of viroids with graft transmissible dwarfing symptoms in Australian orange trees. Phytopathology 77:205-207. Schwinghamer, M. W., and P. Broadbent. 1987. Detection of viroids in dwarfed orange

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trees by transmission to chrysanthemum. Phytopathology 77:210-215. Semancik, J. S. 1986. Separation of viroid RNAs by cellulose chromatography indicating conformational distinctions. Virology 155:39-45. Semancik, J. S., D. J. Gumpf, and J. A. Bash. 1992. Interference between viroids inducing exocortis and cachexia diseases in citrus. Ann. Appl. BioI. 121:577-583. Semancik, J. S., A. G. Rakowski, J. A. Bash, and D. J. Gumpf. 1997. Application of selected viroids for dwarfing and enhancement of production of Valencia orange. J. Hort. Sci. 72:563-570. Semancik, J. S., C. N. Roistacher, R Rivera-Bustamante, and N. Duran-Vila. 1988. Citrus cachexia viroid, a new viroid of citrus: relationship to viroids of the exocortis disease complex. J. Gen. Virol. 69:3059-3068. Semancik, J. S., and L. G. Weathers. 1972. Exocortis disease: an infectious free nucleic acid plant virus with unusual properties. Virology 47:456-466. Solymosy, F., and T. Kiss. 1985. Viroids and snRNAs. p. 183-199. In: K. Maramorosch (ed.), Subviral pathogens of plants and animals: Viroids and prions. Academic Press, Orlando, Fla. Stannard, M. c., J. C. Evans, and J. K. Long. 1975. Effect of transmission of exocortis dwarfing factors into Washington navel orange trees. Australian J. Expt. Agr. Animal Husb. 15:136-141. Stasys, R A., 1. B. Dry, and M. A. Rezaian. 1995. The termini of a new citrus viroid contain duplications of the central conserved regions from two viroid groups. FEBS Lett. 358:182-184. Symons, B. 1989. Pathogenesis by antisense. Nature 338:542-543. Takahashi, T., S. Fujiwara, K. Chiba, and N. Yoshikawa. 1992. Comparison of plant hormone requirements in leaf tissues from hop stunt viroid-infected and uninfected hop plants. Z. Pflanz. Pflanzenschutz 99:62-70. Taylor, R, M. Gleeson, P. Broadbent, D. Hailstones, 1. Barchia, and M. Gillings. 1997. Improved detection methods for citrus viroids. Final Report to Horticultural Research and Development Corporation. CT311. Sydney, Australia. Tribulato, K, G. Continella, and G. La Rosa. 1992. Research on higher density planting for orange and lemon. Proc. Int. Soc. Citriculture 2:702-704. Weathers,1. G., and K C. Calavan. 1961. Additional indicator plants for exocortis and evidence for strain differences in the virus. Phytopathology 51:262-264. Wertheim, S. J. 1985. Productivity and fruit quality of apple in single row and full field planting systems. Scientia Hort. 26:191-208. Wheaton, R A., W. S. Castle, D. P. H. Tucker, and J. D. Whitney. 1978. Higher density plantings for Florida citrus-Concepts. Proc Florida. Sta. Hort. Soc. 91:27-33. Yaguchi, S., and T. Takahashi. 1985. Syndrome characteristics and endogenous indoleacetic acid levels in cucumber plants incited by hop stunt viroid. Z. Pflanzenkrank. Pflanzenschutz 92:263-269.

7

Growth, Development, and Cultural Practices for Young Citrus Trees Frederick S. Davies and James J. Ferguson Horticultural Sciences Department University of Florida Gainesville, Florida 32611-0690

I. Introduction II. Growth and Development A. Seedlings B. Budded Trees III. Tree Selection and Planting IV. Irrigation A. Scheduling B. Reclaimed Wastewater C. Salinity V. Fertilization VI. Freeze Hardiness and Protection A. Freeze Hardiness B. Freeze Protection VII. Pruning VIII. Biotic Factors A. Fungal Diseases B. Virus and Virus-like Diseases C. Bacterial Diseases D. Insects and Mites E. Nematodes F. Vertebrates G. Weeds IX. Summary Literature Cited

Horticultural Reviews, Volume 24, Edited by Jules Janick ISBN 0-471-33374-3 © 2000 John Wiley & Sons, Inc. 319

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I. INTRODUCTION Citrus crops, including oranges, mandarins, grapefruit, lemons, and limes, with total production of 88 million tonnes in 1996, are among the leading world fruit crops (FAG 1996). Moreover, there was a steady increase in planting of new trees during the 1980s, particularly in Florida, Cuba, China, Mexico, and Turkey. The number of new plantings reached a plateau in the 1990s in most regions due to over-supply and generally low prices worldwide, and probably will continue to do so into the twenty-first century. It is difficult to estimate the number of young trees (defined here as nonbearing trees that have been in the orchard less than three years) worldwide. In Florida alone, 6 to 10 million new trees are planted yearly (Florida Agr. Stat. Servo 1996). Furthermore, millions of young trees are planted each year to replace those lost due to diseases, blight, pests, drought, flooding, and freeze damage. For example, about 3 to 5% of the citrus area in Florida is replaced yearly (Ferguson and Taylor 1993). But this percentage is considerably higher in areas such as Venezuela where losses to citrus tristeza virus (CTV) were extensive. Moreover, millions of young trees have been planted in Brazil and in areas of rapid citrus expansion such as China, Turkey, Morocco, and Mexico. Production practices differ for young trees in contrast to those for mature trees. The relatively smaller root, canopy, and trunk sizes make young trees more susceptible to environmental stresses and more vulnerable to defoliation by pests and diseases, and to competition for water and nutrients by weeds. The primary objective in growing a young tree is to develop a canopy and root system and to bring the tree into production as soon as possible after planting. Early production is extremely important in today's economic climate. For example, in Florida the average cost to bring a tree into production, including tree planting, grove preparation, and cultural costs, is $37.73 for the first 3 years in the field (Muraro 1997). The objectives of this review are to discuss environmental factors involved with the growth and development of citrus trees from seedling or budded tree through the first 3 years in the field. In addition, recent research on planting procedures and cultural practices such as irrigation, fertilization, freeze protection, and weed, pest and disease control will be reviewed. Information has been obtained from many citrus-growing regions worldwide, but in some sections the bulk of the information is from Florida.

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II. GROWTH AND DEVELOPMENT

Most commercially grown citrus is produced as a two-part tree consisting of a rootstock and a scion. In the past, trees were grown from seed, but seedlings generally have a long juvenility period and despite the presence of somatic embryogenesis (nucellar polyembryony), the resulting seedlings may not be true-to-type (genetically the same as the maternal tree). Additionally, rootstocks offer obvious advantages in improving production and vigor and reducing losses from soil-borne diseases, viruses, and viroids (Castle 1987a). Therefore, most scion cUltivars are budded or grafted onto a rootstock. Conversely, most citrus rootstocks are grown from seed because nearly all commercially important rootstocks produce a high percentage of nucellar seedlings that are true-to-type. Moreover, most citrus viruses and viroids, with one isolated exception of psorosis, are not transmitted through seed. Thus, the growth and development of the rootstock seedling itself is also of importance, particularly to citrus nursery growers. A. Seedlings

Several factors affect seedling growth and development, the most important of which are temperature, light, and water. Temperature is a very important factor in seed germination and subsequent seedling growth. An early study by Fawcett (1929) in California determined that the optimum temperature for seed germination and seedling growth 90 days after planting was 23° to 29°C for sweet orange and 23° to 26°C for sour orange. Days to emergence of the radicle (primary root) was greatly delayed as temperature decreased below the optimum, with little or no germination at 12.5°C, or at temperatures above 40°C. Plant growth was also abnormal above 35°C. Camp et al. (1933) conducted similar studies in Florida over a 6-year period using sweet and sour orange, rough lemon, and grapefruit seeds. They observed considerable seed-to-seed variation in germination time even within the same temperature range. Optimum soil temperatures were estimated to be 31° to 34°C for sweet orange and grapefruit, 34°C for rough lemon, and 32° to 34°C for sour orange. Minimum temperature for germination was below 15°C and the maximum above 40°C, as observed by Fawcett (1929). Average time until emergence ranged from about 15 days at optimum temperatures to 84 days at 13°C. Temperature ranges for germination were slightly different for 'Cleopatra' mandari~ and trifoliate orange (Mobayer 1980b).

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Emergence rate increased linearly from 15° to 35°C. Moreover, germination time between the first and last seeds within a group decreased with increasing temperatures from 11° to 30°C. Trifoliate orange and 'Cleopatra' have lower optimum (30°C) and minimum (6° to 10°C) germination temperatures than the previously mentioned cultivars, probably because of their more temperate origins. In fact, trifoliate orange is deciduous and, unlike Citrus cultivars, seeds germinate better if exposed to 7°C for 12 weeks prior to planting (Mobayer 1980a). Wiltbank et al. (1995) repeated much of the previous work but used a wider temperature range and more carefully controlled temperatures using a temperature gradient bar. Optimum emergence temperatures were similar, 31° to 34°C, to those reported above and there were slight differences among species (Fig. 7.1). Time to emergence ranged from 9 to 104 days.

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In addition, removal of the seed coats greatly reduced the time to first emergence. Again, days to emergence were increased considerably below 15° or above 40°C. Minimum temperatures for germination varied slightly from previously reported values, but were generally in the same range. Recent work by Rouse and Sherrod (1997) expanded upon Wiltbank's study using the same carefully controlled temperatures. The authors determined optimum germination temperatures for 17 citrus cultivars and related species. As expected, a range of optima occurred from 25°C for Poncirus trifoliata to 33°C for 'Rangpur' x 'Troyer'. Days to germination also varied from 5 to 28 depending on species and temperature. Stoffella et al. (1995) tested the effects of CO 2 -enriched irrigation water on seedling emergence and growth of four citrus rootstocks (rough lemon, 'Carrizo' citrange, 'Cleopatra' mandarin, and sour orange). Germination and seedling growth differences were observed among rootstocks, with rough lemon seedlings having the lowest mean days to germination. 'Carrizo' citrange seedlings had the greatest stem diameters and were tallest 72 days after planting. Daily stem diameter growth increased at 0.03 mm and shoot elongation daily rate was 0.15 mm at 27°/17°C (day/night) temperatures. Citrus seeds have hypogeous germination and usually the radicle is the first organ to emerge (Fig. 7.2A). As seeds germinate, they rapidly take up water for the first 24 to 36 h, with uptake rates reaching a plateau after that time (Mobayer 1980b). Light is not required for germination. The primary shoot is next to emerge (Fig. 7.2B). Initial growth emanates from the shoot and root apical meristem. Development of first foliage leaves and lateral branches (Fig. 7.2C) occurs a few weeks after emergence, depending largely on temperature. Initial shoot growth occurs as a single stem emanating from a single apical meristem until lateral branches form later in development (Fig. 7.2D). Temperature also has a pronounced effect on seedling growth. Peltier (1920), who was actually interested in temperature effects on growth of citrus canker, also measured growth of citrus and citrus hybrids. Optimum growth occurred at 20° to 30°C depending on species, with limited growth below about 15°C or above 35°C. He also astutely identified differences between temperature optima and minima for citrus compared with Poncirus and Poncirus hybrids. Of interest, growth of grapefruit was inhibited above 35°C, whereas Poncirus, a more temperate genus, continued to grow. However, a limited number of trees was used in each of five experiments. Similarly, Girton (1927) found that seedling stem and leaf growth rates of sweet oranges increased as temperature increased from 13° to 31°C. Temperature ranges for seedling growth in this study generally were in agreement with those of previous studies

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Fig. 7.2. Germination and early growth and development of citrus seedlings. (A) Seed with primary root, (B) seed with primary shoot, (C) seedling with first foliage leaves and secondary (lateral) roots, (D) seedling during early development.

for seed germination. The minimum temperature for growth was 12°C and the maximum about 37°C. Young and Peynado (1962) examined effects of constant daytime and variable nighttime temperatures on growth and freeze hardiness of 30 citrus and related species. Minimum nighttime growth temperatures ranged from 13.8° to 15°C for Poncirus to below 8.8°C for C. macrophylla, limes, and lemons. Growth of lemon types at low soil temperatures may contribute to their greater freeze sensitivity compared to Poncirus species and hybrids (Wilcox and Davies 1981). Several studies have demonstrated the important effect of soil (root) temperature on growth of citrus trees. In a classic study, Girton (1927) exposed sour orange seedlings to several root temperatures. Growth rates were low at 13°C; optimum total root length and root elongation

7. GROWTH, DEVELOPMENT, AND CULTURAL PRACTICES FOR CITRUS TREES

325

rate occurred between 23° and 27°C. Root hair index also increased significantly with increasing temperatures until 37°C was reached. In a similar study, Halma (1935) grew rooted cuttings of sweet orange, lemon, and grapefruit at root temperatures of 16° to 27°, 12° to 22°, and 3° to 20°C for nearly a year. Lemon cuttings grew considerably more than the other cultivars and growth increased with increasing root temperature. Orange and grapefruit cuttings showed decreased growth in the 3° to 20°C range. Optimum soil temperatures were estimated to be 31° to 34°C for sweet orange and grapefruit, 34°C for rough lemon, and 32° to 34°C for sour orange. B. Budded Trees

In many commercial citrus areas, seedlings are grown in the field or the greenhouse until the stem attains 5 to 10 mm in diameter. This occurs 3 to 6 months after planting, depending on temperature, light, water, and adequate pest and disease control. The seedling (rootstock) is then budded or grafted using the desired scion cultivar. Inverted or regular T budding is the most commonly used method in most citrus regions (Davies and Albrigo 1994). A vertical slit about 2 to 4 cm long is made in the rootstock stem followed by a horizontal cut at right angles, thus completing the T shape. The bud is then removed from the scion budstick, inserted into the slit, and wrapped with plastic tape. After 1 to 2 weeks the bud has "taken," i.e. the cambium of the rootstock and scion grow together (Jackson 1991). The scion is "forced" to grow in several ways because the new bud will not grow or will grow poorly if the rootstock is allowed to grow normally. Consequently, the stem above the bud is removed entirely, cut partially through and bent over (bending) or just bent over and tied (lopping). Rouse (1988) found that the new bud grew most rapidly if the rootstock was lopped or bent. Williamson et al. (1992) found growth was best when shoots were bent rather than lopped or cut (Fig. 7.3). Photosynthate translocation from rootstock leaves was also greatest for bent vs. cut or lopped treatments, which likely contributed to increased growth. The pattern of shoot growth after forcing is interesting. From day 8 to 32 there was a rapid increase in shoot length, with an average daily growth rate of 0.66 cm for the bent treatment (first growth flush). Shoot growth ceased from day 32 to 75 and then resumed from day 75 to 120 at about 0.62 cm (second growth flush). Guazzelli et al. (1995) also measured shoot growth rate in a greenhouse nursery as part of a fertilizer study. In this instance, daily growth rates ranged from 0.21 to 0.33 cm probably due to differences in temperature between the two studies.

F. DAVIES AND

326

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Fig. 7.3. Scion length of citrus nursery trees following bud forcing by cutting off, lopping, or bending. Data points and bars represent means ± SE (n ranged from 5 to 22 depending on measuring date). Source: Williamson et al. (1992).

Media (root) temperatures less than about 15°C inhibit shoot growth in the nursery, particularly for trees on 'Swingle' citrumelo rootstock. Increasing media temperatures from 15° to 25°C significantly increased percentage budbreak (Al-Jaleel and Williamson 1993). Thus, the authors suggested that bottom or container heating may be useful for nurserymen to improve growth of trees on 'Swingle' citrumelo rootstock. The influence of root temperature on growth was further supported in field studies using budded trees of 'Washington' navel and 'Eureka' lemon. Generally, growth ceased during the season when soil temperatures were below 18°C (Halma and Compton 1936). Air (canopy) temperature was not controlled in either study; obviously it also has an effect on tree growth. Liebig and Chapman (1963) grew 'Washington' navel oranges in the greenhouse on three rootstocks at root temperatures of 14 0, 22° or 30°C. Vegetative growth increased with increasing root temperature, but flowering was greatest at 14°C, with no flowering occurring at 30°C. When plants were moved from 30° to 14°C, they flowered. Rootstock and soil type did not affect growth, possibly due to the short duration of the study (9 months).

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It has long been observed that citrus trees grow in flushes (Reed 1928; Reed and MacDougal 1937). The pattern and periodicity of lemon shoot growth was first defined mathematically by Reed (1928), who identified three growth flushes for mature lemon trees growing in California. Each flush showed a characteristic sigmoidal growth curve. About the same time, Crider (1927) showed that there is a distinct alternation of root and shoot growth. Young citrus trees typically have 3 to 5 growth flushes per year in subtropical areas, with almost continuous growth flushing in low tropical regions due to high heat unit accumulation (Davies and Albrigo 1994). Bevington and Castle (1985) clearly demonstrated the alternation of shoot and root growth using 13-month-old 'Valencia' orange trees growing in large chambers placed in the field; their data further suggest that root and shoot growth occurs in flushes (Fig. 7.4). In winter and spring, shoot and root growth alternate, but by late summer both occur simultaneously, probably due to high average soil temperatures. Mean root elongation rate increased linearly from 17° to 30°C.

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328

F. DAVIES AND J. FERGUSON

There have been several studies on the growth and development of young, budded citrus trees in the field. Dry matter accumulation is probably the most accurate indicator of young tree growth, but such measurements require harvesting the tree, which is very time-consuming and will terminate the study. Thus, growth rate is usually expressed by measuring trunk diameter (trunk cross sectional area), shoot growth, tree height, or canopy volume. Marler (1988) found that trunk diameter had the greatest correlation to dry mass (r = 0.90), while tree height had a much lower correlation coefficient (r = 0.60). Shoot growth and canopy volume are quite variable also, but are often used to quantify growth. Trunk diameter in subtropical areas typically increases slowly after planting until the tree becomes established (Davies et al. 1996). There is then a nearly linear increase throughout the summer until growth slows during the winter months as temperatures decrease. The yearly rate of increase in trunk diameter varies with species. Grapefruit trees increased in trunk diameter by 21 % to 85% (Maurer and Davies 1993b; Maurer et al. 1995), and oranges by 40% (Marler et al. 1987) to 100% (Ferguson and Davies 1995) after the first year in the field under humid subtropical conditions in Florida. The percent change in diameter likely is higher in low tropical and lower in arid subtropical growing regions due to differences in heat unit accumulation, irrigation, or rainfall. Increases in trunk diameter are generally less as a percentage of initial stem size during subsequent years (Ferguson and Davies 1995). Shoot number and length vary with time of growth flushes. Guazzelli et al. (1996) found an average of 100 to 150 new shoots during the spring flush, with only 1 to 30 new shoots being produced during the summer flush for the first growing season. Shoot length, however, was much greater for the second compared with the first flush. This relationship between shoot number and length has also been observed for mature trees (Sauer 1951). In a similar study, Marler and Davies (1990) found that l-year-old 'Hamlin' orange trees produced 950 to 1429 cm of new shoot length and 1.3 m 2 of new leaf area the first year in the field. Canopy volume also increased several-fold during the first 3 years in the field. Root growth, although often overlooked, is also impressive during the first year in the field. 'Valencia' orange trees on rough lemon and 'Carrizo' citrange rootstocks produced more than 152 m and 103 m of fibrous roots with surface areas of 1989 cm 2 and 3137 cm 2 , respectively, in only 13 months after planting (Bevington and Castle 1985). Marler and Davies (1990) observed that total root (tap, structural, and fibrous) dry weight increased by 126 to 191 g, depending on the season, in only 8 months after planting for 'Hamlin' orange trees.

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Root systems of young citrus trees also spread quite extensively laterally and vertically after planting, particularly in sandy soils. Bevington and Castle (1985) studied root distribution of 'Valencia' orange trees on rough lemon or 'Carrizo' citrange rootstocks. Trees were grown in large plexiglass chambers in the field. Within 3 months of planting, new roots had developed from the tap root, which is typically severed during digging from the nursery to a 90-cm depth. Moreover, they had attained a depth of 150 cm within 8 months of planting. Lateral roots spread horizontally from the tap root to distances of 178 cm. Similarly, Marler and Davies (1990) observed an average lateral extension of roots of 137 cm within 8 months of planting. In this case, 70 to 80% of the entire root mass was located within 40 cm of the tap root. In other experiments, they observed that roots were located within only 30 cm of the soil surface and, in fact, that nearly 50% of the total root mass was within 10 to 20 cm of the surface (Marler and Davies 1989). Lateral root spread of 150 cm (Till and Cox 1965) and 160 cm (Aiyappa and Srivastava 1965) was also measured in very different soil types for orange trees in Australia and mandarin trees in India. Certainly, soil type, climatic conditions, and species account for these variations in root distribution among citrus regions. III. TREE SELECTION AND PLANTING

Proper tree selection and planting can greatly improve survival and growth of young citrus trees in the field and are important first steps in establishing a successful orchard. Citrus nursery trees are produced in field nurseries as bare-root or occasionally balled and burlaped trees or in containers in greenhouse and field nurseries. Small container-grown trees are also transplanted into large fabric bags grown in the ground for later transplanting as larger trees. For both containerized and bare-root trees, root growth patterns and field planting methods affect plant establishment, root development, and tree growth in the field. Castle (198 7b) reported that root-binding of container-grown trees severely restricted root growth and expansion in the field. Bare-root trees also grew poorly when roots were pruned or forced into undersized planting holes. Disturbing the root ball of containerized trees at planting (Castle 1987b) and partially or completely removing the planting medium (Marler and Davies 1987) increased growth. Protecting roots of bare-root trees from drying during transplanting also improved plant establishment and growth more than various pruning or irrigation treatments (Grimm 1957). Nevertheless, planting trees at the proper depth, especially in areas where

330

F. DAVIES AND J. FERGUSON

Phytophthora is a problem, probably has the greatest effect on subsequent tree survival (Davies and Albrigo 1994). Propagation of citrus nursery trees on genetically similar nucellar rootstocks may reduce variability due to rootstock as a factor in the growth of young citrus trees. Several workers have studied the effect of nursery tree size or type on tree growth in the field. Gardner and Boranic (1959) reported initial tree size did not affect growth and yield of sweet oranges after 17 years in the field. In later studies focusing on the early growth of bare-root vs. containerized trees, Marler and Davies (1987) reported that trunk cross-sectional area of bare-root trees was greater than that of containerized trees of comparable initial size after 20 months, but that canopy volume was similar. In contrast, Willis et al. (1991), in a young tree fertilization study where the N rate was kept constant, reported no growth differences after one year in the field for trees grown for 1.5 years in the greenhouse nursery and bare-root trees grown for 2 years in the field nursery. Productive orchards have been established using both types of nursery trees. IV. IRRIGATION Citrus trees in general are fairly well-adapted to drought conditions. Leaves have thick cuticles and low stomatal conductance that reduce water loss during drought (Syvertsen 1982; Syvertsen and Lloyd 1994). Nevertheless, growth and development of young citrus trees can be adversely affected by water stress (Kriedemann and Barrs 1981). Therefore, from a commercial standpoint, proper water management is important, especially in arid and semi-arid regions. In fact, improved water management is a major reason for increased early growth and fruiting in many citrus-growing regions. For thousands of years, young citrus trees were irrigated using basin or furrow flooding. These methods are still used in many citrus-growing regions. Flood irrigation provides large amounts of water, much of which is outside the rooting zone of the tree. Often young citrus trees in particular are subjected to periods of water stress and reduced growth when flood irrigation is used because it is applied at irregular intervals. The widespread use of microirrigation has increased frequency of irrigation and thereby increased growth and precocity of young citrus trees. A. Scheduling The key factors involved in proper irrigation include correct timing, proper distribution of water, and applying the correct amount of water.

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Irrigation timing is typically based on soil factors such as amount of available water, tree factors such as visible wilting, or climatic factors such as evapotranspiration (ET). However, many citrus growers also irrigate based on days since the last irrigation or rainfall, the calendar method, or based on the appearance of the soil or tree. Each of the above methods of scheduling irrigation has advantages and disadvantages from both practical and scientific perspectives. Rodney et al. (1977) compared growth of 'Valencia' orange trees on C. macrophylla and 'Troyer' citrange rootstocks over a 5-year period in Arizona. Irrigation was scheduled using tensiometers, although specific soil moisture tension levels were not given. They found that drip irrigation produced larger trees than those receiving flood, basin, or sprinkler irrigation, and that it also used considerably less water for the first 2 years after planting. However, in the ensuing 4 years, growth rates were lower for drip-irrigated trees than those on the other treatments. The authors suggested that this occurred possibly due to accumulation of salts in the root zone of drip-irrigated trees. Leaching of salts occurred using the other methods. Irrigation method also affected root density and distribution, with the greatest overall root mass observed with basin irrigation. Root density was greatest near the emitters, as is typically the case for root distribution in semi-arid or arid regions where microirrigation is used. Smajstrla et al. (1985) also used tensiometers to schedule microsprinkler irrigation for 2-year-old 'Valencia' orange trees in Florida. Tree growth the first year was greatest at 20 kPa (moderate) soil water tension compared with 10 (wet) or 40 (dry) kPa. The importance of maintaining adequate but not excessive (10 kPa) moisture levels in the root zone was emphasized. Irrigation is also scheduled based on soil water content, expressed as % weight of available soil moisture. Marler and Davies (1990) compared growth of 1-year-old 'Hamlin' orange trees maintained at 20, 45, or 65% soil water depletion (SWD) as measured by a neutron probe. Soil water depletion refers to the amount of water removed from the root zone relative to the available water. Root and canopy growth was the same at 20 or 45% SWD, although trees in the 45% treatment received far fewer irrigations and less water than the 20% treatment. Trees irrigated at 65% SWD had significantly less root and shoot growth than trees in the other two treatments (Table 7.1). Therefore, optimum SWD for these sandy soils in Florida ranged from 30 to 45% SWD. Climatic factors may also be used to schedule irrigation. DeBarreda et al. (1984), working in Spain, compared growth of navel orange trees on 'Troyer' citrange rootstock using either drip or basin irrigation. Irrigation was based on class A pan evaporation of 55 or 100 mm per year.

F. DAVIES AND J. FERGUSON

332

Table 7.1. Growth characteristics of young 'Hamlin' orange trees as related to irrigation based on soil water depletion, 1985-87. Z Source: Marler and Davies (1990). Soil water depletion (%)

Canopy volume (m 3 )

Trunk crosssectional area (cm 2 )

Canopy dry weight (g)

Shoot length (em)

Leaf area (m 2 )

1985

20 45 65

0.57 0.52 0.33**

5.1 4.9 4.2**

425 426 337**

951 951 752**

1.3 1.2 1.0**

379 383 300**

950 943 894

1.3 1.3 0.9**

393 259** 230**

1429 937* 872*

1986

20 45 65

0.51 0.54 0.31**

4.9 4.8 4.2* 1987

20 45 65

0.56 0.37 0.36

4.7 3.8* 3.3*

1.4 0.7* 0.5*

ZMeans of 20 (1985), 21 (1986), and 5 (1987) trees/treatment. *,**Response is significant when compared with the 20% soil water depletion treatment by the Williams method at P = 5% or 1%, respectively.

The results are difficult to interpret from the tables, but the authors concluded that for drip-irrigated trees, increasing amounts of water and frequency of irrigation increased trunk diameter the first year in the field, while the weight of feeder roots was much greater, as expected, in the wetted compared with nonwetted areas. These results are typical for trees grown in semi-arid or arid climates with limited rainfall. However, for the basin-irrigated trees, a pan coefficient of 0.15 was best in year 1, increasing to 0.20 and 0.30 in year 3. Increasing frequency of irrigation in this case did not increase growth. In a similar study, Castel (1993) applied four levels of drip irrigation based on evapotranspiration (ET) of 30 to 200% to young 'Nules Clementine' mandarin trees in Spain. He suggested that there was a linear increase in trunk diameter with increasing irrigation rate; yet, the data indicated that initial tree size had more effect on final size than irrigation rate. Canopy volume and amount of the orchard floor covered increased with increasing irrigation level. In a related study, Castel and Buj (1992) compared the growth of l-year-old 'Clementine' trees at two irrigation frequencies and four different application rates (Fig. 7.5). Amount of water applied was based on 130,100,

7. GROWTH, DEVELOPJ\1ENT, AND CULTURAL PRACTICES FOR CITRUS TREES

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334

F. DAVIES AND J. FERGUSON

70, and 40% of pan evaporation adjusted for a citrus crop coefficient. Irrigation frequency (1 vs. 2 times/week) had no effect on growth. Irrigation level, however, did affect growth. Trunk diameter and leaf area index was significantly less at the lowest rate (40%). Optimum growth for the first 2 years in the field occurred with water applied at 70% ET once/week. During the third year of the study, the 130% ET treatment produced the greatest trunk diameter and canopy volume. Several of the above irrigation scheduling methods were compared for young 'Ray Ruby' grapefruit trees in Texas (Swietlik 1992). The soil consisted of 90 to 130 cm of clay or sandy clay with a sandy clay loam subsoil. Trees were irrigated based on class A pan evaporation, soil tension using tensiometers, or SWD using trickle or flood irrigation. Generally a greater amount of water was applied when basing irrigation on pan evaporation compared with tensiometer readings and in most years growth was similar. The most significant finding from the study was that trickle irrigation used 90% less water than flooding without reducing tree growth. Previously, Leyden (1975) had observed a reduction in water use by trickle irrigation of about 82% over flood-irrigated trees. Davies (unpublished) compared growth of 1-year-old 'Hamlin' orange trees using the calendar, SWD, and leaf wilting methods of scheduling irrigation. With the calendar method, trees were irrigated every 2 to 3 days if no rain had occurred regardless of soil water content. This is a common method of scheduling irrigation. The SWD method utilized neutron probe readings to schedule irrigation as described previously by Marler and Davies (1990). A third group of trees was irrigated only when the leaves were visibly wilted. Surprisingly, no differences in tree vigor, trunk diameter, or height occurred among treatments even though trees on the calendar system received 24 irrigations per year compared with 8 for the SWD method and only 1 for the leaf wilting point method. Thus, under the high rainfall and humidity conditions in Florida, young citrus trees appeared less affected by infrequent irrigation than previously believed (Davies et al. 1989). Apparently, young citrus trees adapt well to drought conditions via stomatal closure. Proper coverage of the root system by the irrigation system is also considered necessary to achieve maximum growth and early fruiting. Azzena et al. (1988) measured tree growth and root distribution of young 'Valencia Late' orange trees using various dripper and microsprinkler placements around the tree and concluded that the greatest growth occurred when four drippers were placed around the tree, 1 m from the trunk. However, no growth data were presented. In the moderately fine-textured soils of Spain, drippers were more efficient in delivering water than microsprinklers. Similarly, Bevington (1992) in Australia determined

7. GROWTH, DEVELOPMENT, AND CULTURAL PRACTICES FOR CITRUS TREES

335

that growth was greater during the first 4 years after planting for dripcompared with microsprinkler-irrigated trees. Moreover, drip irrigation used only 16% of the water applied by microsprinklers. In contrast, Marler and Davies (1989) observed no differences in growth of 2-year-old 'Hamlin' orange trees whether they were irrigated using 90° or 180° pattern microsprinklers. Apparently, the 90° pattern covered enough of the root zone to prevent reductions in tree growth. Similarly, Davies et al. (1996) found that growth of 1- to 2-year old interset 'Hamlin' orange trees was unaffected by microsprinkler water distribution pattern. For mature citrus trees in Florida, 50 to 60% of the root system should be covered by the irrigation pattern to obtain optimum yields (Smajstrla and Koo 1984), but the amount of a young tree root system that should be covered to maximize vegetative growth has been suggested in some of the above studies but not determined precisely. Obtaining adequate root zone coverage is also a function of duration of irrigation and soil type. In general, drippers are operated for 1 to 4 h depending on the soil type, which affects lateral movement of water. Microsprinklers should be operated for 2 h or less (this is not always the case in practice) depending on emitter output and pattern and rooting depth (Marler and Davies 1989). Leaching of nutrients and pesticides may occur when irrigation systems are operated longer than necessary. Overirrigating is costly and may actually reduce tree growth in finetextured, poorly drained soils due to a decrease in soil oxygen levels. B. Reclaimed Wastewater In many citrus growing regions, water for irrigation is limited, expensive, or of poor quality. In some of these regions, considerable quantities of municipal reclaimed wastewater (usually secondary- or tertiary-treated) is available that is usually disposed of by discharge into bodies of water or the soil. Reclaimed water in some locations may contain nutrients and heavy metals that can contribute to surface or ground water pollution. As long as 60 years ago, reclaimed water was used to irrigate citrus in Egypt (Omran et al. 1988). Reclaimed wastewater has also been used for citrus irrigation in Spain, Israel, and the United States. Nevertheless, there is concern that high sodium, chloride, and boron levels in reclaimed water may reduce growth of young citrus trees. In addition, reclaimed water is usually applied at very high rates that may produce anoxic conditions in the soil or promote damage by diseases such as Phytophthora, thus reducing growth or causing tree death, especially in fine-textured soils. Maurer and Davies (1993a) observed the effects of reclaimed water on growth of 'Redblush' grapefruit trees in Florida for the first 3 years in the

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F. DAVIES AND J. FERGUSON

field. Reclaimed water was applied 3 times/week regardless of rainfall, at rates of 1.9 to 2.5 cm/week using microsprinkler irrigation. Tree growth was similar over the 3 years for the reclaimed water and standard irrigation (canal water), provided that supplemental fertilization was used, because the reclaimed water itself did not contain sufficient nutrients for optimum growth. Leaf sodium and boron levels were higher in trees receiving reclaimed water compared with canal water, but levels were not toxic. In a related study (Maurer and Davies 1993b), reclaimed water was applied to reset 'Marsh' grapefruit trees at high frequencies and application rates. Again, there were no adverse effects on growth compared with trees receiving irrigation with canal (surface) water. Leaf sodium and boron tended to be higher in the reclaimed water vs. control trees, but no leaf damage was observed. Weed growth and distribution were increased with frequent application of reclaimed water. Slightly different results were obtained when reclaimed water was used to irrigate 'Hamlin' orange and 'Orlando' tangelo trees growing on deep, well-drained sandy soils of central Florida (Wheaton and Parsons 1993). High application rates and frequencies of application were also used. In this instance, high irrigation rates with reclaimed water increased tree growth over standard irrigation practices. Similarly, Koo and Zekri (1989) observed increased growth of 2- to 3-year-old orange trees receiving reclaimed water compared with well water, although cultivars and cultural practices also differed between the two treatments. Differences in growth responses among the three studies are likely due to quite different soil types and drainage patterns. Application of reclaimed water also affects nutrient concentrations in soils. In Egypt, heavy metals accumulated in soils with application of reclaimed water, but not to toxic levels in virgin, sandy soils (AbdEl-Naim and El-Awady 1989). Nitrogen, calcium, and boron levels also increased when reclaimed water was applied to citrus soils in Spain, but no adverse effects were observed (Estellar et al. 1994). There is concern that nitrates in particular may accumulate in groundwater where high rates of reclaimed water are used, but this claim has not been substantiated. Like municipal wastewater, disposal of effluent from citrus processing plants is also a problem in some citrus areas. It is typically high in sodium as a result of using sodium hydroxide for cleaning processing plants. Koo (1973) applied citrus effluent to young citrus groves in Florida and observed that treated effluent had no adverse effects on the growth of several citrus cultivars over a 2-year period, but long-term effects of irrigation with effluent are unknown. Processing plant effluent is not commonly used for irrigation of citrus.

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Irrigation scheduling methods are well-established for young citrus trees, particularly in arid and semi-arid regions where water is a limiting factor for growth. Microirrigation is becoming or has been a widely used method due to limitations on water use in many citrus-growing regions.

c.

Salinity

The salinity (NaCI) level of irrigation water is of concern in many citrusgrowing regions. This is especially true in semi-arid and arid growing areas such as Australia, Israel, Mexico, and southern California and the maritime regions of Florida and Cyprus where salt-water intrusion occurs. In these areas, high salt levels may severely reduce growth of young citrus trees. Citrus trees are generally salt-sensitive, although a range oftolerance exists within the genus and related genera (Maas 1993). Salinity damage and growth reductions occur due to osmotic (Lloyd et al. 1987; Walker et al. 1983), toxic ion (Cooper 1961), or ion imbalance (Walker and Douglas 1983) effects. Salinity also reduces nitrate uptake in citrus due to reduced water uptake (Lea-Cox and Syvertsen 1993). Several other studies on the effects of salinity on growth and development of mature trees have been summarized by Maas (1993) and will not be covered here. The effect of salinity on growth and development of young citrus trees under field conditions has not been extensively studied. Alva and Syvertsen (1991) applied three different levels of total dissolved salts to 'Valencia' orange trees on sour orange or 'Carrizo' citrange rootstocks. The trees were grown outdoors in large lysimeter tanks in native, sandy soil. Root density increased at the highest salinity levels (2.6 dS m-I ) for both rootstocks, suggesting that water stress may increase root growth. Application of saline water also increased sodium concentration in fibrous roots and decreased foliar potassium levels. The effects of salinity on growth were not reported. In contrast, Syvertsen and Yelenosky (1988) observed that salinity decreased root dry mass of 13-month-old 'Pineapple', 'Cleopatra', and trifoliate seedlings. The levels and duration of salinity treatments differed between the two studies, however. Boman (1993) conducted a detailed factorial experiment comparing growth of 1-year-old 'Ruby Red' grapefruit trees using several rootstocks, salinity levels, and fertilizer rates. For each rootstock, there was a linear decrease in trunk cross-sectional area and canopy volume with increasing salinity levels (Table 7.2). Canopy volume was decreased approximately 7% for each 1.0 dS m- I increase in salinity. Trees on 'Cleopatra' mandarin rootstock were more salt tolerant than

338

F. DAVIES AND

J. FERGUSON

Table 7.2. Mean change in trunk cross-sectional area and December 1989 canopy volume by water salinity for each rootstock (n 48). Source: Adapted from Boman (1993). Rootstock Water salinity (dS/m-1 )

Carrizo citrange

Cleopatra mandarin

Sour orange

Swingle citrumelo

Trunk cross-section area change (%) 0.7 2.3 3.9 5.5 Linear z Quadratic

297 252 244 240

310 273 263 244

252 241 231 220

NS

NS

NS

364 373 321 366 NS NS

Canopy volume (m 3) 0.7 2.3 3.9 5.5 Linear z Quadratic

0.54 0.37 0.37 0.32

0.66 0.55 0.47 0.49

0.25 0.24 0.17 0.18

0.54 0.49 0.40 0.36

NS

NS

NS

NS

ZLinear and quadratic contrasts are nonsignificant (NS), or significant at P = 5% (*) or P = 1 % (**) level.

those on 'Swingle' citrumelo, 'Carrizo' citrange, and sour orange, respectively. Conversely, trees on sour orange grew more than those on 'Carrizo' or 'Cleopatra' mandarin. Sour orange is a widely used rootstock in saline soils (Cooper 1961). It has long been observed that rootstock affects salinity tolerance of citrus (Cooper et al. 1952; Cooper 1961) and several papers on the subject were summarized by Maas (1993).

V. FERTILIZATION Surveys of Florida citrus nurseries indicated that annual N application rates varied from 168 to 448 kg/ha (Castle and Ferguson 1982; Williamson and Castle 1989). Subsequent reports of annual N rates in greenhouses ranged from 194 to 531 kg/ha and 357 to 410 kg/ha for field nurseries (Castle and Rouse 1990). However, some reports suggest that much lower optimum rates should be used. In the surveys cited above, nutrient solutions in citrus greenhouse nurseries ranged from 100 to

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339

400 mg N/L (Castle and Ferguson 1982; Williamson and Castle 1989) in media composed of peat, perlite, and vermiculite. In early work on the effect ofnitrate-N rate on the growth of sweet orange seedlings, California researchers (Chapman and Liebig 1937) reported that seedlings grown for 5 months in a washed sand culture at 7 mg NIL had 76% the dry weight of seedlings grown at 70 mg/L. Research from South Africa on rough lemon rootstock grown in pine bark (Lea-Cox 1989) and 'Hamlin' orange on 'Carrizo' citrange and 'Cleopatra' mandarin rootstocks grown in sand in Florida (Maust and Williamson 1991) indicated that 10 and 15 to 20 mg/L, respectively, were the critical N levels in the media solution. But when 'Hamlin' orange was grown on 'Swingle' citrumelo rootstock in a commercial medium of peat moss and perlite, optimum N rates were calculated to be 165 mg/L, suggesting differences in rootstock nutrient requirements and nutrient availability in different planting media (Guazzelli et al. 1995). Fertilization rates for young citrus trees are generally based on tree age and growing conditions, which may include geographic location and planting site, e.g., newly cleared or old citrus land and interset or reset trees. Numerous studies have been conducted, involving different fertilizer sources, rates, frequencies, durations, and scion-rootstock combinations (Table 7.3). Some experiments were begun after trees had been in the field for 1 year (Alva and Tucker 1993) to 3.5 years (Smith et al. 1953; Calvert 1969; Ferguson 1994) or were 2-year-old trees at planting (Zekri and Koo 1991a). Other research was conducted for only 1 year after planting (Ferguson et al. 1988; Obreza 1990; Boman 1993; Maurer and Davies 1994; Ferguson 1994). In some studies from Florida, fertilization rate had no effect on tree growth during the first year (Rasmussen and Smith 1961; Obreza and Rouse 1993) or even in the second year (Rasmussen and Smith 1961; Ferguson et al. 1988; Obreza 1994; Willis et al. 1991) in the field. However, increasing fertilizer rates during the first 3 years after planting increased yields during the fourth year (Obreza 1993), suggesting a cumulative effect of fertilizer treatments. Mineralization of soil organic N may account for lack of growth response, potentially amounting to 0.22 kg available N/ha/day in the top 0.3 m of orchard soil, especially in converted pastureland (Obreza 1990). High nutrient amounts typically applied to nursery trees may continue to sustain growth for the first year in the field. Guazzelli et al. (1996) reported that leafN levels (1.4 to 4.6%) in nursery trees had little impact on subsequent growth responses to different fertilizer rates in the field during the first 2 years, but N storage in the trunk, stem, and roots may provide an important N source. An annual N rate of 0.17 kg/tree produced maximum growth during the second year (Guazzelli et al. 1996).

w

~

Table 7.3.

Fertilizer rate, source, and application frequency studies for young citrus trees.

0

Cultivar

Rootstock

Tree age (yr)

Valencia orange Hamlin, Valencia, Pineapple oranges Valencia orange Orlando tangelo

rough lemon rough lemon

3.5 1-3

rough lemon trifoliate orange

2-8? 1-2

Hamlin orange

sour orange

1-2

Hamlin orange

sour orange

1

Hamlin orange

Carrizo

1

Hamlin orange

Carrizo, sour orange

1

Valencia orange

Carrizo

2-5

Hamlin orange

Carrizo

1-4

Pineapple orange

Swingle

2-3

Ruby Red grapefruit

sour orange Carrizo Cleopatra mandarin Swingle Swingle sour orange

1

Marsh grapefruit

3 1

Source Z Liquid Dry CR Dry Dry CR Dry CR Dry CR Dry CR Dry Liquid Dry CR Dry CR Dry CR Dry Liquid

Canal water RW Dry

N/tree-yr (kg) 30,80,210 ppm 0.03-0.14

Fertilizer frequency per year

Reference

2x/wk 3-6x dry lxCR 4x 4x dry 1-2 CR 4x dry 4xCR 6x dry 1-2x CR 6x dry 1-3x CR 5x dry 5, 10, 30x liquid 4-6x dry 2xCR 2-5x

Smith et al. 1953 Rasmussen and Smith 1961 Calvert 1969 Jackson and Davies 1984

Alva and Tucker 1993

0.24 0.01-0.15

4x dry lxCR 5x dry 40x liquid

0.22-0.45

5x dry

0.11-0.33 0.07-0.14 0.07-0.29 0.08 0.02-0.06 0-0.22 0.06-0.23 0.27-0.54 0.06-1.44 0.02-0.31

Marler et al. 1987 Ferguson et al. 1988 Obreza 1990 Willis et al. 1990 Zekri and Koo 1991b Obreza 1993

Boman 1993

Maurer et al. 1995

Hamlin orange

Carrizo

1-3

Ambersweet hybrid Grapefruit

Swingle Swingle

2-3 2

Hamlin orange

Swingle

1

Hamlin orange

Carrizo

1-5

Hamlin orange Marsh grapefruit Ambersweet orange Marsh grapefruit

Swingle

1-4

Swingle Sour orange Swingle

Redblush

Dry CR wetCM Dry CR CR PCM PCM/dry dry CR PCM PCM/dry CM

0.16-1.31 0.87-01.0 0.37 0.24 0.16 0.24-0.37 0.24-0.37 0.27 0.13 0.13-0.27 0.06-0.27 0.27

eN

H::-

Obreza and Rouse 1993 Ferguson 1994

dry CR dry CR

0.03-0.76

1-3

dry

0.22-0.45

5-6x dry

Maurer et al. 1995

1-3

Reclaimed wastewater dry liquid dry CR dry dry CR

> 0.01

Liquid> 50x

Maurer and Davies 1993b

0.22-0.34 0.22-0.45 0.07-0.57

5x dry liquid> 50x 3x dry 2x CR 6x dry 5x dry lxCR

Flame grapefruit

Swingle

1-4

Hamlin orange Hamlin orange

Swingle Swingle

1-2 1-2

0.04-0.52

0-0.34 0.23-0.34

ZCR: Controlled-release; CM: Chicken manure; PCM: Processed chicken manure.

I-'

4-6x dry 2-3x CR lxCM 6x dry 2xCR lxCR 3xPCM 3x PCM/dry 6x dry 2x CR 3xPCM 3x PCM/dry 6xCM 4-6x dry 2-3x CR 3-5x dry lxCR

Obreza 1994 Ferguson and Davies 1995

Obreza 1995 Guazzelli et al. 1996 Davies et al. 1996

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F. DAVIES AND J. FERGUSON

Given this variability, field studies of at least 3 years may be necessary to determine accurately the effect of fertilizer rate on young tree growth (Obreza 1990). Different responses to similar fertilization rates may result from variations in soil type (Obreza and Rouse 1993), tree age, size, location in orchard (Calvert 1969), rootstock (Wutscher 1989), amount of stored reserves (Legaz et al. 1995), or type of nursery trees (Marler and Davies 1987). Growth response of young citrus trees on the same rootstock and fertilized at the same N rate also varied according to soil calcium carbonate levels (Obreza 1995). Moreover, young grapefruit trees fertilized at the same N rate grew differently during the first year, which was related to different rootstock tolerances to total dissolved salts, which range from 500 to 3800 mg/L in irrigation water (Boman 1993). Trees on 'Cleopatra' mandarin had the greatest canopy volume, while those on 'Carrizo' citrange and 'Swingle' citrumelo had 15 to 20% less volume and trees on sour orange had from 55 to 66% less growth. Trunk crosssectional area was greatest for 'Swingle' citrumelo, followed by 'Cleopatra' mandarin, 'Carrizo' citrange and sour orange, illustrating the problem of quantifying growth responses of young citrus trees related to fertilization. Controlled release (CR), dry, water soluble, dry and liquid fertilizers have been applied at different annual N rates (0.01 to 0.76 kg/tree) and frequencies (1 to 40 times per year) to young citrus trees as summarized in Table 7.3. Application rate of CR fertilizers generally varied from 1 to 2 times per year, with one report (Jackson and Davies 1984) that more frequent applications increased growth. Controlled-release materials have also been compared with dry fertilizers, with CR fertilizers at low rates and frequencies stimulating growth comparable to that of dry fertilizers at high rates and application frequencies (Marler et al. 1987; Alva and Tucker 1993; Ferguson and Davies 1995). A combination ofCR and dry fertilizers applied 2x/year also provided a continuous nutrient supply and promoted young tree growth increases comparable to that obtained with more expensive CR fertilizers applied at the same N rate (Zekri and Koo 1991b). Controlled-release fertilizers are particularly useful for interset or reset trees in a mature orchard because they save on application costs without compromising tree growth (Davies et al. 1996). When urea-N is used in CR fertilizers, mild biuret toxicity may occur. Leaf chlorosis on young citrus trees associated with urea soil and foliar applications was first reported in Florida in 1954 as caused by biuret impurities (Oberbacher 1954) and mild biuret toxicity on young citrus trees fertilized with CR urea fertilizers was also reported more recently by Zekri and Koo (1991a) and Ferguson and Davies (1995).

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Studies from Florida (Willis et al. 1990) and Texas (Swietlik 1992) indicate that fertigation frequency or the use of dry, granular compared with liquid fertilizer had no differential effect on tree growth. However, fertigating trees 30x/year increased growth over 5x/year for trees on 'Carrizo' citrange growing in sandy soils. Willis et al. (1991) reported no difference in growth of l-year-old 'Hamlin' orange bare-root or containerized trees fertilized annually with 0.23 kg N/tree as dry fertilizer applied 3 or 5 times per year or liquid fertilizer applied from 3, 10, or 30 times per year. Bare-root trees that received 0.06 kg N/tree per year were smaller than trees receiving 0.11 kg N/tree per year either as a dry fertilizer 5 times per year or as a liquid 5, 10, or 30 times per year. Reclaimed water, as discussed previously, has been used to irrigate young citrus trees in several citrus-growing regions and has been used as a supplementary nutrient source because it often contains moderate levels ofN, K, and P, as well as high levels ofB and Na. Reclaimed water plus fertigation at N rates ranging from 0.22 to 0.34 kg/tree per year stimulated growth more than well water plus fertigation, dry fertilizer or reclaimed water plus dry fertilizer at the same rates (Maurer and Davies 1993a). Maurer et al. (1995) also applied 0.22, 0.34, and 0.45 kg N per tree per year to reset 'Marsh' grapefruit trees along with canal water and reclaimed water. Growth was comparable for all treatments. The difference in response to reclaimed water between the studies may have been due to water source, soil type, or growing region. Most of the fertilizer studies discussed above were conducted in Florida, where soils are generally sandy and may not be representative of other citrus areas with more finely textured soils. In several growing regions, fertigation is being widely used to reduce costs and improve application efficiency. Certainly, many areas still fertilize individual trees by hand as the preferred method. VI. FREEZE HARDINESS AND PROTECTION

A. Freeze Hardiness Freeze damage has been a major cause of young tree losses in Florida, Texas, northern Mexico, and central China (Davies and Albrigo 1994) and has caused limited damage in Spain, Italy, Japan, Turkey, Greece, Australia, and Israel in some years (Yelenosky 1985). Young trees tend to be more severely damaged by freezes than larger, older trees because they have less mass and will reach critical temperatures sooner. Young trees also tend to have a higher proportion of new growth that is often less freeze-hardy than older, mature growth. The inherent freeze hardiness of

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mature tissues of young and mature trees is similar. The term "freeze hardiness" will be used instead of frost or cold hardiness in this review, as proposed by Rieger (19S9). Citrus trees are rarely damaged by frosts, with the exception of flowers. Cell damage mainly occurs with the phase change of water to ice, which results in intra- and intercellular freezing. Most true citrus species evolved in subtropical or tropical regions of Asia and have the capacity to withstand temperatures as low as _5° to -7°C without considerable damage. Citrus survives sub-zero temperatures by supercooling and tolerating intercellular ice formation (Yelenosky 19S5). Furthermore, young citrus trees are capable of acclimating and deacclimating, which affects the freeze tolerance of the tree. Young (1969) observed that citrus trees begin freeze-acclimating as temperatures fall below 12°C. Subsequently, Maurer and Davies (1994) measured seasonal changes in leaf freeze hardiness for young 'Redblush' grapefruit trees in Florida. Leaves began freeze-acclimating as average air temperature fell below 12°C in December, reaching maximum hardiness of -SoC, after which they deacclimated as temperatures increased, followed once again by acclimation during February as temperatures again decreased. These data indicate the rapid effect that temperature changes can have on leaf freeze hardiness and illustrate the important influence of prefreeze temperatures on freeze hardiness and tree survival. Furthermore, recent studies suggest that Poncirus trifoliata, a citrus relative, may acclimate within 6 h at 10°C and under long days (Tignor et al. 1997), suggesting that acclimation and deacclimation may occur much more rapidly than previously believed. Freeze hardiness of young citrus trees is also affected by tree-water relations. Yelenosky (1979) found that water stress increased the extent of leaf supercooling over nonstressed trees at -5.5°C, although no differences between treatments occurred at -6.7°C. Wilcox et al. (1983) also found that reducing soil temperature and xylem water potential (increasing stress) increased freeze hardiness of 'Valencia' orange trees. Both of the above studies were done with containerized trees under controlled conditions. Recently, Tignor et al. (1998) found that leaf freeze hardiness could be increased by regulating microirrigation of 1-year-old 'Hamlin' orange trees in the field. Trees that were maintained at 45 % SWD during the fall were more freeze-hardy than those at 20% SWD. The results were not consistent from year to year, however, and the use of irrigation to regulate freeze hardiness in the field merits further study. There has been a good deal of controversy concerning the effects of tree nutrition on freeze hardiness. Several studies on mature citrus trees suggest that deficiencies of Zn, Cu (Lawless 1941), and Mn (Swietlik and

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345

LaDuke 1985) may decrease citrus tree hardiness. In addition, during the 1960s some believed that increasing K levels in the leaf would increase hardiness. This notion was later disproved in controlled freeze chamber tests on 'Pineapple' orange seedlings (Jackson and Gerber 1963). Similarly, Koo (1985) observed that P, K, and N deficiency may also decrease hardiness. The universal dogma is that healthy trees are more freeze hardy than mineral-deficient trees. However, it appears that severely nutrient-deficient young trees in some cases may be more hardy than healthy trees, which is likely related to cessation of growth more than to physiological factors. No differences in freeze hardiness related to N application rate were observed for first-year trees in the field (Marler and Davies, unpublished). In contrast, in a very extensive fertilizer study on young trees, Smith and Rasmussen (1958) found that high rates of N applied in the fall decreased tree hardiness in central Florida. Maurer and Davies (1994) found that leaf N concentration had no effect on hardiness during acclimation; however, high leafN levels decreased hardiness during deacclimation in the spring. Therefore, N effects on freeze hardiness may vary depending on time of year, which may account for the contradictory reports on its role in citrus freeze hardiness. B. Freeze Protection While young citrus trees are more susceptible to freeze damage, they also are easier to freeze-protect than mature trees. Several freeze (cold) protection methods have been developed and tested over the years. The most important of these include soil banking, tree wraps, and irrigation. In addition, several types of spray materials have been tested with inconsistent results. The value of mounding soil around young trees has been recognized for many years (Hume 1904). In the late 1800s, citrus growers in Florida mounded soil over entire trees or filled wooden molds with soil around the trunk to insulate it from freezes. Soil banking was a standard practice, especially for citrus growers in north Florida, and is also a commonly used method in central China. Considerably later, Yelenosky (1965) compared grove heaters with soil banking as methods of protecting young trees from freeze damage. Heaters provided effective protection during radiative (low wind) freezes if positioned near the tree, but were less effective during advective (windy) freezes. The small canopy of a young tree intercepts far less radiation than that of a larger, mature tree. Consequently, canopy temperature decreases much more rapidly. Furthermore, an orchard of young trees does not delay radiative losses as effectively as an orchard of mature trees. Soil banking considerably

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F. DAVIES AND J. FERGUSON

delayed the rate of trunk temperature decrease under the bank through its insulating value and provided excellent freeze protection. Similar studies by Jackson et al. (1983) and Davies et al. (1986) found that trunk temperatures in the soil bank were 5° to 11°C greater than air temperature. More importantly, the lower portion of the trunk was protected from freeze damage even under severe advective freezes. Soil banks do not protect the upper canopy of the tree (Davies and Jackson 1985). Tree regrowth from the area under the bank is usually rapid, and the cost of banking is far less than that of removing and replanting trees. Soil banking is typically done in early winter either by hand or with custom-made banking equipment. Trees are unbanked in early spring to reduce the risks of insect or fungal damage. Banking and unbanking may cause physical damage to the bark, predisposing the tree to Phytophthora and insect, especially ant and termite, damage (Davies and Jackson 1985). In many citrus regions, the trunks of young trees are wrapped with a variety of insulating materials for protection from low temperatures and wind. In California and Arizona, trees were wrapped with corn stalks or other plant materials, which were actually more effective in preventing wind damage than freeze injury because of their low insulating properties (Turrell 1973) . Use of mineral soil banks, which were first tested in the 1950s, provided as much as an 8°C trunk temperature increase over unprotected trees during the 1962 freeze in Texas (Leyden and Rohrbaugh 1963). Other types of insulating wraps, including fiberglass and polyurethane, also gave adequate protection to the trunk and inhibited sprouting (Hensz 1969). Similarly, Rose and Yelenosky (1978) found that fiberglass wraps provided a 2.2° to 2.8°C increase in trunk temperature over nonwrapped trunks, but were far less effective than soil banking. In a later study, Davies et al. (1984) compared the effectiveness of several types of insulating wraps for freeze protection of 1-year-old citrus trees in Florida. Tree wraps with the highest insulating values (lowest thermal diffusivity) logically delayed the rate of temperature decrease the most. Moreover, addition of packets of water within the wrap significantly decreased thermal diffusivity and improved freeze protection of standard polystyrene or fiberglass tree wraps (Rieger et al. 1988a). Tree wraps alone are not as effective for freeze protection as soil banks, but they also prevent sprouting and herbicide damage to the trunk and do not have the disadvantages of banks as outlined previously. Wraps may also be left on the trees for the entire season or for several seasons, although in some cases ant and Phytophthora problems may arise. Water has been used effectively for many years for freeze protection of young citrus trees in the nursery via overhead sprinklers or in the field via flood irrigation. Generally, large volumes of water are applied.

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Temperature is increased in the orchard via sensible heat and the heat of fusion as water changes to ice (Rieger et al. 19Ssb). In many areas, flooding is impractical and overhead sprinklers may cause limb breakage of young trees in the field. In the early 19S0s, microsprinkler irrigation began to be tested for freeze protection of 1- to 2-year-old trees in Florida. Parsons et al. (19S2) found that microsprinklers provided effective freeze protection in a high-density planting of young citrus trees when high application rates were used. Similarly, Davies et al. (19S4) found that a combination of tree wraps and microsprinklers positioned to the northwest of the tree, which is the primary wind direction during advective freezes in Florida, provided effective freeze protection for l-year-old trees even at -10°C air temperatures under advective conditions. Furthermore, tree wraps provided freeze protection to the trunk even if irrigation was discontinued due to a system failure. Parsons et al. (1985) also determined that microirrigation effectively protected young citrus trees when 90° emitters were used and positioned to the northwest of the tree. Several different types, patterns, and placements of emitters had been used over the years with varying degrees of success. As a consequence, the effect of emitter pattern, position, and precipitation rate was studied in detail by Rieger et al. (19S6). In general, trunk temperatures were greater using a 90° pattern compared with a 360° pattern, and the highest irrigation rate, as would be expected, produced the highest increase in trunk temperature. However, the most efficient increase in trunk temperature, per unit of water applied, occurred using a 90° pattern at a moderately low rate of 38 L/h. Davies and Rippetoe (19S9) also tested several different emitter patterns and locations in the tree canopy and observed the greatest freeze protection either using a 90° pattern or by positioning cone-type emitters in the canopy. Elevating emitters into the tree canopy has provided freeze protection for moderate-sized (1 to 2 m in height) trees in Florida (Parsons et al. 1991) and Louisiana (Bourgeois and Adams 1987). However, Martsolf and Hannah (1991) found that elevated emitters caused limb and trunk breakage of young citrus trees, although there is some debate as to whether this occurs under all freeze conditions.

VII. PRUNING Hand pruning is used to shape young citrus trees in fresh fruit growing areas such as those in parts of the Mediterranean region, South Africa, Japan, and China. In contrast, hand pruning and shaping is rarely used

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F. DAVIES AND J. FERGUSON

in the United States due to high labor costs. However, sprouts originating from the lower trunk, especially from the rootstock, are typically removed by hand or prevented from developing by using tree wraps. In Spain, citrus trees, especially mandarins, are pruned to 2 to 3 major scaffold limbs (Zaragoza and Alfonso 1981). In South Africa, young soft citrus (mandarin) trees are shaped by hand during the first 3 years after planting (Wahl and Rabe 1991). During the first year, all growth is removed below a 40 cm height. At the end of the first year 3 or 4 scaffold branches are selected between a 40 and 80 cm height. Branch angles range from 45° for 'Clementine' mandarins to 55° for more vigorous cultivars. Branch angles are formed by using weights or spacers, as is the practice with some deciduous crops. Secondary scaffold branches are formed about 1 m above soil level in the third year. Meticulous hand pruning is also done on young citrus trees in China to shape the tree and remove unwanted or diseased limbs. Hand pruning may also be necessary in some tropical regions to shape the tree and reduce upright, vigorous growth that is typical in these regions. Nevertheless, the economic benefit of hand pruning of young citrus has not been thoroughly studied. VIII. BIOTIC FACTORS Young citrus trees are susceptible to many of the same pathogens and pests that affect mature trees. However, some bacterial, fungal, and virus diseases and pests, originating either from the nursery where the trees were grown or present in the planting site, can significantly affect the establishment and decrease early growth of citrus trees. The major fungus and bacterial disease problems for young citrus trees are listed in Table 7.4. A. Fungal Diseases

Phytophthora spp. occur in most citrus-producing regions of the world, with damping-off of citrus seedlings, foot rot, and root rot causing serious damage in citrus nurseries and young tree plantings (Klotz 1978a). Pre- and post-emergent damping-off of citrus seedlings in citrus nurseries has been associated with Phytophthora spp. as well as other soilborne fungi such as Pythium spp. Foot rot affects the bark and wood of susceptible rootstocks and scions, causing exudation of gum, cracking of the bark and girdling of the trunk, leaf yellowing, twig dieback, and sometimes tree death (Whiteside et al. 1988). Rot of fibrous roots can also reduce growth and cause general decline, especially when not enough fibrous roots are produced to replace roots killed by Phytophthora (Lutz

7. GROWTH, DEVELOPMENT, AND CULTURAL PRACTICES FOR CITRUS TREES

Table 7.4.

349

Important diseases of nursery stock and young citrus trees.

FUNGAL DISEASES Alternaria brown spot; Alternaria leaf spot Aerolate leaf spot Black rot of seedlings Botrytis blight Damping off of seedlings Foot rot Fusarium wilt Greasy spot Malsecco Mushroom root rot Root rot Sour orange scab Sweet orange scab Tyron's scab

Alternaria citri Ell & Pierce Pellicularia filamentosa (Pat.) Rogers Thielaviopsis basicola (Berk & Br.) Ferr. Botrytis cinerea Pers. Ex Fr. Phytophthora spp., Pythium spp., Rhizoctonia solani Kuhn Phytophthora nicotianae Breda de Haan Fusarium oxysporum (Schlecht.) Mycosphaerella citri Whiteside Phoma tracheiphila (Petri) Kantsch. & Gil. (syn. Deuterophoma tracheiphila Petri) Armillaria mellea (Vahl ex Fr.) Kummer; Clitocybe tabescens (Scop.) Bres. Phytophthora nicotianae Breda de Haan (syn. P. parasitica Dastur) Elsinoe fawcetti Bitanc. & Jenkins Elsinoe australis Bitanc. & Jenkins Sphaceloma fawcetti var. scabiosa (McAlp. & Tryon) Jenkins

VIRUS AND VIRUS-LIKE DISEASES Citrange stunt, crinkly leaf, exocortis, infectious variegation, psorosis, ring spot, satsuma dwarf, tatter leaf, tristeza, cachexia BACTERIAL DISEASES Blast Citrus bacterial spot

Citrus variegated chlorosis Canker Greening

Pseudomonas syringae van Hall Xanthomonas axonopodis Starr & Garces emend. Vauterin et al. pv. citrumelo Gabriel et al. Xylella fastiodiosa Wells et al. Xanthomonas axonopodis Starr & Garces emend. Vauterin et al. pv. citri (Hasse) Dye Spiroplasma citri Saglio et al.

and Menge 1986). Agostini (1989) demonstrated that the presence ofimmature fibrous roots, susceptible rootstocks, and favorable soil moisture favored the development of root rot and that soil populations of Phytophthora nicotianae were generally correlated with the susceptibility of specific rootstocks to this pathogen. In subsequent greenhouse studies, Graham (1995) showed that trifoliate orange and 'Swingle' citrumelo seedlings were more tolerant to fibrous root rot caused by P. nicotianae

350

F. DAVIES AND

J.

FERGUSON

than 'Carrizo' citrange, sour orange, 'Ridge Pineapple' sweet orange, and 'Cleopatra' mandarin. Tolerance was expressed as the greater capacity of tolerant rootstocks to regenerate roots under certain conditions or rootstock influences on pathogen population levels. The incidence and severity of root rot disease on Phytophthora-susceptible seedlings also increased in relation to the amount of root damage caused by larvae of the root weevil, Diaprepes abbreviatus, but Phytophthora-tolerant seedlings showed little or no increase in disease severity with increasing insect injury (Rogers et al. 1996). Cohen et al. (1964) suggested that foot rot in young orchard trees could be traced back to the nursery. Zitko et al. (1987) isolated Phytophthora from 8 of 15 Florida field nurseries but only 1 of 13 container nurseries, presumably because of the use of soil-less media and fungicides in the latter. River water used to irrigate young citrus has also been shown to be a source of Phytophthora innoculum in South Africa. Addition of organic amendments such as composted municipal solid waste to the planting hole may increase Phytophthora spore production and infection of young citrus trees (Widmer et al. 1996). Mushroom root rot, most commonly caused by Armillaria mellea and Clitocybe tabescens, occurs in major citrus-growing regions in the United States, Europe, and Africa (Knorr 1965). Losses of young trees from mushroom root rot are usually due to direct contact of infected plant debris with healthy citrus roots, especially on poorly cleared land where trunks and large roots of native vegetation and citrus have not been completely removed (Broadbent 1981). Fungal diseases affecting the foliage and fruit of mature trees can also affect the growth of young trees by causing leaf distortion, necrosis and drop, as well as twig death. Alternaria citri is the causal agent of brown spot of mandarins, tangors, and tangelos. This disease has also recently been reported on 'Sunburst' tangerine, 'Marsh', and 'Flame' grapefruit (Timmer and Peever 1997). Brown spot symptoms range from large, necrotic areas to small, circular spots, with necrosis extending into leaf veins. Leaves infected in the spring abscise before the end of winter (Whiteside et al. 1988). Alternaria leaf spot of rough lemon, caused by a host specific strain of Alternaria citri, is also characterized by lesions varying from large, necrotic areas to small, circular spots. Colletotrichum gloeosporioides (Penz.) Sacc., a secondary pathogen, is commonly found along with Alternaria in concentric rings that form an extensive chlorotic halo. Stem infection and defoliation commonly cause shoot dieback and extensive branching of rootstock seedlings, retarding growth, and promoting excessive branching and development of bushy plants that are difficult to bud. As a consequence, rough lemon seedlings were often budded low because bud-insertion sites were not available higher up on

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the stem, increasing susceptibility of trees to foot rot (Whiteside and Knorr 1978). Areolate leaf spot, caused by Pellicularia filamentosa, occurs in seedbeds and nurseries in humid tropical areas of South America. Symptoms include light-colored necrotic areas with dark, concentric rings on leaves and, when severe infection occurs, leaf drop. Sour orange rootstocks and grapefruit, orange, and mandarin cultivars are susceptible (Whiteside et al. 1988). Other fungal diseases causing nursery tree losses include: black root rot of greenhouse seedlings, caused by Thielaviopsis basicola; botrytis blight of citrus twigs, leaves and bark, caused by Botrytis cinerea, which infects young lemon trees, especially under prolonged wet, cool conditions; damping-off of recently germinated seedlings caused by Rhizoctonia solani, Phytophthora, and Pythium species; and Fusarium wilt, especially of 'Mexican' lime, caused by Fusarium oxysporum (Timmer et al. 1979). Greasy spot, caused by Mycosphaerella citri, is perhaps the most important disease affecting leaf longevity in humid, subtropical and tropical climates where nearly 100% relative humidity and high temperatures occur simultaneously for sustained periods. Leaves of all commercial cultivars are affected, but the disease is usually more severe on grapefruit, lemons, and early-maturing orange cultivars than on 'Valencia' orange and mandarins. Up to 30% of the area of individual leaves can be affected (Martinez-Soriano et al. 1981) and up to 30% of the canopy of mature trees can be defoliated (Whiteside 1977). Since ascospores from fallen, infected leaves are the major source of inoculum, young trees in replant situations in infected groves may be more susceptible than trees in plantings on newly cleared land. Nevertheless, greasy spot is usually a less severe problem on young trees than it is on mature trees. Mal secco (dry disease), caused by Phoma tracheiphila, occurs primarily in the Mediterranean Basin, around the Black Sea, and in Asia Minor. Veinal chlorosis is the most common symptom, followed by leaf drop and twig dieback. Lemon trees are very susceptible, followed by bergamot, some mandarins, tangelos, and tangors. Susceptible rootstocks include rough lemon, limetta, alemow, sour orange, and 'Troyer' and 'Carrizo' citranges (Whiteside et al. 1988). Citrus scab, including sour orange scab caused by Elsinoe fawcetti, sweet orange scab caused by E. australis, and Tryon's scab caused by Sphaceloma fawcetti var. scabiosa, occurs in citrus-growing areas that usually have postbloom rainfall but not in areas with an arid or semi-arid climate. Sour orange scab has an economic impact primarily on the foliage and shoots of rough lemon, 'Milam' lemon, sour orange, 'Rangpur'

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lime, trifoliate orange, and 'Carrizo' citrange nursery seedlings, and it can distort leaves and twigs of budded trees (Whiteside 1978). Copper fungicides are commonly used to control fungal diseases of citrus foliage and fruit. Consequently, accumulation of as much as 540 kg extractable Cu per ha in the top 15 cm of old orchard soils in Florida has been documented (Zhu and Alva 1993). High soil Cu levels have been associated with chlorosis of young citrus trees and iron deficiency, especially on Cu-sensitive rootstocks like 'Swingle' citrumelo (Alva 1993). Stunting of young citrus trees and P deficiency was also correlated with high soil Cu levels and attributed to a reduction in growth of arbuscular mycorrhizal fungi in the soil (Graham et al. 1986). B. Virus and Virus-like Diseases

Historically, virus and virus-like diseases have been the most destructive diseases of citrus worldwide. Some diseases, such as citrange stunt, crinkly leaf, citrus exocortis viroid (CEV), infectious variegation, psorosis, ring spot, satsuma dwarf, stubborn, tatter leaf, cachexia (xyloporosis), and citrus tristeza virus (CTV) , can be transmitted by budding or grafting (Rauchaudhuri and Ashlawat 1984; Carpenter and Calavan 1969; Weathers 1969) and may be nursery related. Additionally, pathogen-free nursery stock may also reduce the spread of vector-transmitted diseases like CTV, especially in areas where natural spread is slow. Registration, certification, and indexing programs to prevent the spread of virus and virus-like diseases have been established in major citrus-producing areas (Calavan et al. 1978) and can reduce the spread of viral diseases. Such programs provide true-to-type nursery stock, including superior scion cultivars, as free as possible from pests and pathogens by using tissue culture and shoot-tip grafting methods to produce virus-free mother trees and to detect CTV and other graft-transmissible pathogens (Mathews et al. 1996). Stunting of young citrus trees, especially on sour orange rootstock in the nursery and the field, leaf cupping, vein clearing, and chlorosis are the major symptoms of CTV affecting young citrus trees. Sour orange rootstocks budded with scions infected with decline-inducing CTV isolates become stunted and chlorotic in the nursery (Whiteside et al. 1988). CTV infects almost all citrus cultivars but is a major problem for sweet orange, grapefruit, and mandarin trees on sour orange rootstock. Visual symptoms, indexing on indicator plants, and serology can be used to detect CTV infection. Enzyme-linked immunosorbent assay (ELISA) and biological indexing are being used to distinguish between severe and mild strains (Irey et al. 1988). CTV is transmitted by budding from

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infected scion trees and by several aphid vectors; the brown citrus aphid, Toxoptera citricidus, the most efficient vector, is distributed throughout Asia, South Africa, South America, and Florida. Cachexia viroid symptoms (pits and channels in the wood) have been reported on inoculated 2- and 3-year-old sweet orange trees on 'Palestine' sweet lime and 'Orlando' tangelo rootstocks in Iran (Habashi 1984). CEV was also isolated, but no symptoms were reported 7 months after navel orange trees on five rootstocks were inoculated in Brazil (Fernandes et a1. 1984). Typical CEV bark scaling symptoms, especially on trifoliate orange, some citranges, 'Rangpur' lime, and sweet lime, do not usually appear before trees are 3 years old and may not appear until after 8 years, but trees may be stunted by severe strains (Knorr 1973). Once trees on sensitive rootstocks attain considerable size, infection by mechanical means is less detrimental than if trees were infected in the nursery (Garnsey and Weathers 1972). Symptoms of the tatter-leaf citrange stunt complex (chlorotic spots and ragged margins on distorted leaves) were also reported only 4 to 6 weeks after inoculation in 'Rusk' citrange, 'Peiyu' pummelo, and rough lemon seedlings grafted with infected buds in Taiwan (Su and Cheon 1984). Visual symptoms of citrus blight, a disease of unknown etiology also referred to as young tree decline, sand hill decline, and declinio, do not generally appear in citrus trees for 5 to 10 years after planting (Young et a1. 1978; Gould et a1. 1987) but root extracts and leaf extracts can be used to identify blight in symptomless 2-year-old young trees (Derrick et a1. 1992). Furthermore, neither rootstock seed nor budwood source appear to be related to the incidence of blight on mature trees (Pelosi et a1. 1987; Wutscher and Smith 1988), ruling out nursery origin as a source for this disease. Blight has been transmitted from mature to young trees by root grafting (Timmer et a1. 1992) but is generally not considered a problem for trees less than 3 years old. C. Bacterial Diseases

Bacterial canker diseases of citrus are caused by different strains of Xanthomonas axonopodis (syn campestris pv. citri). Canker A occurs in Asia, some Pacific and Indian Ocean islands, and some South American countries, and attacks the largest number of citrus species, including grapefruit, 'Key' lime, lemons, sweet oranges, and mandarins. Other forms of citrus canker are identified by host range, cultural and physiological characteristics, and laboratory tests (Whiteside 1988; Gabriel et a1. 1989). Canker B occurs in Argentina, Uruguay, and Paraguay, and is more restricted in host range, attacking mainly lemons and 'Key' lime.

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Canker C is known to be pathogenic only to 'Key' lime. The D strain has been associated with Mexican bacteriosis, with the E strain causing citrus bacterial spot, responsible for an epiphytotic in the 1980s in Florida citrus nurseries. Over 23 million nursery and young citrus trees were destroyed in Florida's citrus canker eradication campaign (Schoulties et al. 1985; Schoulties et al. 1987). Spread of citrus bacterial spot (CBS) within citrus nurseries occurred by mechanical pruning, hedging, introduction of infected plant material, budding, planting, other nursery operations, and natural spread by wind and rain (Gottwald and Graham 1991). The problem was particularly severe for 'Swingle' citrumelo and trifoliate orange seedlings (Graham and Gottwald 1991). However, the incidence of CBS decreased when infected trees were transplanted to the field (Gottwald and Graham 1990; Gottwald et al. 1992), indicating degrees of aggressiveness of different CBS strains under different nursery and grove conditions. The Asian strain of citrus canker was detected in Florida in 1997 and quarantine areas have been established. Blast, caused by Pseudomonas syringae, is characterized by lesions on leaf wings and petioles resulting in leaf drop. Infection occurs on shoots and leaves injured by wind, heavy rain, hail, thorns, etc., especially under conditions of prolonged rain or fog at temperatures between 8° and 20°C (DeWolfe et al. 1966). When young trees are infected with the greening organism, a phloemlimited bacterium vectored by two species of psyllids, complete yellowing appears soon, whereas symptoms vary widely in mature trees infected in the orchard (Aubert et al. 1984). Three-year-old 'Valencia' orange trees naturally or experimentally infected with stubborn, caused by a mycoplasma-like organism, Spiroplasma citri, were smaller than uninfected trees, had smaller leaves and less than half the total foliage weight of healthy trees (Carpenter and Calavan 1969). Trees affected by citrus variegated chlorosis (CVC), first recognized in Brazil, show foliar chlorosis on mature leaves resembling zinc deficiency, gummy leaf lesions, defoliation, twig and branch dieback, and tree stunting (Lee et al. 1991; Rossetti 1993). CVC is caused by a xylemlimited bacterium, Xylella fastidiosa, which can be spread by contaminated budwood, vectored by sharpshooter leafhopper, and affects orange cultivars primarily. The earlier young trees are infected, the more severe symptoms will be, with some trees dying before 2 years. Long-term infection results in symptoms throughout the canopy, but recent infections may result in symptoms being expressed in only one sector of the canopy. Once infected young trees are introduced into a

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planting, the disease can spread rapidly. Use of disease-free budwood, pruning diseased branches, management of leafhopper populations, and cultural practices that alleviate water stress have shown promise in controlling eve. D. Insects and Mites Most major citrus pests that cause damage to mature citrus trees (Table 7.5) also frequently affect young citrus trees. Simanton (1976) conducted monthly surveys of Florida citrus for 16 years and reported the following pests to be most prevalent: citrus red mites, Panonychus citri, which feed on epidermal leaf cells of young citrus trees, causing leaf dehydration and death, especially after severe wind or cold stress; aphids that stunt new growth and delay development of trees to bearing size; grasshoppers, including the American grasshopper, Schistocera americana, and the eastern lubber grasshopper, Romalea microptera, which defoliate young citrus trees; and cottony cushion scale, Icerya purchasei, which infests young citrus in newly planted groves and nurseries. Table 7.5.

Important pests of nursery stock and young citrus trees.

American grasshopper Easter lubber grasshopper Black scale Cottony cushion scale Brown citrus aphid Citrus Leafminer Deer Eastern subterranean termite Imported red fire ant Leaf-cutting ants Mealybug Burrowing nematode Citrus nematode Lesion nematode Sting nematode Red mite Rust mite Texas citrus mite Root weevils Orange dogs (swallowtail butterfly)

Schistocera americana (Drury) Romalea microptera (Palesot de Beauvois) Sassetia meglecta (Delotto) Icerya purchasei Maskell Toxoptera citricidus Kirk. Phyllocnistis citrella Stainton Family Cervidae, Odocoileus virginian us Goldman and Kellogg (Miller and Kellogg) Reticulitermes flavipes (Kollar) Solenopsis invicta Buren Atta and Acromyrmex species PlanococcllS citri (Risso) Radopholus citrophilus Huettel, Dickman & Kaplan Tylenchulus semipenetrans Cobb Pratylenchlls spp. Belonolaimus longicaudatlls Rau Panonychus citri (Risso) Phyllocotmta oleivoera (Ashm.) Eutetranychus banksi (MeG) Diaprepes spp., Prepodes spp, Pachnaeus Htus Germar. PapiHo cresphontes Cram.

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Cases of unusual young tree susceptibility to pests include 'Sunburst' mandarin, which is more susceptible than other cultivars to Texas citrus mite, Eutetranychus banksi, the citrus rust mite, Phyllocotruta oleivora, and which supports higher populations of black scale (Sassetia neglecta), aphids, and citrus mealybugs (Planococcus citri) than found on adjacent cultivars (Albrigo et al. 1987). Citrus leafminer (CLM), Phyllocnistis citrella, a major citrus pest in Asia, Australia, Africa, and North America (Claussen 1933; Heppner 1993; Pena and Duncan 1993) causes leaf distortion, chlorosis, necrosis, leaf drop, and reduction in photosynthetic area, especially for citrus nursery trees (Pena and Duncan 1993), but leaf drop rarely occurs and the amount of damage needed to reduce tree growth appears to vary with tree size and vigor. Villanueva-Jimenez (1998) found no effect of low CLM populations on growth of grapefruit trees in the nursery. In contrast, Pena and Schaffer (unpublished) suggest that high populations may decrease growth of young 'Tahiti' lime trees. Initial injury is confined to the epidermal leaf cells, but if the fragile mine covering is ruptured before a new, cuticular layer is formed, favorable conditions may be created for infection by pathogens (Achor et al. 1997). As the number of CLMs and duration of mining increased, logically, leaf damage increased (Schaffer et al. 1997). The percent leaf damage was somewhat inversely correlated with net photosynthesis until high levels of damage were done. CLM feeding sites can later become invaded by aphids, mealybugs, and red mites (Pena and Duncan 1993). Several natural parasites exist that may control CLM populations, the most important of which is Aegeniopsis citricola (Hoy and Nguyen 1994). The imported red fire ant (Solenopsis invicta) feeds on the bark, cambium, new leaves and shoots, flowers, and developing fruit, with tree mortality 5.5 to 6.6 times greater in untreated areas than sections of citrus groves treated with insecticides (Smittle et al. 1988; Banks et al. 1991). In a survey of central Florida groves, Banks et al. (1991) reported foraging fire ants in from 31 to 38% of surveyed sites, with 367 nests per ha. They also reported fire ants tending scale insects and aphids and observed that on trees older than 4 to 5 years, the fire ant had less impact on tree growth. Fire ants may also be a hazard to grove personnel. Approximately 40 species of leaf-cutting ants, found in the New World from Argentina to Texas, cut plant leaves, flowers, fruit, and other material and carry it back to the nest where smaller worker ants prepare it as a substrate for a fungus on which the ants feed (Wetterer 1995). Atta and Acromyrmex species feed on a range of agronomic and horticultural crops, including citrus, cocoa, manioc, coffee, maize, and

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cotton, sometimes completely eliminating citrus crops in infested areas (Cherrett and Jutsum 1982; Cherrett 1986). Root weevil larvae feed on the roots and trunk of young citrus trees, including most commercial rootstocks and intergeneric hybrids, causing root damage and death (Grosser and McCoy 1996). Adults feed on and notch leaf edges, but overall have little direct impact on the tree, except in young trees infested by unusually large numbers of adults. When severe leaf damage occurs, leaf water loss increases proportionally more than the decline in photosynthetic rate per leaf for a given amount of leaf damage, suggesting that drought stress may be especially important in heavily infested groves (Syvertsen and McCoy 1985). Damage from these insects is particularly severe in Florida and the Caribbean, where fiddler beetles (Prepodes spp.) and sugarcane root stalk borer (Diaprepes spp.) predominate. Systemic insecticides applied as a soil drench controlled neonatal larvae of the citrus root weevil in containergrown plants in citrus nurseries for a minimum of 10 weeks. This provided some assurance that trees coming from the nursery were not already infested with the citrus root weevil (McCoy et al. 1995). Montes et al. (1981) also reported control of the root weevil, Pachnaeus litus, in Cuban citrus nurseries by inoculating soil with suspensions of the nematode genus Neoplectana. Entomopathogenic nematodes such as Steinernema and Heterorhabditis species applied through the irrigation system can also provide protection from root weevil larvae in the soil (Downing et al. 1991). Grasshopper nymphs migrating from pastures and low marshy lands to adjacent citrus plantings can occasionally defoliate young citrus trees. Griffiths and Thompson (1952) observed young citrus groves with 0,11 and 17% of the trees infested by the American grasshopper, Schistocerca americana, and related percent infestation to defoliation of young citrus trees. Up to 95% control could be obtained 1 day after application of pesticides as compared with no treatment. Clean cultivation can deter oviposition and reduce the number of nymphs that survive after hatching by eliminating host plants and reduce populations below levels that are damaging to young citrus trees (Griffiths et al. 1947). Discing or chopping when grasshoppers are half grown can force migration onto adjacent citrus trees. Termites also can cause tree losses under circumstances similar to those that promote the spread of fire ants. Remnant termite populations survive on buried pine residues, especially in newly cleared pine and palmetto woodland, for 5 years or more. The eastern subterranean termite, Reticulitermes flavipes, has caused extensive losses in localized areas of young citrus planted in southwest Florida by girdling trees between the

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crown roots and the soil line. Shallow planting depth and soil removal at the tree base to the level of the crown roots reduced the risk of termite damage. Insecticides applied to the soil surface immediately adjacent to the trunk or inside tree wraps reduced damage and promoted healing (Stansly et al. 1992). Although Phytophthora lesions were not observed on trees girdled by termites, a significantly higher incidence of Phytophthora propagules was detected in samples of soil and feeder roots from girdled trees than from trees that were not girdled (Stansly et al. 1991). Larvae of the swallowtail butterfly (Papilio cresphontes), commonly called orange dogs, can defoliate young citrus trees. Bullock and Pelosi (1991) found that two orange dogs consumed an average of 331 cm 2 of foliage during their 20-day developmental period. E. Nematodes

Approximately 14 migratory endo- and ectoparasitic and sedentary endoparasitic nematodes have been reported as citrus pathogens (Inserra and Vovlas 1977; Van Gundy and Meagher 1977), with some having a wide host range. General symptoms include root destruction and distortion, stunting and reduction of tree vigor, and occurrence of nutrient deficiencies and excesses (Van Gundy and Martin 1961). Nematodes are most commonly spread on infested nursery stock, but many citrusproducing areas have established regulatory programs to certify citrus nurseries as nematode free. Moreover, quarantine programs have been established to prevent the spread of nematode infestations in citrus orchards (Calavan et al. 1978). In a survey conducted in Florida in 1984, the citrus nematode, Tylenchulus semipenetrans, was found in 45 of 50 mature citrus groves (Ferguson et al. 1995). However, approximately half of the infested groves contained trees beneath which no citrus nematodes were found. Many infested groves contained a patchy distribution of infested trees, suggesting random inoculation events possibly related to planting of infested nursery trees or movement of infested trees and soil. The citrus nematode can be especially important in replant situations and in areas previously planted to citrus, where they can survive from several months to 9 years in remnant citrus roots after infested citrus trees have been removed (Baines et al. 1977). The burrowing nematode, Radopholus citrophilus, causes spreading decline of citrus. It can travel in the soil as much as 15.2 m per year under favorable conditions (Suit 1947), but is usually not a problem in young orchards. Lesion nematodes, Pratylenchus spp., have been reported in

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Florida, Brazil, and Taiwan. Pratylenchus coffeae does not occur widely in Florida but can cause stunting and reduce leaf size (Whiteside et al. 1988). The sting nematode, Belonolaimus longicaudatus, was also reported in Florida (Kaplan 1985) as causing root damage and reduced growth of nursery and young field trees on rootstocks, including 'Cleopatra' mandarin, trifoliate orange, sour orange, 'Carrizo' citrange, and 'Swingle' citrumelo. F. Vertebrates New citrus plantings established in wildlife areas are occasionally damaged by deer feeding on leaves and twigs and rubbing trees to remove antler velvet. Browsing damage to young citrus trees was reported in California (Biehn 1951) and Florida, especially after growth flushes (Stanberry undated; Stith 1969). In a survey (1963 to 1965) of Florida citrus groves, Stith (1969) reported that 8% of young trees were killed at 16 locations. Damage included decreased growth rate, reduced crops, and unseasonal growth flushes predisposing young trees to freeze damage and tree death. G. Weeds Extensive weed growth can severely decrease growth of young citrus trees in both newly planted groves and reset situations through competition for water, nutrients, and light. However, some weed growth beneath the canopy for a limited time did not reduce tree growth of 1to 2-year-old trees (Futch 1997). Weeds can also create favorable conditions for diseases, insects, rodents, and other animal pests as well as disrupting grove management and increasing freeze damage (Jordan and Day 1973). Perennial vines can damage young citrus trees, especially in replant situations. Parasitic flowering plants such as Cuscuta and Cassytha species, found in California, Mexico, Florida, the West Indies, and South America, germinate in the soil but penetrate tree bark with rootlike extensions to secure nutrition. These dodders grow abundantly on branches and twigs, smothering seedlings in seed beds and young trees in the nursery and the field. Mistletoes in the genera Loranthus, Dedropemon, and Struthanthus are minor pests in South Africa and Mexico (Klotz 1978b). Milkweed or strangler vine, Morrenia odorata (Hook. & Arn.) Lindl., originally imported from South America as an ornamental, has been a major pest in Florida citrus groves. A Florida isolate of Phytophthora palmivora that is not pathogenic to citrus has been

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used as a biological control, although its effectiveness varies (Ridings et al. 1982). Weed management strategies and chemical control may also be different in containerized and field nurseries for rootstock seedlings and grafted trees, reset and interset trees, and newly planted groves where herbicides are applied in the water ring or as a band application. Pre-emergence herbicides applied at low rates controlled weeds in citrus containerized nurseries without phytotoxicity (Singh and Tucker 1983; Singh and Achhireddy 1984; Singh and Tucker 1984b). In groves without permanent irrigation systems, pre-emergence herbicides applied in irrigation water to the water ring around young trees also suppressed weed growth without apparent phytotoxicity (Singh and Tucker 1984a). Both mowing and tillage can cause injury to young tree trunks and roots, increasing susceptibility to diseases and insect pests. For shallow rooted trees in bedded groves, cultivation can destroy feeder roots in the top soil layers. Sod or mixed vegetation culture between rows and on ditch banks, combined with chemical weed control within the tree row, is common in Florida. For young orchards planted in terraced fields on slopes varying from 5° to 15° in Japan, sod culture is also maintained with weeding or mulching around the base of the tree (Ito et al. 1981; Suzuki 1981). Herbigation (delivery of herbicides through the irrigation system) is used in Israel (Oren and Israeli 1977) and Florida (Wondimagegnehu and Singh 1989), especially during the first years in the field when the tree root zone approximates the wetting zone of the irrigation system. Reclaimed wastewater has been used for irrigation and as a supplemental nutrient source for young citrus trees, as discussed previously. Increased irrigation levels also increased weed growth, possibly requiring higher weed management costs, especially for resets (Maurer et al. 1995). Soil moisture reduction, particularly during the dry season in arid and semi-arid regions, may reduce young tree growth and increase irrigation expenses. When annual weeds and bermuda grass were controlled at 0, 50 and 100% levels in mature California citrus orchards, soil moisture increased at 23-, 46-, and 92-cm depths (Jordan 1981). Kamarudzaman (1955) also reported that, in a 2-year-old planting of 'Valencia' oranges on sour orange rootstock, areas with grass cover used 50% more water than areas with no cover crop. Similarly, Smajstrla et al. (1985) found the presence of sod increased irrigation frequency compared to clean cultivation. Weeds have also been controlled in young orchards in Arizona (Jordan and Day 1973) or in arid and semiarid regions by irrigating only the basin between two ridges of soil, with weeds in non-irrigated areas controlled with foliar herbicide applications or discing.

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Organic and plastic mulches may control weed establishment from seed and may control perennial weeds in a limited area. Bredell et al. (1976) reported that a black polyethylene mulch around drip-irrigated trees increased tree diameter by 12% and canopy surface area by 25% after 4 years, compared with trees mulched with organic materials or without mulch. He suggested that higher temperatures under the plastic mulch may have extended the root growing season of young trees with small canopies that provide little shading. He also demonstrated that plastic mulching reduced evaporation by as much as 66% compared with that of a bare soil surface. Watergrass (Cyperus spp.) was the only weed species that grew in plots with plastic mulch (Bredell 1973). In contrast, black polyethylene sheets and fiberglass pads placed within the dripline suppressed weed growth but did not increase young tree growth during the first 2 years after planting in Florida, compared with bare soil (Jackson and Davies 1984). IX. SUMMARY

Citrus trees undergo tremendous changes in growth and morphology during their first few years of development in the orchard. During this period they are very susceptible to freeze and water stresses, disease and pest damage, and competition from weeds. Therefore, growers must be more vigilant in their cultural programs than they are for mature trees. The emphasis of a young tree program is to bring the tree into production as soon as possible by promoting rapid vegetative growth. This has been achieved in many citrus regions through the use of improved fertilization and irrigation practices that minimize stress and thus increase growth. Consequently, in some regions trees begin producing as soon as 2 years after planting. In some areas such as Florida, there is some concern that the growers "push" their trees too hard, causing them to be excessively vegetative and less productive in subsequent years. This observation has not, however, been substantiated experimentally. Replanting trees to replace dead or damaged ones also offers a challenge to growers. Automated and computerized irrigation systems, use of controlled-release fertilizers, and more effective spray materials has greatly improved survival and growth of these replants. In the future, advances in computer software, expert systems, and GIS systems for more accurate orchard mapping will continue to improve management practices for young citrus trees.

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LITERATURE CITED Abd-El-Naim, M., and R. M. El-Awady. 1989. Studies on heavy metal removal from sewage water used in sandy soils. p. 219-230. In: Toxic substances in agricultural water supply and drainage. 2nd Pan-Amero ICID Regional Conf., Denver, CO (Abstr.). Achor, D. S., H. Browning, and L. G. Albrigo. 1997. Anatomical and histochemical effects of feeding by Citrus leafminer larvae (Phyllocnistis citrella Stainton) in Citrus leaves. J. Am. Soc. Hort. Sci. 122:829-836. Agostini, J. P. 1989. Influence of citrus rootstocks on soil populations of Phytophthora parasitica Dastur. Ph.D. diss., Univ. Florida, Gainesville. Aiyappa, K. M., and K. C. Srivastava. 1965. Studies on root system of Coorg mandarin seedling trees. Indian J. Hort. 22:122-130. Albrigo, L. G., C. W. McCoy, and D. P. H. Tucker. 1987. Observations of cultural problems with the 'Sunburst' mandarin. Proc. Fla. State Hort. Soc. 100:115-118. Al-Jaleel, A., and J. G. Williamson. 1993. Effect of soil temperature and forcing method on scion budbreak and growth of citrus nursery trees. Proc. Fla. State Hort. Soc. 106:62-64. Alva, A. K. 1993. Copper contamination of sandy soils and effects on young 'Hamlin' orange trees. Bull. Envir. Contam. Toxicol. 51:857-864. Alva, A. K., and J. P. Syvertsen. 1991. Soil and citrus tree nutrition are affected by salinized irrigation water. Proc. Fla. State Hort. Soc. 104:135-138. Alva, A. K., and D. P. H. Tucker. 1993. Evaluation of a resin coated nitrogen fertilizer for young citrus trees on a deep sand. Proc. Fla. State. Hort Soc. 104:4-8. Aubert, B., A. Sabine, and P. Picard. 1984.Epidemiology of the greening diseasejn Reunion Island before and after the biological control of the African and Asian citrus psyllas. Proc. Inter. Soc. Citriculture 2:440-442. Azzena, M., P. Deidda, and S. Deltori. 1988. Drip and microsprinkler irrigation of young 'Valencia' orange trees. Proc. 6th Int. Soc. Citriculture 2:747-751. Baines, R. c., S. D. Van Gundy, and E. P. Du Charme. 1977. Nematodes attacking citrus. p. 321-347. In: W. Reuther, L. D. Batchelor, and H. S. Webber (eds.), The citrus industry. Vol. 4. Univ. California Press, Berkeley. Banks, W. A., C. T. Adams, and C. S. Lofgren. 1991. Damage to young citrus trees by the red imported fire ant (Hymenoptera: Formicidae). J. Econ. Entom. 84:241-246. Bevington, K. B. 1992. Initial growth and yield of high density plantings of 'Valencia' orange is influenced by microsprinkler and drip irrigation. Proc. Int. Soc. Citriculture 2:709-711. Bevington, K. B., and W. S. Castle. 1985. Annual root growth pattern in relation to shoot growth, soil temperature and soil water. J. Am. Soc. Hort. Sci. 110:840-845. Biehn, E. R. 1951. Crop damage by wildlife in California, with special emphasis on deer and waterfowl. Ca. Bur. Conserv., Game Bul. 5. Boman, B. J. 1993. First-year response of 'Ruby Red' grapefruit on four rootstocks to fertilization and salinity. Proc. Fla. State Hort. Soc. 106:12-18. Bourgeois, W. J., and A. J. Adams. 1987. Low-volume scaffold branch irrigation for citrus freeze protection. HortScience 22:48-50. Bredell, G. S. 1973. Response of eitrus trees to plastic mulching. Congo Mun. de Citricultum 1:387-394. Bredell, G. S., C. J. Barnard, and C. Human. 1976. Soil mulching practices: the effect of soil mulching on the root development of avocados and 'Valencias'. Citrus & Subtrop. Fruit J. 516:17-19, 21. Broadbent, P. 1981. Armillaria root rot of citrus in New South Wales, Australia. Proe. Int. Soc. Citrieulture 1:351-353.

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Rose, A. J., and G. Yelenosky. 1978. Citrus trunk wrap evaluations. Proc. Fla. State Hort. Soc. 91:14-18. Rossetti, V. 1993. Citrus variegated chlorosis, a new disease in Brazil, a review. Proc. Conf. Int. Org. Citrus Virol. 12:449-452. Rouse, R. K 1988. Bud forcing method affects budbreak and scion growth of citrus grown in containers. J. Rio Grande Valley Hort. Soc. 41:69-73. Rouse, R. K, and T. B. Sherrod. 1997. Optimum temperature for citrus seed germination. Proc. Fla. State Hort. Soc. 109:132-135. Sauer, R. M. 1951. Growth of orange shoots. Austral. J. Agr. Res. 2:105-117. Schaffer, B., J. K Pena, A. M. ColIs, and A. Hunsberger. 1997. Citrus leafminer (Lepidoptera: Gracillariidae) in lime: assessment of leaf damage and effects on photosynthesis. Crop Protection 16:337-343. Schoulties, C. 1., K 1. Civerolo, J. W. Miller, R. K Stall, C. J. Krass, S. R. Poe, and K P. Ducharme. 1987. Citrus canker in Florida. Plant Dis. 71:388-395. Schoulties, C. 1., J. W. Miller, R. K Stall, K 1. Civerolo, and M. Sasser. 1985. A new outbreak of citrus canker in Florida. Plant Dis. 69:361. Simanton, W. A. 1976. Populations of insects and mites in Florida citrus groves: a summary of 16 years of monthly inspections. State Doc. 7, Univ. Fla., Gainesville. Singh, M., and N. R. Achhireddy. 1984. Tolerance of citrus rootstocks to preemergence herbicides. J. Environ. Hort. 2:73-76. Singh, M., and D. P. H. Tucker. 1983. Preemergence herbicides for container-grown citrus. HortScience 18:950-952. Singh, M., and D. P. H. Tucker. 1984a. Evaluation of herbicides for water ring treatments in citrus. Proc. Fla. State Hort. Soc. 97:51-53. Singh, M., and D. P. H. Tucker. 1984b. Herbicide evaluation for weed control in Florida citrus nurseries and groves. Proc. Int. Soc. Citriculture 1:181-185. Smajstrla, A. G., and R. C. J. Koo. 1984. Effects of trickle irrigation methods and amounts of water applied on citrus yields. Proc. Fla. State Hort. Soc. 97:3-7. Smajstrla, A. G., 1. R. Parsons, K. Aribi, and G. Velledis. 1985. Response of young citrus trees to irrigation. Proc. Fla. State Hort. Soc. 98:25-28. Smith, P. F., and G. K. Rasmussen. 1958. Relation of fertilization to winter injury of citrus trees. Proc. Fla. State Hort. Soc. 71:170-175. Smith, P. F., W. Reuther, and G. K. Scudder. 1953. Effect of differential supplies of nitrogen, potassium, and magnesium on growth and fruiting of young 'Valencia' oranges in sand culture. Proc. Am. Soc. Hort. Sci. 61:38-48. Smittle, B. J., c. T. Adams, W. A. Banks, and C. S. Lofgren. 1988. Red imported fire ants: feeding on radio labeled citrus trees. J. Econ. Entomol. 81:1019-1021. Stanberry, F. W. Undated. Deer damage to agricultural crops-Florida. Fla. Game and Fresh Water Fish Comm. Special Report. Stansly, P. A., R. K Rouse, and S. B. Davenport. 1992. Protection of young citrus trees from damage by subterranean termites. Proc. Fla. State Hort. Soc. 105:7-10. Stansly, P. A., R. KRouse, R. J. McGovern, and S. B. Davenport. 1991. Chemical deterrents to girdling of young citrus by subterranean termites. Proc. Fla. State Hort. Soc. 104:156-159. Stith, 1. G. 1969. Deer damage to citrus trees and control with repellant sprays. M.S. thesis, Univ. Florida, Gainesville. Stoffella, P. J., Y. Li, R. R. Pelosi, and A. M. Hamner. 1995. Citrus rootstock and carbon dioxide enriched irrigation influence on seedling emergence, growth and nutrient content. J. Plant Nutr. 18:1439-1448. Su, H., and J. Cheon. 1984. Occurrence and distribution of tatter-leaf citrange stunt complex on Taiwanese citrus. Proc. Int. Soc. Citriculture 2:426-427.

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Suit, R F. 1947. Spreading decline of citrus in Florida. Proc. Fla. State Hort. Soc. 60:17-23. Suzuki, K. 1981. Weeds in citrus orchards and their control in Japan. Proc. Int. Soc. Citriculture 2:489-492. Swietlik, D. 1992. Growth, yield and mineral nutrition of young 'Ray Ruby' grapefruit trees under trickle or flood irrigation and various nitrogen rates. J. Am. Soc. Hort. Sci. 117: 22-27.

Swietlik, D., and J. V. LaDuke. 1985. Nutritional status and growth responses of freezeinjury citrus trees to mineral foliar sprays in the first year of recovery. J. Rio Grande Valley Hort. Soc. 38:51-58. Syvertsen, J. P. 1982. Minimum leaf water potential and stomatal closure in citrus leaves of different ages. Ann. Bot. 49:827-834. Syvertsen, J. P., and J. J. Lloyd. 1994. Citrus. p. 65-99. In: B. Schaffer and P. C. Andersen (eds.), Handbook of environmental physiology of fruit crops, Vol. II: Subtropical and Tropical Crops. CRC Press, Boca Raton, FL. Syvertsen, J. P., and C. W. McCoy. 1985. Leaf feeding injury to citrus by root weevil adults: leaf area, photosynthesis and water use efficiency. Fla. Entomol. 68:386-393. Syvertsen, J. P., and G. Yelenosky. 1988. Salinity can enhance freeze tolerance of citrus rootstock seedlings by modifying growth, water relations, and mineral nutrition. J. Am. Soc. Hort. Sci. 113:889-893. Tignor, M. K, F. S. Davies, and W. B. Sherman. 1998. Irrigation and nutrient application frequency effects on freeze hardiness and growth of 'Hamlin' orange trees in Florida. Proc. Fla. State Hort. Soc. (in press). Tignor, M. K, F. S. Davies, W. B. Sherman, and J. M. Davis. 1997. Rapid freeze acclimation of Poncirus trifoliata seedlings exposed to 10 D C and long days. HortScience 32:854-857.

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8 Fruit Growth Measurement and Analysis* Lin us U. Opara Bioproducts Quality Research Center for Postharvest & Refrigeration Research Institute of Technology and Engineering, Massey University, Palmerston North, New Zealand I. Introduction A. General B. Historical II. Fruit Developmental Stages A. Growth B. Maturation C. Ripening D. Senescence III. Indices of Fruit Growth A. Microstructural B. Macrostructural C. Biochemical D. Changes in Seeds E. Developmental Indices of Fruit Growth 1. Chronological Indices 2. Environmental Indices IV. Growth Measurement Techniques A. Destructive Fruit Growth Measurement 1. Physical Measurements 2. Microstructural Measurements 3. Biochemical and Physiological Measurements *The author acknowledges the financial support of the former New Zealand University Grants Committee (UGC) through the award of a Doctoral Scholarship. I thank the three referees and editor for their valuable comments and suggestions that led to a substantial improvement of the manuscript, and Drs. Ian Warrington, Nihal de Silva, and Paul Austin at HortResearch, Palmerston North, New Zealand, for providing me with copies of their publications on apple fruit growth.

Horticultural Reviews, Volume 24, Edited by Jules Janick ISBN 0-471-33374-3 © 2000 John Wiley & Sons, Inc. 373

374

1. OPARA B. Non-destructive Fruit Growth Measurement C. A Mixed Model Analysis for Fruit Sampling

V. Approaches to Fruit Growth Analysis A. Graphical Analysis of Growth Pattern 1. Sigmoidal Growth 2. Double-sigmoidal Growth 3. Exponential or Curvilinear Growth Pattern 4. Effects of Growth Measurement Interval and Type of Primary Data 5. Conflicting Literature Evidence B. The Classical Approach 1. Simple Ratios 2. Absolute Growth Rate 3. Relative Growth Rate 4. Mean Relative Growth Rate C. The Functional (Curve Fitting) Approach 1. Empirical Functional Models 2. Mechanistic Functional Models 3. Expolinear Functional Models 4. Stochastic Functional Models 5. Other Functional Models D. Biological Significance of Functional Growth Parameters E. Computers and Electronics in Fruit Growth Analysis VI. Applications of Fruit Growth Data VII. Some Problems in Fruit Growth Measurement and Analysis VIII. Summary and Prospects A. General Review B. Prospects for Rationalized Techniques and Units 1. Instantaneous versus Mean Values of Growth Data 2. Units of Measurement 3. Symbols and Notations C. Future Research Literature Cited

I. INTRODUCTION

A. General

Fruit size and size distributions are important horticultural characteristics. In general, large fruits fetch better prices, although there are rare exceptions, such as cucumber for pickling. In many fruit crops, high yields of small fruit may be economically worthless, hence the extensive practice of fruit thinning. As a consequence of the key role of fruit size, there are extensive research efforts to uncover the mechanism of growth in order to develop strategies, both genetically and culturally, to regulate fruit size. This review strives to identify the various indices of fruit growth and development, and summarizes the methodologies for their measurement and analysis. Destructive and non-destructive

8. FRUIT GROWTH MEASUREMENT AND ANALYSIS

375

techniques for fruit growth measurements are presented and the classical and functional approaches to fruit growth analysis are reviewed. Finally, a rational system of fruit measurement is proposed. The emphasis in this review is on growth based on changes in size and form in time. Because fruit growth is often associated with that of the seed, growth of seed will be covered where it provides additional information on fruit growth. Detailed discussions on indices for whole plant growth can be found in Watson (1947a, 1952, 1958), Richards (1969), Evans (1972), Causton (1977), and Hunt (1978, 1990). A definitive and in-depth advanced coverage of plant growth analyses can be found in Evans (1972), while Kvet et al. (1971) present an excellent and wideranging review, covering both classical and functional approaches. The botanical fruit refers to a mature ovary and other flower parts associated with it plus receptacle, withered remnants of the petals, sepals, stamens, and stylar portions of the pistil. It also includes any seeds contained in the ovary. There are many kinds of fruits that are horticulturally important, including dry or fleshy, and simple, multiple, or compound (Fig 8.1). The derivation of common fruits from various plant tissues is shown in Fig 8.2 (Coombe 1976). The horticultural literature is dominated by studies on the growth and development of fleshy fruits (apple, pear, peach, and grape), as is this review, but information on non-fleshy fruits will also be covered here. Growth is an essential element of the life cycle of all living organisms. It occurs as a change in size, form or shape, and number. The potential size of a plant and its organs such as fruit is genetically determined (Strickberger 1976). Thus, growth rate, size, and composition depend on the interaction between the genetic constitution, the needs for metabolism, and on the environment (Lawlor 1991). In fruit, growth generally refers to the increase in length, diameter, and mass. This is macroscopic (whole) fruit growth. Fruit growth also occurs at the microstructural (cellular) level, which manifests as increases in cell size and number, as well as cell wall thickness and size of intercellular air space (Opara and Bollen 1995). In this review, the term fruit development is used to refer to the series of processes from the initiation of growth to death (Watada et al. 1984). When the term growth is applied to the performance of horticultural plants, it is used primarily to describe the irreversible increase in physical attributes of a developing plant or plant organ (Watada et al. 1984). The physical growth attributes are size, changes in form (shape), and sometimes includes changes in number (Hunt 1978). Thus, plant growth may be classified into two main levels: (a) the whole plant and (b) the constituent parts such as root, stem, leaf, and fruit.

L. OPARA

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8. FRUIT GROWTH MEASUREMENT AND ANALYSIS

377

Fig. 8.2. Outlines of sections of the fruits of 11 species showing the diversity of tissues that can develop into fruit flesh (Coombe 1976).

Hardwick (1984) defined growth analysis as the procedure of analyzing plant growth rate by expressing it as the algebraic product of a series of factors. This definition focuses unnecessarily on the plant alone without reference to the components such as fruit, embryo, seed, or root, which are often of major interest in growth studies. Fruit growth analysis is therefore a quantitative approach, using simple basic data such as mass, diameter, and length, to determine the magnitude or pattern of changes in growth attributes during the time course of fruit development. In growth analysis, the results derived from the principal growth data can be expressed as mean values or instantaneous values. The mean values are calculated by the classical approach, while the instantaneous values are obtained by the functional approach. The definitions and formulae used for these calculations are discussed in Section V. Throughout this review, the term growth rate will be used broadly to include all indices used for quantification, including absolute

378

L. OPARA

growth rate, relative growth rate, and mean relative growth rate. In some cases, to avoid misinterpretation, other terminologies related to fruit will be used according to the original research. B. Historical In a series of classical experiments on maize (Zea mays, cv. 'Badischer Fruh') carried out at Poppelsdorf in Germany in the 1870s, Kreusler et al. (1879) demonstrated a growth pattern which has since been found to be typical among annual plants and plant organs such as fruit. The key features of this pattern of growth includes the great variation in the magnitude of the growth quantities, the symmetry of the growth curve, and the time scale which it occupies (Hunt 1978). During the early part of this century, studies by Noll and his pupils in Germany (Gericke 1908; Kiltz 1909; Blackman 1919,1920; Kidd and West 1919; Evans 1972) and Gresseler (1907) on sunflower growth also contributed to the development of modern concepts of growth analysis. Noll and his pupils defined a simple ratio called the plant's Substanzquotient (substance quotient), which gave a measure of the Assimilationsenergie (assimilation energy) of a plant at different periods of its life. This quantity was obtained by determining the quantity of dry substance of a plant at equal intervals and relating each weight thus obtained to the previous one, dividing the former by the latter. Thus, the magnitude of the quotient is related to both the rate of plant growth or its constituent and the length of the time interval between the two growth measurements. Earlier researchers who applied Noll's Substanzquotient in growth analysis used a constant interval of 7 days, although it was not originally defined in relation to a specific time interval (Hackenberg 1909). Noll's quotient is no longer used in growth studies, and is now replaced by various growth rates which I briefly review in Section VB. The publication of Blackman's (1919) paper entitled The Compound Interest Law and Plant Growth marked a significant turning point in mathematical analysis of growth data. Blackman recalculated Gressler's (1907) data on sunflower growth, and used Eq. (1) to calculate an "efficiency index" (R) of dry weight production:

[1] where X is the weight at time T, R is the rate of interest, termed the "efficiency index," which is a measure of the efficiency of dry weight production. Further applications and refinement of Blackman's concept of "efficiency index" led to the proposition of "relative growth

8. FRUIT GROWTH MEASUREMENT AND ANALYSIS

379

rate" (RGR) (West et al. 1920) as an alternative summation or integration of all the developmental processes that contribute to growth. Today, the term RGR is the most widely used index in growth analysis studies. One of the earliest studies that measured fruit growth throughout the growth period was reported by Anderson (1895), in which the mass of Cucurbita pepo (summer squash) fruit was measured by a se1£registering balance. With increasing interest in commercial horticulture (Chandler 1925), the study of fruit growth became widespread during the first two decades of this century, notably in the apple (Whitehouse 1916; Magness and Diehl 1924), peach (Connors 1919), tomato, cucumber, muskmelon, and squash (Gustafson 1926), and citrus (Waynick 1927). In these studies, fruit growth was assessed by increase in length, width, and mass in detached fruit, or the volume of a fruit was measured by the displacement of water in a calibrated container. II. FRUIT DEVELOPMENTAL STAGES

The chronological development of fruit from flowering to maturity and senescence involves a sequence of physical and biochemical changes, both at the macro- and micro-levels. The molecular, cellular, and physiological mechanisms involved in fruit development (Bollard 1970; Coombe 1976; Gillaspy et al. 1993), the regulation of assimilate supply to fruit (Ho 1992; Marshall and Grace 1992), and the role of endogenous auxins (Varga and Bruinsma 1976; Miller 1990) in developing fruit are well documented. The entire period during which these developmental changes occur can be broadly classified into four stages: growth, maturation, ripening, and senescence. Often in horticultural literature, and in fruit growth studies in particular, there is a great deal of confusion and misuse of the concepts that describe distinctive stages from "birth" to "death" of fruit (Watada et al. 1984; Kader et al. 1985). A. Growth The period of growth commences at the onset of fruit development (Fig 8.3), and is generally accompanied by increase in size, change in shape, and mobilization of food materials from other parts of the plant. The initiation of the floral primordia, which develops concurrently with the flower, occurs prior to fruit development (Janick 1986). Fruit growth commonly involves (1) the enlargement of the ovarian tissue, which is generally stimulated by pollination, and (2) the enlargement of the receptacle, or both. Enlargement of the ovary results in fruit set, while the

L. OPARA

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enlargement of the receptacle accounts for most of the growth during the later periods of fruit development. The entire period of fruit growth is accompanied by significant changes in cellular structures, which result in observed changes in fruit size and shape. These changes involve both cell division and cell enlargement, but the early growth of the ovary occurs primarily due to cell division, with little cell enlargement

8. FRUIT GROWTH MEASUREMENT AND ANALYSIS

381

(Janick 1986). The growth of the ovary may cease at the time of, or before, anthesis, or continue for a time after anthesis and prior to pollination. Following pollination, the ovary grows rapidly and this marks the beginning of fruit development. In many species, the entire fruit growth pattern is characterized by an initial period of rapid cell division and development of cell walls, followed by a long period of cell expansion primarily by vacuolation. The duration of cell division during fruit development and the extent to which cell division contributes to whole fruit growth vary considerably among species (Ho 1996; Cowan 1997). In fruit such as blackcurrant (Ribes nigrum) , cell division (except in the embryo and endosperm tissue) ceases at anthesis, while in tomato (Lycopersicom esculentem) , cell division is completed at the time of pollination. In these species, final fruit size is dependent on the number of cells in the ovary at anthesis. In some species (such as apple, cucurbits, and citrus) there is a brief period, while in others (such as strawberry and avocado) there is a rather extended period of cell division after pollination. In the avocado, for instance, cell division continues throughout fruit ontogeny, albeit at a slower rate during the latter stages of development (Zilkah and Klein 1987; Cowan 1997). Thus, in many species, cell division is completed at or shortly after flower opening (anthesis), and later growth of the fruit following pollination is predominantly due to increases in cell size rather than in cell number (Wareing and Phillips 1970). It is therefore common for the early stages of fruit growth to involve not only cell expansion but also cell multiplication, highlighting the inability of cell enlargement processes alone to account for fruit set and fruit growth (Leopold 1964). In some fruits such as mature watermelon (Citrullus vulgaris), the cells may enlarge to tremendous sizes to the extent that they can be distinguished individually by the naked eye. B. Maturation From a physiological perspective, maturation refers to the processes associated with completing natural growth and development and the attainment of full size. At physiological maturity, a plant organ such as fruit will continue ontogeny even if detached (Watada et al. 1984). On the other hand, horticultural maturity has been defined as the stage of development when a plant or plant part possesses the prerequisites for utilization by consumers for a particular purpose (Watada et al. 1984; Reid 1992). Thus, fruit may be horticulturally mature in the early stage, mid-stage, or late stage of development, depending on the intended use.

382

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The term horticultural maturity is synonymous with commercial maturity, which generally refers to the maturity of fruit for eating (Kingston 1991; Abbot et al. 1997; Behboudian and Mills 1997). Watada et al. (1984) proposed a framework for defining the horticultural maturity of several crops in relation to developmental stages of the plant (Fig. 8.3). In postharvest technology, maturation is commonly defined as "that stage at which a commodity has reached a sufficient stage of development that after harvesting and postharvest handling, its quality will be at least the minimum acceptable to the ultimate consumer" (Reid 1992). Several maturity indices and techniques are available for assessing the maturity of temperate and tropical fruits (Arthey 1975; Watada et al. 1984; Reid 1992; Studman and Yuwana 1992). C. Ripening Stage

The term ripening refers to the processes that qualitatively transform the mature fruit as it reaches the end of its growth period (Leopold 1964). These changes in ripening fruit are well documented and generally include tissue softening, with the associated changes in coloration and flavor, hydrolytic changes, which usually result in the formation of sugars, increased permeance of the cuticle to gases, respiratory climacteric and other quality changes, such as production of flavor (Biale 1950; Beevers 1961; Burg and Burg 1962a,b; Watada et al. 1984; Dadzie 1992; Maguire 1998). The ripening of fleshy or succulent fruits is a complex process that ultimately terminates in the senescence and decay of the tissue. The biochemical pathways that are implemented during ripening vary among fruit species, and fruits can be classified as climacteric or non-climacteric based on the occurrence of a respiratory climacteric during ripening. These pathways represent a highly coordinated sequence of events (Roberts and Hooley 1988), and thus ripening is not considered a degenerative process arising out of cell autolysis, but an example of a precisely regulated developmental program (Brady 1987). The changes that occur during ripening are driven by large expenditures of respiratory energy, and the significant role of ethylene in fruit ripening has been extensively studied (Nelson 1940; Hansen 1942; Biale et al. 1954; Ulrich 1958; Biale 1960; Varner 1961; Burg and Burg 1962a). D. Senescence

Senescence is used collectively to refer to the degradative changes that naturally lead to death of whole plants or organs. From a mathematical viewpoint of growth analysis, senescence has been defined as the period

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during which growth rates recede after the principal period of growth (Robertson 1923). The period of senescence is therefore characterized by depressed growth rates. Fruit senescence begins during ripening of both fleshy and non-fleshy fruits. The generalization that senescence is not inevitable as long as cell growth can proceed (Sax 1962) can be extended to say that termination of growth sets the stage for senescence (Leopold 1964). Several biochemical and physiological changes in fruit accompany senescence, but a biophysical basis may be the deterioration of structural integrity within the aging fruit. These changes in fruit manifest as loss of firmness, color degradation, and increased skin membrane permeability. From a developmental perspective of the whole plant, the advantages of senescence have been documented (Sax 1962), including the potential to permit a more rapid evolutionary development through a more speedy genetic turnover, such as allowing nutrient partitioning to seeds. The precise mechanism of senescence in plants or organs is not adequately known. Sklensky and Davies (1993) provided an extensive review on senescence in relation to reproduction and nutrient partitioning in whole plants with monocarpic growth habits, and concluded that there was no unequivocal evidence of any factor exported from flowers or fruits of annual plants that causes initiation of senescence. In his classical study of cytomorphosis, Minot (1908) conceptualized aging as the progression of senescence, claiming that the rate of senescence increases from birth. Thus, during fruit growth and development, senescence marks the period during which fruit has lost the power of growth. The period of senescence in fruit is also marked by rapid loss of shelf life, and in some fruit such as the apple, senescence also results in a loss of eating quality. III. INDICES OF FRUIT GROWTH Fruit growth is accompanied by changes in both internal cellular structures and composition, as well as changes in external (macro) whole fruit structures. Accordingly, these microstructural and macrostructural changes provide indices of growth in fruit. A. Microstructural Fruit Growth Indices

Fruits, like other plant organs, are collections of cells that function in a coordinated manner to undertake all the developmental processes needed for growth. The growth of fruits involves both increase in cell

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number due to cell division, and increase in cell size due to vacuolation. These two phases in fruit growth are usually separated in time, giving rise to an early phase when cell division predominates, followed by a second stage during which there is active increase in cell size. Since growth involves increases in cell number, cell size, and size of intercellular air space, these criteria may be used as indices of fruit growth. In many species, such as cherry and apple, fruit size is correlated with cell size and cell number (Bain and Robertson 1951), but is more closely related to cell number (Goffinet et al. 1995). In fruit such as the apple, the development of air spaces during the period of cell enlargement also contributes to the total growth. Bain and Robertson (1951) showed that fruit weight was well correlated with cell number in 'Granny Smith' apple, and during growth there was a greater increase in fruit volume than in fruit weight, indicating the development of air spaces as part of fruit development. B. Macrostructural (External) Fruit Growth Indices Although fruit growth is often accompanied by significant changes in cellular and biochemical attributes, it is often difficult and inconvenient to quantify these changes. Firstly, most microscopic and biochemical measurement techniques are destructive, and require specialized instrumentation and tedious sample preparation and handling protocol. Secondly, advanced image capture and analysis may be required for detailed and accurate quantification of cellular attributes. Since fruit growth results from increases in cell number and cell size, which manifests as increase in dimensions and mass, it is often convenient to measure increases in fruit diameter, length, and mass over a chosen time interval. Most fruit growth studies use one or more of these primary data as indices of fruit growth. Two assessments are often required to carry out a simple growth analysis (Radford 1967; Beadle 1993): (1) a measure of the material present, and (2) a measure of the magnitude of the assimilatory system of the plant material. The assimilatory system is usually measured in terms of the leaf area, but it can be the leaf protein, leaf nitrogen, or chlorophyll content. In fruits, the basic measures of material present are the total mass (fresh or dry), and size (length or diameter). In some growth studies, these basic measures are often converted into derived units of growth such as area, volume, shape, and density. The use of total weight in growth analysis assumes that dry matter increase is the result of net photosynthetic activity of the whole plant. Although dry weight is commonly used in growth analysis to reflect the actual amount of new organic material synthesized

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by the plant or organ, a change in dry weight is not always a satisfactory measure of growth, since plant tissues may increase in dry weight due to the accumulation of reserve materials, such as starch and fat, even though the organ may not be growing (Wareing and Phillips 1970). C. Biochemical Fruit Growth Indices Biochemical changes have also been measured as indices of growth (McKee et al. 1955; Wright 1956; Schenk 1961). Because the concentrations of these substances is generally correlated with fruit growth, horticulturists have successfully used some of them, such as the hormones, to alter fruit size, shape, and the timing of physiological events such as fruit abscission. The choice of any attribute for growth studies depends on the type of fruit and the purpose of growth measurement. Often, more than one attribute is required to adequately characterize the growth of fruit. Schenk (1961) illustrated the separate growth rates for parts of the peanut fruit, and found that an initial preferential growth of the shell yielded later to a growth of skins and later the kernel. The transfer of mobilization sites resulted in extensive losses from former mobilization centers. Chapman et al. (1991) measured the physical and chemical changes during the maturation of peaches and reported that increased levels of volatiles closely paralleled seed and mesocarp growth. D. Changes in Seeds as Indices of Fruit Growth Seeds, including embryo and endosperm, play important roles in fruit development (Nitsch 1950; Visser 1955). In parthenocarpic fruit that develop without seeds, it is well known that in many instances the presence of seeds markedly alters fruit size (Leopold 1964). For instance, fruits of some squash cultivars are oblong when seedless, but when seeds are present they are pear-shaped. Similarly, parthenocarpic pears are oval in shape, and they develop the pyriform shape only when seeds are present. The number and position of the seeds affects fruit shape, and in stone fruits, abortion of the embryo often results in fruit abscission from the tree. Because of their influence as sources of growth-stimulating signals in fruit, researchers have measured some physical and biochemical developmental changes in embryos and endosperm as indices of growth. This approach to analysis is particularly useful in understanding the mechanisms by which the mobilization of substances (food materials such as nutrients) occurs. Carr and Skene (1961) studied the increase in fresh weight of the pod and seed of the bean fruit, in which the pod develops

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first, followed by the embryo and seed. The seeds exhibited a double sigmoid growth curve, while the whole fruit followed a single sigmoidal curve. Tukey (1933) reported that the pericarp tissues of 'Elberta' peach followed a double sigmoid growth, and that the cessation of the first growth period coincided approximately with the completion of nucellar (nucellus plus integnum) growth and onset of embryo growth. Schenk (1961) studied the differential growth of the parts of the peanut fruit based on protein content (mg per fruit) and showed that extensive loss of protein mobilization sites in the shell and skins resulted in transfers to the kernels. The data on pea fruit (Vida faba) by Emmerling (1880) and cited by McKee et al. (1955) clearly illustrates the mobilization of nitrogenous materials from the leaves into the hull and then into the seeds during fruiting. The potential application of somatic embryogenesis for propagating forest trees has focused research on seed and embryo development (Durzan 1988; Wann 1988; Janick et al. 1991). Physical attributes such as embryo dry weight and length, and biochemical quantities such as fatty acid and sucrose gradients have been used to quantify the changes that occur during seed development. In loblolly pine (Janick et al. 1991) embryos could be macroscopically observed on or about day 30, and embryo density (based on sucrose density gradient) decreased from day 30 to 50 by 10% sucrose equivalent. Similarly, embryo dry weight, length, and fatty acid accumulation increased until about day 50 and then remained constant. E. Developmental Indices of Fruit Growth

One of the fundamental aspects of growth analysis is the selection of a developmental index to characterize the period growth duration. These indices can be used for experimental design and evaluation, and they can also provide a means of extrapolating fruit developmental processes and anatomical events among different seasons (years) and growing regions (Gage and Stutte 1991). In fruit growth studies, several chronological and environmental indices are commonly used to describe the course of fruit development. Selection of a developmental index is preferably based on a simple measurement that changes in a predictable and quantifiable way during development and which can be recorded for each plant or fruit to be studied or analyzed (Erickson 1976). 1. Chronological Indices. These can be based on days after a specific mor-

phological event (such as flowering, full bloom, anthesis, petal fall), the Julian date, the calendar date, or the time (usually days) after pollination

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387

or a specific treatment. The days after a specific physiological event (such as pit hardening, split-pit, internal ring-cracking, stem-end splitting) can also be used as a chronological index of fruit growth (Opara 1993). The date of full bloom, the time of maximum blossom opening, is the most widely used index in fruit growth measurement, but it is considered something of a misnomer (Gage and Stutte 1991) because of severallimitations. Populations of blossoms may open and become fertilized over several days, thereby making precise assessment of full bloom difficult (Ragland 1934). The date of full bloom can vary considerably from year to year (Weinberger 1948), and the fact that the determination is somewhat subjective introduces an inherent variability into the chronological characterization of subsequent developmental events (Fischer 1962). This criticism also applies to the chronology used by Worrell et al. (1998) to study the growth of breadfruit, which is based on "the earliest detectable stage." Gage and Stutte (1991) have presented a good analysis of the pros and cons of other chronological indices of fruit growth. 3. Environmental Indices. These indices are based on the dependence of fruit growth on important environmental factors, especially temperature (Fischer 1962; Munoz et al. 1986; DeJong and Goudrian 1989; Topp and Sherman 1989; Gage and Stutte 1991). Growing degree days and heat accumulation are the two environmental indices for fruit growth, and they are particularly applicable in the early stages of development (Fischer 1962; Batjer and Martin 1965; Topp and Sherman 1989). These indices are objective and rely on a measurable parameter, temperature, which has quantitative effects on growth and development. However, the development of a universally suitable environmental index is difficult because of the temperature interactions with photosynthesis, water potential, respiration, and other physiological processes (Gage and Stutte 1991). Furthermore, because of the complex nature of tree architecture, fruit micro-environment, and fruit physiology, no single environmental index explains the variability that often occurs in fruit growth. Often, the environmental indices of fruit growth are related to a chronological index, such as "degree-days after flowering" (Pavel and DeJong 1993a,b). The diversity of chronological and environmental indices available for characterizing fruit growth reflects the range of factors and developmental events that influence fruit growth. The corollary is that several factors can influence the choice of time indices for fruit growth studies, including the type of fruit, time available for measurement, and length of the growing season. The objective of the study (for instance, quantification of season-long fruit growth or diurnal changes) is also a critical factor.

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IV. GROWTH MEASUREMENT TECHNIQUES

Several procedures have been employed to measure fruit growth. These procedures are either (1) destructive and involve the removal of fruit from the plant for field/laboratory measurement, or (2) non-destructive and involve the selection and tagging of individual fruit on the tree on which repeated growth measurements are made. Whichever technique is used, the time during the 24-h period when samples are collected and/or measured must be standardized to minimize any errors that may arise due to diurnal fluctuations in fruit growth (Tukey 1956, 1960; Diaz-Perez and Shackel 1991). A. Destructive Fruit Growth Measurement

This involves periodic removal, at appropriate time intervals, of fruit samples that are measured later (usually within 24 h) to obtain primary growth data for subsequent analysis. Measurement techniques must be standardized to ensure that results obtained can be interpreted in the context of existing literature. A major disadvantage of such destructive measurement of growth is that it does not reveal the fruit growth rhythm and diurnal fluctuations that may be of particular interest in understanding the environmental and physiological basis of the measured growth. 1. Physical Measurements. Following harvest, physical properties of

fruit such as length, diameter, and mass can be measured using suitable devices such as ruler, calipers, and desk-top balance. Many analogue and digital types of devices are available and most can provide accuracy to 1 %, which is often sufficient for most applications. Electronic instrumentation is also available which enables automatic data-logging of the measuring device to the computer to facilitate data handling and analysis (Jaffe 1976; Jaffe et al. 1985; Johnson et al. 1992; Kerckhoffs et al. 1997). Fruit mass is usually expressed on a dry weight or fresh weight basis. 2. Microstructural Measurements. Microscopy techniques such as light microscopy, electron microscopy, and confocal laser scanning microscopy are used to measure changes in microstructural attributes, such as cell size, cell number, and intercellular air space, as indices of fruit growth (Gpara and Bollen 1995). Schechter et al. (1993a) combined physical measurements (and their relative growth rates) and rates of cell division to obtain a more complete picture of the growth pattern of apple fruits.

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3. Biochemical and Physiological Measurements. Certain physiological and biochemical measurements provide indices of changes in fruit growth and development. These include respiration, chlorophyll content, pH, acidity, sugar gradients, starch, hemicellulose, relative density as sucrose gradient, and fatty acid content in fruit, seed, or embryo (Watada et al. 1984; Janick et al. 1991). Yamaguchi et al. (1996) studied the changes in the amounts of NAD-dependent sorbitol dehydrogenase (NAD-SDH) (enzyme code, 1.1.1.14) and its involvement in the development of apple fruit and found a weak correlation between enzyme protein and the NAD-SDH activity. The changes in the amounts ofNADSDH protein did not show the same pattern as those in relative growth rate, which may be used to express sink activity, especially in young fruit (Archbold 1992).

B. Non-destructive Fruit Growth Measurement

By strict definition, non-destructive growth measurement techniques involve continuous or periodic measurement of the same intact growing fruit. However, some researchers have used the term "non-destructive" measurement to include the measurement of the volume and dimensions of harvested fruit from which the mathematical relationships were developed for predicting other growth attributes such as fresh mass (Marcelis 1992). The most common approach is to randomly select a sample of fruit, tag them, and manually measure fruit diameter and length at marked positions on each individual fruit with a caliper (Gustafson 1927; Schapendonk and Brouwer 1984; Tazuke and Sakiyama 1984; Gpara 1993; Nerd and Mizrahi 1998; Worrell et al. 1998). From these data, fruit volume can be estimated and a regression equation is often used to estimate fresh mass from the volume or dimensional data (Schapendonk and Brouwer 1984; Tazuke and Sakiyama 1984). Alternatively, sensors can be used to measure the growth of attached fruit based on gross changes in dimensions (Higgs and Jones 1984; Tazuke and Sakiyama 1984; Johnson et al. 1992) or volume (Lang and Thorpe 1989). Fruit volume can be measured by water displacement (Gustafson 1926; Struik et al. 1988; Magein 1989) or by measuring the buoyant force on a fruit immersed in fluid based on Archimedes' principle (Lang and Thorpe 1989; Lang 1990). Photographic techniques have also been used to measure fruit growth (Erickson 1976; Wehner and Saltveit 1983). Photographic analysis of developing cucumber fruit at 2- or 3-day intervals over a 3D-day period beginning from pollination showed that all sections of the fruit grew in length at a constant rate. The pattern of growth

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was fairly uniform, except that there was slightly more growth in the center section than at the ends. The diameter of intact growing tomato fruit has been measured using electronic calipers, based on the use of a computer-linked linear voltage displacement transducer (Grange and Andrew 1993; Johnson et al. 1992; Pearce et al. 1993a,b). In comparison with destructive growth measurement techniques, the non-destructive approach has the advantage of reducing fruit-to-fruit variation and providing detailed information of the growth of fruit such that the effects of important events (both external and internal of fruit) on fruit development can be quantified. Another advantage of nondestructive measurement is that fewer plants and fruit are necessary for measurements, and this saves time and space, particularly in controlled environment experiments. The choice of a particular non-destructive measurement technique will depend on the feasibility of its use for the type of fruit, growing environment, simplicity, accuracy, rapidity, and cost. Further reductions in the cost of computers and electronic devices in general should facilitate the increased application and adoption of these techniques for both research and production horticulture. C. A Mixed Model Analysis for Fruit Sampling One of the difficulties in fruit growth studies is the selection of an appropriate sampling strategy for estimating fruit size so as to adequately account for the variation within the plant. Traditionally, fruits are sampled at the lower, middle, and top parts of the tree. However, vines such as kiwifruit may present additional difficulty due the architecture of the plant or training system used. De Silva and Ball (1997) developed a mixed model analysis that effectively described the patterns of variation and spatial dependencies within the kiwifruit vine. Simulation of different sampling strategies aimed at estimating the vine mean fruit mass under two vine training systems (cane and T-bar) highlighted appropriate modifications to the existing systematic sampling plan, which should result in an efficient and unbiased estimator of vine mean fruit mass. V. APPROACHES TO FRUIT GROWTH ANALYSIS Quantitative analysis of fruit growth is a powerful tool in horticultural research. It requires the knowledge and application of fruit science, mathematics, and statistics for data handling and interpretation. Often the results of fruit growth analysis are reported as plots of changes in growth attributes over time or as tables containing values of growth data.

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Fruit growth analysis can be carried out in three broad ways: plot of the growth curve and determination of the distinct growth stages occurring, application of classical models that describe the rate of change of measured growth attribute with time, and the use of functional or dynamic models that enable measured growth data to be curve-fitted into existing mathematical functions. Radford (1967) presented an excellent review on the pitfalls of the indiscriminate use and abuse of these models and recommendations were given on the selection of appropriate ones in a given situation. The relative merits of the classical and functional methods of analysis were also covered. In this section, I will outline the basis for each technique, and discuss some of the strengths and limitations in fruit growth studies. The use of computers in fruit growth analysis is also discussed, including the dramatic shift from the "pocket calculator revolution" to modern integrated data acquisition and growth analysis systems. A. Graphical Analysis of Growth Pattern The first stage in the analysis of fruit growth is to plot the data graphically and examine the pattern or shape of the growth curve. The graph could be a plot of the raw growth data versus time, or the percentage of the maximum value of the growth quantity versus percentage of total growth period. For each type of fruit, the shape of the plot of measured growth data over time follows a distinctive pattern that includes identifiable stages of development. In general, the growth pattern of fruits is either sigmoid or double-sigmoid. 1. Sigmoidal Growth. Similar to most cells, tissues, and organisms, many fruits have a sigmoidal growth pattern (Figs. 8.4 and 8.5). In this type of growth, fruit exhibits a fast exponential or geometric growth phase in the earlier stages until a maximum rate is reached, which is then followed by a stable growth phase, without apparent change in dimension until fruit is fully ripe. Fleshy fruits such as apple, tomato, strawberry, pineapple, and papaya (Leopold 1964) and many others like sweet pepper (Marcelis and Hofman-Eijer 1995) have single-sigmoid growth patterns. Recently, McGarry et al. (1998) reported the growth pattern of saskatoon fruit, an emerging horticultural crop across the Canadian prairies. The authors showed that fruit fresh mass increased in three stages, typical of a sigmoidal growth pattern. If the fruit remains on the shrub past harvest maturity, fruit growth slows and eventually ceases (Rogiers and Knowles 1997). For most fruit with sigmoidal growth, the pattern for fruit fresh weight is similar to that for length and diameter,

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although the period during which maximum fresh weight increase occurs is often preceded by the attainment of maximum dimensions (Calegario et al. 1997). 2. Double-sigmoidal Growth. In double-sigmoidal triphasic growth, there are two periods of rapid growth increases that are separated by a period of slow or suspended growth (Figs. 8.4 and 8.5). Most stonefruits such as

393

8. FRUIT GROWTH MEASUREMENT AND ANALYSIS

I

size

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SIGMOID Full bloom

Time

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Fig. 8.5. Stages of the growth of fruit for (top) sigmoid growth pattern, and (bottom) double-sigmoid growth pattern (Jackson 1986).

peach, cherry, plum, almond, nectarine, and apricot have double-sigmoid growth patterns. Some non-stony fruits such as grape, blueberry, fig, currant, and grape (Coombe 1976), and compound fruits such as breadfruit, soursop, and pineapple also have double-sigmoidal growth curves (Worrell et al. 1994, 1998). In many stonefruits with double-sigmoidal growth pattern, the period of suspended growth often coincides with pit hardening.

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3. Exponential or Curvilinear Growth Pattern. In exponential growth, fruit exhibits rapid growth initially and also maintains rapid growth during later stages in development, resulting in a pattern similar to the second phase of a sigmoid curve. Blumenfeld (1980) showed that the growth pattern of loquat fruit (Rosaceae) as judged by cumulative weight or volume was exponential (Fig. 8.6), which is distinct from those of other members of the Rosaceae such as apple and pear (sigmoidal) and stonefruits (double sigmoid). This peculiar growth pattern was attributed to the rapid growth that occurred toward ripening when temperatures rose in spring. The growth curve for dry weight of fleshy fruits such as papaya follows an exponential pattern (Calegario et al. 1997), and continues at a higher rate after maturity (Qiu et al. 1995; Calegario et al. 1997). The increase in rate of dry matter accumulation was attributed mainly to photosynthates, directed mostly to the pulp, resulting in increases in cell wall volume due to cell divisions and parenchyma cell expansion (Roth and Clausnitzer 1972). Studies on fruit development and ripening in 'Yellow' pitaya fruit showed that fruit growth, expressed as an increase in length or diameter, was curvi-,

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Fig. 8.6. Loquat fruit exponential growth pattern (diameter). Arrows show when irrigated. Fruit was ripe on day 20 (Blumenfeld 1980).

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linear (Weiss 1995; Nerd and Mizrahi 1998). When growth ceases, fruit peel color turns from green to yellow. Seed fresh mass growth in saskatoon fruit (a pentalocular pome) increased linearly up to 670 growing degree days (total growing degree days 750) (McGarry et al. 1998). 4. Effects of Growth Measurement Interval and Type of Primary Data.

The time interval during which growth is measured affects the shape of the growth curve obtained. In fruit that is characteristically sigmoidal or double-sigmoidal, omitting a percentage of the total time during the entire growth period could result in a linear or exponential growth curve. For instance, in a study of fruit growth in 'Gala' apples starting from about 45 days after full bloom (DAFB), Opara (1993) obtained an exponential growth of fruit dimensions, while fresh mass followed a sigmoidal pattern. While the results for fresh mass corroborates with previous evidence for apples (Volz 1991), the growth pattern of fruit diameter and length did not follow the expected sigmoid pattern for apples (Pearson and Robertson 1956; Coombe 1976; Lotter et al. 1985; Hussein and Slack 1994), presumably because growth measurement was started after the completion of a larger part of the first phase of rapid growth. Volz (1991) showed that this first phase only lasted up to 40-45 DAFB for 'Granny Smith' and 60-70 DAFB for 'Royal Gala' apple. These results highlight the need for adequate description of period of growth measurement and a clear specification of the chronological index of growth when reporting fruit growth data. The type of data used for plotting growth (e.g. primary raw data vs. growth rate data; length vs. mass; fresh mass vs. dry mass) also influences the shape of the growth curve (Ryugo 1988; Magein 1989; Opara 1993; Calegario et al. 1997; Worrell et al. 1998). Worrel et al. (1998) demonstrated this disparity between growth kinetics when the measurement of growth using linear parameters was compared with that assessed by fresh and dry weight of breadfruit. The authors found that whether linear measurements were taken in situ or from fruit harvested for weight determinations, the single sigmoidal growth curve resulted, thus discounting the possibility that the differences in growth curves could be attributed to differences in fruit samples. Magein (1989) observed that the growth of 'Cox's Orange Pippin' and 'Golden Delicious' apple fruit followed a single-sigmoidal pattern using fruit diameter, and when the absolute growth data was used the growth pattern was double sigmoidal. These disparate results justify the caution by Coombe (1976) that different interpretations can emerge if growth data are calculated on

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different bases or if rates are made relative to a previous reference. Similarly' Ryugo (1988) and Opara (1993) cautioned against the use of linear growth attributes alone to follow the growth of an organ and extrapolating therefrom. Opara (1993) demonstrated the relevance of this advice during his investigations on the chronological development of stern-end splitting in relation to fruit growth of apples. When fruit growth pattern was plotted using raw lineal growth data, there was no distinct growth phase in relation to the onset of fruit splitting; however, the onset of fruit splitting coincided with the attainment of final fruit shape and asymmetrical growth rates. 5. Conflicting Literature Evidence. Although there is considerable evi-

dence in the literature that suggests that many fruit types may be classified on the basis of the shape of their growth curves, several researchers have reported conflicting results on growth patterns for the same type of fruit even when the same growth quantity was used. For instance, Silva (1995) and Calegario et al. (1997) reported that in papaya, fruit of 'Improved Sunrise Solo Line 71/12' showed a single-sigmoid growth curve based on changes in fruit length and diameter, while Ghanta (1994) observed a double-sigmoid curve for 'Ranchi'. Several factors can contribute to this anomaly, including cultivar differences, sampling errors, different orchard management factors, and environmental factors (Walton and De Jong 1990). Although the growth of apple fruit has been extensively studied since the 1930s, there is still some controversy in the literature on the exact growth pattern. Until the 1980s, most research concluded that apple fruit growth follows a sigmoid curve (Tetley 1931; Smith 1950; Blanpied and Wilde 1968). However, many researchers have identified a short or temporary period of reduced fruit growth (Zucconi 1981; Magein 1989), which resulted in a double-sigmoid curve. On this basis, fruit development was divided into three distinct stages, because a period of slowing down of growth (stage 2) appears between two periods of more rapid growth (Magein 1989). Lakso et al. (1995) commented on the putative double-sigmoid growth curve of apple fruit that "the erratic nature of the timing and amplitude of the short 'lag period' in the data presented casts doubt on the soundness of this proposal." The data of Lakso et al. (1995) support the expolinear growth model for apple based on fruit fresh weight. Clearly, part of the anomaly must be due to the use of different indices of growth by researchers. For instance, based on evidence from Bain and Robertson (1951) and Denne (1963), Volz (1991) noted that "the fresh weight of apple fruit is described as sigmoidal," and

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commented further that "However, a recent study on apple fruit tagged on the tree and measured periodically throughout the season suggests that the growth curve may be double sigmoid." The author, however, failed to inform the reader that the study referred to (Magein 1989) was based on measurement of fruit diameter. Furthermore, the conclusion by Magein (1989) was based on a plot of absolute growth rate (rate of change in size) over time as opposed to a plot of the primary cumulative lineal growth data. In the article, Magein (1989) admitted that the curve of cumulated growth of the apple fruits suggested a sigmoid growth pattern, as reported by many other researchers. This discussion on the apple may also apply to other fruit, such the pineapple, in which researchers have reported both sigmoidal (Leopold 1964) and double-sigmoidal growth patterns (Worrell et al. 1998). It should be noted that growth curves do not seem to be distinctive for the different morphological types of fruits, because there are berries, pomes, and simple and accessory fruits in each type (Leopold 1964). Compound fruit such as breadfruit and soursop have double sigmoid growth just like stonefruit, grape, and fig, and others. Thus, growth curve kinetics are not dependent on fruit type, nor do taxonomic relatedness or continental origins prescribe the pattern of fruit growth (Leopold 1964; Worrell et al. 1998).

B. The Classical Approach A description of total growth purely in terms of lineal dimensions clearly leaves out a great deal of information (Fogg 1963), because it takes no account of changes in form which invariably accompany growth. Thus, quite different interpretations can emerge if data are calculated on different bases or if rates are made relative to a previous reference (Coombe 1976). Furthermore, when fruit growth is analyzed by a simple plot ofprimary data against time on an arithmetic scale, a common difficulty with interpreting the results is that often very little of the first phases of development is revealed (Hunt 1990). To provide detailed insights on fruit growth dynamics, the primary data need further analysis, which can be carried out using several classical and functional relationships. In classical analysis of fruit growth, the course of development is followed through a series of relatively infrequent, large harvests with many replications. Simple ratios, including morphogenetic index shape (length/width) and growth rates (absolute, relative, mean relative), and other dependent variables are then calculated over the period of growth measurement.

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1. Simple Ratios. The ratios of some basic growth data are often used to quantify a derived growth attribute. In horticulture, the most commonly used quantitative index of fruit shape or form is the ratio of fruit length to diameter (LID) (Westwood 1978; Wehner and Saltveit 1983; Opara 1993; Marcelis 1994). According to Westwood (1978), this expression of shape may be thought of as relative fruit length; the higher the value, the more elongated is the fruit. Watson and Gould (1993) described the shape of developing kiwifruit using a shape index (S), representing the proportionate difference between maximum (D max ) and minimum (D min ) diameters, where:

[2]

The difference between fruit diameter and length (D - L) has been used as an index of fruit shape in apples (Opara 1993), and according to the author, this also provided a good measure of the deviation from the initial shape at a reference time after full bloom. Similarly, stem-end cavity shape was quantified as the ratio of cavity depth to fruit diameter (C/D) and cavity depth to fruit length (elL) ratios. Nerd and Mizrahi (1998) used the relative dimensional changes in fruit length and diameter to demonstrate the growth of tagged intact 'Yellow' pitaya fruit during development and ripening. The authors found that the dimensions of fruit showed small changes during ripening; the diameter increased 8.5% and the length decreased 2.5% from the beginning of color change (= 80 days from anthesis) to full color. 2. Absolute Growth Rate (AGR). This is the simplest derived index of fruit growth. It represents a rate of change in size, or an increment in size per unit time. In whole fruit growth studies, the common primary data are fruit diameter (longitudinal or latitudinal) and mass. For a primary growth datum, X, the instantaneous growth rate over an interval of time, t, is defined as: ACR= dX dt

[3]

and the mean value over the interval t n and t n - 1 is given by: MACR = (X n

X n- 1 )

tn - 1

[4]

399

8. FRUIT GROWTH MEASUREMENT AND ANALYSIS

i!

.!!

E

e

f=

f=

~

...

~

...0-0

0 0

.!! :»

.2:-

G

..

~

0

til

0::

I

..0

«

1 .. A

TIme

.

Time

B

Fig. 8.7. Changes in (A) absolute growth rate [dX/dt] and (B) relative growth rate [(dX/dt) (l/X)], where the dry weight changes follow the types in Figs. 8.4 and 8.5 (top) (Adapted from Wareing and Phillips 1970).

Generally, fruit growth rate decreases gradually, and the AGR is given by the slope of the growth curve at any time. A plot of the changes in AGR with time typically gives a bell-shaped curve (Fig. 8.7), which shows that the growth rate reaches a maximum (at the point of inflection of the sigmoidal growth curve) and reduces to zero at the end of the growth period. Historically, AGRs are most useful where knowledge of the absolute size of fruit at a particular time (say at maturity) is required. It is also a valuable comparative tool when used within bodies of like data such as samples of the same fruit cultivar grown under different orchard management practices. However, when used to compare unlike systems of data, say between cell number per unit area and mean area per unit time, their usefulness declines. Other growth analysis techniques such as the relative growth rate are needed to compare the overall performance of such unlike systems. 3. Relative Growth Rate (RGR). A major criticism of AGR results is that they do not account for the original difference in size, which clearly can

1. OPARA

400

limit the amount of growth that is possible. This is particularly relevant when comparing the growth of plants or organs such as fruits which have the same AGR over specified periods of time, but which had different initial size at the start of the measurement period. In the early part of the century, Blackman (1919) proposed an "efficiency index," analogous to the interest in financial investment, as a measure of the efficiency of the plant or organ as a producer of new material. Values of the efficiency index, R, provided a more informative comparison on the relative performance, X, of plants or organs over a time interval - T1 • Blackman's equation, which follows a "Compound Interest Law" fairly closely, may be written as: [5]

where: X = weight of plant or constituent at time T z , Xo initial weight of plant or constituent at time T 1 , and e = the base of natural logarithm (2.7182).

If Eq. 5 is expressed in the form y = a + bx, we should obtain a straight line when log X is plotted against time for a sigmoidal growth: log X = log X o + RTlog e

[6]

The term "relative growth rate" (RGR) was suggested by West et al. (1920) as a replacement for the efficiency index (R), and the authors

pointed out that RGR must not be viewed as a constant since it varied markedly at different growth stages. Other researchers have also used the name "specific growth rate" for R (Hunt 1978), which is most simply expressed as an instantaneous value (Fisher 1921). RGR expresses growth in terms of a rate of increase in size per unit initial size. In practice, the relative growth rate (RGR) is the most frequently used index for quantitative analysis of fruit growth. Taking into account the original fruit size at the onset of growth measurement allows more equitable comparisons than does use of AGR (Hunt 1990). RGR is therefore defined as the change in fruit size per unit of initial size per unit of time. Expressing rate of change of size in terms of changes in fruit diameter or mass (X): RCR = ~. dX = d(loge X ) X dt dt

[7]

The instantaneous values of RGR usually change smoothly with time and the ontogenetic drift may be followed approximately by deriving the

8. FRUIT GROWTH MEASUREMENT AND ANALYSIS

401

mean RGR for successive harvest intervals. As the harvest intervals get longer, the mean RGR follows instantaneous RGR only crudely, and as the intervals become shorter, the correspondence between instantaneous RGR and mean RGR becomes progressively smaller (Hunt 1978). A plot of fruit RGR against time shows that the RGR remains fairly constant initially but later declines (Fig. 8.7). The RGR may also decline steadily from the onset of growth, in which case a true exponential phase does not occur (Wareing and Phillips 1970). Fruit RGR is very sensitive to growing conditions and varies considerably among fruit types. Studies on growth of 'Gala' apples showed that RGR was quite erratic during the entire season, indicating the sensitivity of RGR to conditions in the orchard (Opara 1993). The simultaneous decline in both AGR and RGR indicates a loss in the capacity of the fruit to grow. RGR is particularly useful for comparing the growth rates of different fruit types. As highlighted in Section I, "growth analysis" is often used synonymously with RGR. The preference for RGR over AGR for describing growth rates is mainly because it accounts for differences in the individual sizes, and in whole plant growth analysis it also "provides a convenient integration of the combined performances of the various parts of the plant" (Hunt 1982a,b), although it does not reveal anything about the causal processes that contribute to the performance of the plant. 4. Mean Relative Growth Rate (MRGR). The absolute variability in fruit growth rate increases as they increase in size, and often logarithmic transformation of the primary growth data (X) homogenizes this variability with time to provide a mean RGR over the interval. Mathematically, the MRGR is obtained by integrating Eq. 7 between the limits t i and t z , and rearranging:

[8]

Several units of fruit relative growth rates have been reported in the literature (Hunt 1978) but each has the form: size per size per time (size • size-I. time-I). Each of the most commonly used units has an advantage over the others, but often the most critical factors are convenience and degree of accuracy. Units of percentage per time (Hunt 1978), or simply per time (Staudt et al. 1986) have also been used, and their major disadvantage is that further reference must be made to the primary unit of growth data for complete understanding of the analysis and comparison of growth data.

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1. OPARA

C. The Functional (Curve Fitting) Approach The calculation of growth attributes based on classical analytical techniques was the standard approach for over 50 years following the earliest study on the subject at the end of the last century. The estimation of mean values over the period of time between two harvests was direct and simple, and judicious experimental design enabled researchers to improve the accuracy of results obtained. During the second half of this century, horticultural scientists have embraced the developments in computer technology and utilized these to develop mathematical functions to analyze fruit growth data. In functional or dynamic growth analysis, smooth curves are fitted to growth data obtained from few fruit samples at more frequent intervals. Using these growth curves, fitted values of data are extracted and then used to derive the various growth attributes that may subsequently be plotted as fitted instantaneous values (Hunt 1978). Several benefits of this approach to growth analysis have been documented in the literature (Radford 1967; Hughes and Freeman 1967; Hunt 1973). Functional fruit growth analysis can be classified into three types: empirical, mechanistic, and stochastic. 1. Empirical Functional Models. Although the word "functional" is

commonly used without particular reference to empiricism, it must be taken in the mathematical sense of a relation between variables rather than in the physiological sense of a mode of action or activity (Hunt 1990). In its simplest form, an empirical functional model of fruit growth relates the loge of size (X) to time (t) (Hunt 1978; Hughes and Freeman, 1967): [9]

The slope of the function, f (t), gives the instantaneous value of relative growth rate (RGR). One of the simplest possible functions, a linear regression or first order polynomial, can be derived in the form (Grime and Hunt 1975): [10]

Many researchers have employed polynomial regressions to fit curves to logarithmic experimental growth data, including second-order polynomials (quadratic curves) and third-order (cubic) regression, respectively (Vernon and Allison 1963; Hughes and Freeman 1967; Hunt and Parsons 1974):

8. FRUIT GROWTH MEASUREMENT AND ANALYSIS

log e X == a + bt + ct 2

403

[11] [12]

Grossman and DeJong (1995a,b) used the functional approach to successfully model the growth of peach fruit during the season and following resource limitation by thinning. Fruit growth curves were obtained by fitting cubic polynomial functions and cubic splines to logtransformed dry weight data for each fruit using the method of leastsquares regression. The following form of splines was used, with knots at x i and x == j: y== a + bx+ cx 2 + dx 3 + (x> i)e(x

iP + (x> j)f(x- jJ3

[13]

De Silva et al. (1997c) used the same approach and developed a three-parameter piecewise power function and a five-parameter spline exponential function for predicting fruit fresh mass of 'Royal Gala' apple during the season. It was considered that the model precision was adequate to meet industry requirements for monitoring fruit mass during the season, although there was evidence of seasonal and regional differences in the estimated bias. In another study, De Silva et al. (1997b) investigated the potential to predict apple fruit diameter distributions early in the growing season by fitting statistical distributions to fruit sizes. Lognormal density function was a better descriptor of apple size distribution at varying times right through the season, while the Johnson's (Johnson 1949) SB density function proved marginally better to model early season fruit diameters. The normal distribution did not fit the fruit size data because of skewness and the changing shape of fruit diameter distribution with time. A major disadvantage of empirical growth functions is that difficulties sometimes occur when trying to interpret a whole body of data in which regressions of varying order have been applied objectively, according to the needs of individual sets of data (Hurd 1977; Hunt 1978). Variability of growth data could also pose problems with functional empirical analysis of growth. Often the question arises as to which degree of polynomial should be fitted to logarithmically transformed data when employing the functional approach to growth analysis. Elias and Causton (1976) showed that when growth curves were fitted to data of low variability, unrealistically high degrees of polynomial were quite likely to be selected if purely objective criteria alone were used. However, when variability was high, the "best fit" was more likely to be a lower-order polynomial. The authors therefore concluded that regression should be fitted using harvest mean

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1. OPARA

values where "the test of adequacy of fit is independent of the underlying population variability." 2. Mechanistic Functional Models. These models are conceived in terms of the mechanism of the system, or how parts of the system work together as they might in a machine (Hunt 1978). Richards (1969) provided an extensive review of the process of dry weight increase from a mechanistic perspective. A mechanistic model commonly used in fruit growth analysis is the "mono-molecular" function, which originated in physical chemistry, and has the form:

[14]

Here, the absolute growth rate is k(X - Xl)' which is proportional to the amount of growth yet to be made, and declines linearly in value with increase in Xl (Opara 1993). Mechanistic modeling of fruit growth has received greater attention in recent years, presumably due to greater recognition by researchers of its usefulness in explaining the underlying mechanism of fruit development. Most mechanistic models are based on carbon (assimilate) supply and demand during fruit development. Bustan et al. (1995) showed that prevalence of source limitation during the season was clearly revealed by higher RGR, and higher AGR suggested a higher daily carbohydrate demand on a single fruit basis. Bruchou and Genard (1995) developed a carbon-based model of fruit growth with emphasis on the transfer of assimilates in fruit-bearing shoots in peach. Fruit growth (~Gf) was estimated as the difference between assimilate supply (~Fe) and respiration losses (~Mf' ~Rf): [15]

where: ~Gf = dry weight of fruit, Me = carbohydrate input from the metamer into the cytoplasm, ~Mf = carbon losses needed by fruit maintenance respiration, and ~Rf = carbon losses needed by fruit growth respiration. According to the authors, the above source-sink concept provided considerable explanation of the variability in fruit growth within the tree. It was thus proposed that the model might be used to test the effect of the non-homogenous distributions of sources and sinks on fruit growth and to test hypotheses to explain fruit growth limitation (sink activity versus the availability of assimilates).

8. FRUIT GROWTH MEASUREMENT AND ANALYSIS

405

Some researchers have considered the fruit as a single compartment receiving sap flow by its peduncle and losing water by transpiration through the skin, such that the solution flow received by the fruit and the water loss per unit surface area varied with time. Lee (1990) developed a mathematical bulk flow model of fruit growth based upon a unidirectional flux of solution into the fruit, relying entirely on the processes of diffusion and water evaporation driven by radiant energy. The growth (G) was defined as: [16] where: J = maximum rate of delivery of fluid through the pedicel or peduncle of the fruit (m 3 • h-1), A f = total surface of the fruit (m 2 ), and L pc = permeability of the cuticle (m • h-1). The model explained both the rate of fruit growth and the final size of several varieties of cherries (Prunus avium) and tomatoes (Lycopersicon esculentum). Following Lee (1990), Genard et al. (1995a) considered that the fruit receives a daily sap flow from the plant (F) and loses water by transpiration (T) and by carbon respiration (R). Growth (dXldt, in g. day-1) was given by: dX =F dt

R

T

[17]

Mathematical relationships were presented for calculating F, R, and T based on fruit physical and moisture transfer properties. The authors suggested that the model was suitable for fleshy fruits having a higher transpiration, such as the peach, whereas fruit having low transpiration, such as the tomato, would probably fit a "resistance model." In a separate study, Genard et al. (1995b) and Genard and Souty (1996) used the same carbon flow approach to develop a dynamic, deterministic model of sugar accumulation and synthesis in peach fruit flesh. The model was designed to be driven by the flesh dry-weight growth curve, flesh water content, and temperature. Model calibration and testing gave good agreement between predictions and experimental data. Gandar et al. (1995a) used a system of ordinary differential equations (ODEs), in which rates of change of fruit properties are expressed as functions of the state of the fruit and of environmental forcing factors, to develop some "top-down" models which can be fitted into sigmoid and double-sigmoid growth curves. This approach involved asking what sort of equation, or system of equations, might have solutions that match

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L. OPARA

measured growth data. The authors used a combination of ODE-based expolinear (Lakso et al. 1995), Gompertz (1825), and logistic functions to derive deterministic models of fruit growth. Both mechanistic and empirical models rarely fit experimental growth data well; firstly, because fruits seldom behave as simple machines and, secondly, because empirical models are designed expressly (and only) with accuracy of fit in view (Hunt 1978). Thornley (1976) persuasively summarized the relationship between empirical and mechanistic modeling in the general area of plant growth analysis and concluded that there was no clearly defined dividing line between the two methods, and that most modeling exercises contained both empiricism and mechanism in varying admixtures. Hunt (1979) presents a comprehensive treatise on the rationale behind the use of fitted mathematical models in plant growth analysis. 3. Expolinear Functional Model. Lakso et al. (1995) proposed the expolinear growth model of Goudrian and Monteith (1990) as a new model for the inherent growth pattern of fruit of apple, defined as growth pattern under apparently non-limiting conditions. The model was originally developed to describe seasonal crop plant growth patterns that show an initial exponential increase in crop dry weight, followed by a linear growth phase. Tests by Lakso et al. (1995) showed that the model fitted the growth patterns of two apple cultivars differing in the rate of growth in the exponential phase due to differences in crop load. The Gourdian-Monteith expolinear equation has three parameters:

[18] where: X = plant (or fruit) weight, em = maximum absolute growth rate (in weight gain per day reached in the linear phase), R m = maximum relative growth rate (in weight gain per unit weight per day), and tb = xaxis intercept of the linear growth phase (termed the "lost time"). 4. Stochastic Functional Models. Hall and Gandar (1995) and Gandar et al. (1995b) developed a unifying framework for modeling individual fruit growth based on ordinary and stochastic differential equations (Fokler-Planck Equation) and models of fruit-size distributions based on partial differential equations. The modeling framework used the mean and variance of fruit growth rates to model the changing fruit size distribution through the growing season.

5. Other Functional Models. Other mathematical expressions, more complex than the empirical and mechanistic models, have been formulated

407

8. FRUIT GROWTH :MEASURE:MENT AND ANALYSIS

as general-purpose models for fitting growth curves and from which other growth quantities may be derived. Richards (1959) proposed a generalization of the logistic equation: 1

X

= a(l + be-ktr;

[19]

About a decade later, Causton (1967) wrote a computer program for fitting this function. Widely accepted and applied by many researchers, the Richards logistic function was claimed to be the single most widely useful growth function in plant growth studies (Hunt 1978). Examples of mathematical functions commonly used in fruit growth analysis can be found in Marcelis et al. (1989), Marcelis (1992), and Takishita et al. (1993). Hunt (1990) provides an excellent checklist to assist in selecting a suitable functional model to analyze growth data. Other models have also incorporated important environmental factors such as temperature. For instance, Marcelis (1992) proposed a regression model for estimating the dry matter percentage (% of fresh weight) of cucumber fruit as a function of temperature sum, temperature, and fruit size: DM = 13.60 - 0.03665X + 0.9513 x 10- 4 X2 - 0.7100

X

10-7 X3 - 0.4893T + 0.009030T2

-0.7270 x 10- 4 TX + 0.1569 x 10- 5 VX (r 2 = 0.968)

[20]

where: DM = dry-matter percentage (% fresh weight), X = temperature sum after flowering (d °C) = t(T-10), t = time after flowering (d), T = temperature (OC), and V = calculated volume (cm 3 ). Jenni et al. (1997) used the model of Currence et al. (1944) to accurately predict the volume of developing eastern-type muskmelon based on measuring length and diameter in the field (r 2 = 0.997): Y = 0.984KD2L/1000

[21]

where: Y = fruit volume (em 3), K = shape factor, D = fruit diameter (mm), L fruit length (mm), K = 0.1528 (DIL) + 0.4252, if (DIL) ::;, and K = -0.2204 (DIL) + 0.7872, if (DIL) ~ 1. Takishita et al. (1993) analyzed the growth of several apple cultivars in Japan using the Gompertz and logistic curves. They found that the Gompertz curve was suitable throughout the growing season; however, the logistic curve was more suitable than the Gompertz curve during the period from the middle of July to harvest time. Many researchers have incorporated early-season temperatures in growth models to predict fruit size with variable success. Welte (1990)

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L. OPARA

implemented a complex differential dynamic simulation model for predicting final fruit size of apples that included effects on growth rate within certain temperature ranges, daylength effects, crop load effects, and other empirical factors. The model was effective in empirically fitting yearly growth curve variations; however, it did not consider the fundamental pattern of apple fruit growth. Schechter et al. (1993a,b) developed step-wise linear models for the seasonal growth curve with two linear parts approximating the periods of cell division and cell expansion in apples. The linear part late in the season fitted well, but the early season growth appeared to be curvilinear. Austin et al. (1999) developed a compartmental growth model for fruit diameter and the effect of early-season temperatures on potential size at harvest, and results showed close agreement between predicted and measured diameter of 'Delicious' apple fruit. Genard and Bruchou (1993) reported a new approach to studying fruit growth in peach that combined a functional description of growth curves, multivariate exploratory data analysis, and graphical displays. Growth curves were compared using principal component analysis (PCA). The authors suggested that the approach was useful for comparing growth curves fitted to a parametric model. The potential to accurately predict fruit size and size distribution using models that incorporate size-determining factors such as temperature, crop load, and vigor and physiological growth processes is still limited. This is partly due to the complexity of the inter-relationships between these processes and fruit growth, and the inconsistent relationship between fruit growth and temperature in some instances (Jackson 1987; Atkinson et al. 1995). Thus, despite the well-known positive effects of temperature on growth (Berg 1990; Warrington et al. 1998; Austin et al. 1999), attempts by some researchers to relate either initial fruit size or growth rate with final size were not successful (Jackson 1987). Many of the existing models lack the necessary accuracy and have not been fully tested for industry application. D. Biological Significance of Functional Growth Parameters

The functional approach to growth analysis assumes that the fitted growth model adequately describes the trends in the raw data; this in turn depends largely on the assumption that the raw data adequately characterize what is really happening in the growing plant or organism (Hunt 1978). Thus, the researcher must rely on statistical guidance to determine whether a chosen functional model (and the resulting growth curve) is adequate for the growth data measured.

8. FRUIT GROWTH "MEASURE"MENT AND ANALYSIS

409

In the functional approach to growth analysis, several parameters are often required for complete analysis of fruit growth. These parameters have no biological significance in terms of the overall pattern of development of the individual fruit. Thus, one of the inherent but continuing challenges in fruit growth studies using the functional approach is to derive a biological interpretation for the parameters in the model. E. Computers and Electronics in Fruit Growth Analysis

In fruit growth analysis, each derived growth attribute (e.g., AGR, RGR, MRGR) can be calculated as a mean value across a harvest interval by directly substituting measured raw data into a formula. Similarly, where instantaneous growth is of interest, the values may also be calculated by substitution. This approach is only suitable for simple growth experiments and only when a few values of growth attributes are required. However, with increasing complexity of experimental growth studies and the collection of large amounts of raw growth data, computer analysis of growth becomes essential. Rapid advancement in computer hardware and software technology and their declining cost have had a significant impact on approaches to general plant growth analysis. It is now possible to have sufficient computing capacity to support small experimental growth studies. In addition to calculating the various growth rates and ratios, both the raw data and derived growth quantities can be stored and used later for advanced statistical analysis. Computers are now frequently used for collecting fruit growth data in situ (Wagenmakers 1996). Displacement transducers enable diurnal fluctuations in fruit growth pattern to be measured non-destructively and continuously over a period of time (Lang 1990). One of the benefits of such a computer-instrumented measurement system is that it enables the researchers to monitor more closely the effects of external environment or orchard management practices on fruit growth and development. The reduced cost of information systems will soon enable fruit growth measurement and analysis to be carried out interactively away from the orchard. VI. APPLICATIONS OF FRUIT GROWTH DATA Most researchers study fruit growth to answer several important questions in horticulture. Which experimental treatment or orchard management

410

L. OPARA

practice gives the best range of fruit size, shape, yield, and marketable quality? Which fruit cultivar is most suited to a particular growing region? What form does growth take in new fruit species? What are the most critical stages during fruit development that may be related to the incidence of physiological defects and disorders? Over the past century, a number of researchers have conducted studies to help answer such questions. Early-season fruit growth can be measured and fitted to a model to predict final fruit size for planning of marketing programs (de Silva et al. 1997a,b,c). Hunt (1978) provides a list of publications in which the techniques of plant growth analysis have been used to advance knowledge in a variety of fields, particularly during the late 1960s to mid-1970s. Results from growth measurement and analysis have several applications in experimental and commercial horticulture. In addition to quantifying yield and the rate of accumulation of edible parts, they are also useful in understanding the relationships between the edible part and the entire plant, as well as the effects of management practices and environmental factors on growth and development. Growth attributes provide a measure of crop response to environmental factors such as temperature. Several studies have demonstrated a positive correlation between temperatures immediately after bloom and early fruit growth and size (Tukey 1956,1960; Berg 1990; Warrington et al. 1998; Austin et al. 1999). Warmer night temperatures generally accelerated fruit sizing, and may influence fruit shape, vegetative growth, flowering, and the time of embryo growth. Berg (1990) and Warrington et al. (1998) showed that the rate of fruitlet growth during the first 40 DAFB depended largely on the temperature during this period, but Berg (1990) also noted that fruit size at about 40 DAFB was not necessarily related to fruit size at harvest. When growth data is expressed in the same units, it can be used to compare inter-specific differences in growth of different plant edible organs. Blackman (1919) reported the mean RGR per day of different crops to demonstrate these differences: maize 0.071, sunflower (Helianthus macrophyllus giganteus) 0.18, hemp 0.13, and tobacco 0.21. Growth rates are commonly used to determine the rate of assimilate partitioning in above-ground dry matter as affected by environmental and physiological factors. Many studies have used relative growth rate to define how various environmental perturbations impact on growth. Creasy and Lombard (1993) showed that pre-veraison berry growth rate (change in diameter per day) and deformability provided good indicators of grape berry water stress. Rechel and Novotny (1996) used RGR to quantify the effects of harvest traffic on growth of alfalfa.

8. FRUIT GROWTH :tvIEASURE:tvIENT AND ANALYSIS

411

Fruit growth rate is affected by the availability of nutrients, and conversely the demand for nutrients may depend on growth attributes such RGR (Porter and Lawlor 1991). Through its relationship with other growth attributes (Hunt 1978; Ceesay 1980), fruit RGR provides a convenient measure of the integrated performance of the various parts of the plant. This is particularly useful for comparing species and treatment effects on a uniform basis. On a whole plant basis, RGR does not enable us to understand the causal processes that contribute to the plant's generation of bio-mass. It is therefore useful to calculate RGR for each subcomponent of the plant, such as fruit, root, shoot, leaves, or stem. As demonstrated in eco-physiological studies comparing the response of different species (Rorison 1991; Robinson 1991), the measurement and analysis of growth can help to refine our ideas about strategies for optimizing fruit growth and subsequent quality in response to nutrient supply and other orchard management practices. For instance, during breeding, fruit may be grouped according to their growth rates to distinguish between fast-growing and slow-growing cultivars, as these may have implications on the length of the season, as well as in selecting the appropriate time to apply certain treatments such as nutrients or practices such as thinning. The use of growth rates of individual organisms as phenotypic indicators of potential reproductive success has been expounded by many experimental biologists (Calow and Townsend 1981; McGraw and Wulff 1983; Givinish 1986). Growth analysis also permits better understanding of intra-specific differences in crop species. Grossman and DeJong (1995a) compared the seasonal patterns of relative growth rate of fruits on unthinned and heavily thinned peach trees and found that source-limited fruit growth occurred during distinct periods on early-maturing and late-maturing cultivars. Source-limited fruit growth occurred from 300 degree-days after bloom until harvest on the early-maturing cultivar, and occurred from 200-900 and 1600-1900 degree-days after bloom on the later-maturing cultivar. Since resource availability for fruit growth was similar in both cultivars, this result indicated that selection for early maturing fruits did not change the patterns of resource availability for fruit growth. Prediction of fruit size and size distribution, maturity, yield, and quality is essential for the success of commercial horticulture. Fruit developmental stage at harvest affects quality and postharvest life, and export marketing of fruits requires that they be graded into acceptable standards based on size and other attributes. To achieve these requirements, horticulturists need reliable information on fruit growth attributes, size, and other maturity indices to assist in deciding when to begin and end harvest and guarantee that fruit have reached appropriate stages of

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L. OPARA

development and maturity, such that acceptable quality is guaranteed. Accordingly, models have been developed for predicting early-season and final fruit size and size distribution (de Silva et al. 1997a,b,c; Austin et al. 1999), and to predict the effects of thinning (Grossman and DeJong 1995a,b). Fruit size and shape have been associated with susceptibility to vari0us forms of cracking and splitting in fruits (Frazier 1947; Thompson et al. 1962; Fogle and Faust 1976), presumably due to the inter-relatedness between size, shape, growth rates, and growth stress (Gpara et al. 1994). Accordingly, researchers have studied fruit growth in relation to splitting incidence in apple (Gpara 1993), nectarine (Fogle and Faust 1975), grape (Considine 1979), and peach (Davis 1933, 1940; Ragland 1934) to determine the factors contributing to the problem. Results of the studies by Fogle and Faust (1975) showed that relative growth rates at certain stages of fruit development might explain differences in the amount of both macro- and micro-cracking. Gpara et al. (1994) showed that the onset of splitting in 'Gala' apples coincided with the attainment of final fruit shape, and considered this finding vital in developing suitable orchard management strategies for controlling the problem. Regulated deficit irrigation (RDI) is widely acknowledged as an effective irrigation management strategy in deciduous orchards under some conditions (Chalmers 1989; Kilili et al. 1996; Behboudian and Mills 1997), and one of the design objectives is to minimize negative effects on fruit growth and final yield while reducing shoot growth and tree size. Fruit growth measurement and analysis is therefore an important tool in assessing the impact ofRDI (Jerie et al. 1989; Mitchell et al. 1989; Durand 1990; Pola et al. 1991; Behboudian et al. 1994; Mills et al. 1996). In addition to using fruit growth data to assess the impact of RDI, researchers have also monitored fruit growth as a tool to schedule irrigation in fruit trees (Ebel et al. 1995). Because fruit size and quality vary greatly with cultivar and environmental and orchard management factors, knowledge of fruit growth attributes can be insightful for many horticultural procedures, such as selecting superior genotypes, establishing breeding programs, and optimizing orchard management practices (Westwood and Blaney 1963; Gpara 1993; McGarry et al. 1998). This is particularly relevant in developing new and emerging horticultural crops with commercial potential. For instance, McGarry et al. (1998) showed that chemical management of diseases and pests can be scheduled before the exponential growth phase (stage III) of saskatoon fruit to avoid applications close to harvest, and irrigation can be scheduled at the onset of stage III to maximize fruit size before harvest.

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Modeling the changes in fruit quality attributes during development and postharvest handling is a major task for physiologists, horticulturists, and agricultural engineers. Based on the positive relationship between flesh sugar content at harvest and fruit growth (Genard et al. 1991), a dynamic and deterministic model, driven by flesh dry weight growth curve, flesh water content, and temperature, was developed for predicting sugar content (Genard and Souty 1996). Simulation runs suggested that increasing the maximal AGR increased the total sucrose content of the fruit but had only a weak influence on the sucrose concentration of the flesh at harvest. The date of maximal AGR was the most influential parameter associated with sucrose concentration. Based on primary data from both destructive and non-destructive fruit growth, a number of mathematical models have been developed to characterize the interrelationships among the primary growth data on the one hand, and also between the primary data and other derived growth indices (Frechette and Zahradnik 1966; Cooke 1970; Galbreath 1976; Magein 1989; Takishita et al. 1993; Clayton et al. 1995). Skene (1966) used Huxley's (1932) allometric growth formula to study the distribution of growth in apples based on changes in fruit mass, volume, diameter, or length. These models are particularly useful in providing estimates of fruit growth where experimental measurement of some growth attributes cannot be carried out for logistical and economical reasons. They are often used in postharvest applications where the interest may be on derived growth attributes, such as fruit surface area, which influence heat and mass vapor transport and gas exchange properties of fruit (Amos 1995; Maguire 1998; Tanner 1998; Zou 1998). The concepts of absolute and relative growth rates have also been successfully applied to non-biological systems such as predicting the deterioration of equipment and ammunitions (Sohn and Mazumbar 1991; Sohn 1996). Sohn (1996) reported a random effects growth curve analysis which was used to develop a logistic prediction model for ammunition deterioration in terms of depot and vendor information. The study showed that by comparing the expected deterioration rates of ammunition stored in different locations one can improve depot maintenance policy and vendor control. VII. SOME PROBLEMS IN GROWTH MEASUREMENT AND ANALYSIS The concepts and techniques of fruit growth analysis described in this review enable horticulturists and researchers to quantitatively describe

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and interpret fruit growth in a fairly precise manner given only the simplest of measured growth data. However, these measurements and analytical techniques are only part of a wider gamut of horticultural activity in which the overriding aim is to relate fruit development to genotype or orchard management practices and to the growing environment. This raises some pertinent questions about the relevance, interpretation, and general application of the results of fruit growth analysis found in the literature. Some of these problenls include the choice of a chronological index of growth, the selection of the time interval for measuring growth, and the choice of the period during the day when measurement is carried out. The choice of a suitable time interval for growth measurement will largely influence the usefulness of the results of quantitative fruit growth analysis. The concepts and conventional measurement techniques are particularly applicable if the interest is in the growth of fruit over a period of days, weeks, or months, especially if comparisons among cultivars or treatments are involved. Thus, the methods may be too crude to be of much practical relevance in studies dealing with fruit growth over a short period of time, say in seconds, minutes, or hours. However, this problem can be overcome or minimized by increased application of non-destructive measurement techniques in fruit growth studies. Depending on the type of fruit and study objectives, many researchers have used different chronological indices for growth (Section lIIE). This poses particular problems in interpreting research results and comparing data from existing literature. The adoption of a standard chronological index of growth, at least for the same fruit type, will enable comparison of treatment and environmental effects on a developmental basis. For example, Gage and Stutte (1991) proposed an anatomical framework that can be correlated with developmental growth indices of peach based on days after full bloom. Similar work is warranted for other fruits.

VIII. SUMMARY AND PROSPECTS A. General Review

One of the challenges facing the horticultural industry world-wide is the economic production and delivery of top-quality fruit to the consumer. This task is further complicated by the need to quantify the seasonal and regional variability of fruit size and supply volumes. The tools of fruit growth measurement and analysis enable researchers and producers to address some of these problems by providing quantitative data on the

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chronological development of fruit size and shape, as well as the total quantity of crop at harvest. Despite considerable advances in the last twenty years in improving the yield and quality of fruit, a large proportion of fruit is still under-sized, and thus does not meet export marketing standards. Over-cropping in any one year must be avoided, as this tends to encourage biennial bearing with all its adverse effects on fruit quality and yield (Abbott 1960; v'Vebb et al. 1980). Furthermore, over-sized fruit that result from lightly cropping trees are more susceptible to physiological storage disorders (Sharples 1964; Martin and Lewis 1952). Fruit growth measurement and analysis is an important tool for the horticulturist and orchardist seeking to develop orchard management strategies for solving these problems. Fruit growth analysis results provide accurate measurements of the cumulative performance of the fruit across significant intervals of time. Estimating this from purely physiological observations is fraught with problems and involves many shaky assumptions. On the other hand, a major disadvantage of fruit growth analysis is that it provides no indepth mechanistic information about the environmental responses of fruits, even though valuable clues may sometimes emerge from the application of some growth models. The two main distinct approaches to fruit growth analysis are the classical and the functional (dynamic). The division between these two approaches evolved in the 1960s following the work ofD. R. Causton and others (Hunt 1990). The two approaches are not mutually exclusive if time and space are available (harvests may be large and frequent), but such a scheme seldom makes the most efficient use of the material available (Hunt 1990). Thus, the researcher must decide earlier which approach to take, since this will influence the design and implementation of the experiments. Although numerous researchers have demonstrated that very different interpretations can be made when analyzing the components of fleshy fruit if data were calculated on different bases, or if fruit growth rate was calculated by different methods (Coombe 1976; Dejong and Goudrian 1989; Opara 1993; Schechter et al. 1993b), many researchers still report fruit growth based on increments in fruit diameter or mass only. Based on the inconsistency already evident in the literature, a minimum standard approach should include the analysis of increments in fruit diameter, mass, and calculation ofRGR over the growing season. Mathematical modeling (both empirical, mechanistic, and semimechanistic) of size and shape have played a prominent role in the evolution of fruit growth analysis. More recently, developments in computer technology and instrumentation have swiftly extended our capability to

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simultaneously collect, store, and manipulate large amounts of data. It is now possible to measure fruit growth non-destructively, and with rapid advances in global positioning and geographical information systems (GPS and GIS), it is conceivable to set up fruit growth experiments and dial in from many kilometers away to receive data on growth rates and general plant performance. Detailed information about the fruit growth pattern and possible circadian rhythms may be obtained more rapidly and precisely, and with less potential for the thigmomorphogenic phenomena that may result by touching the plant or fruit during measurement, resulting in an altered growth behavior (Jaffe 1976). B. Prospects for Rationalized Techniques and Units 1. Instantaneous versus Mean Values of Growth Data. Analysis of growth

data provides both mean values (generated by the classical approach) and instantaneous values (by the functional approach). Usually the derived indices of growth are defined as instantaneous values (i.e., at a single point in time), and many researchers traditionally displayed both mean and instantaneous results as single points on a progression plotted against time. Only instantaneous values may be properly represented as such single points, and mean values should appear as a histogram, with a class interval equal to the harvest interval. This requirement is often ignored by many researchers, as noted by Hunt (1990), a situation which can lead to incorrect interpretation of growth patterns. Using data on the increase in diameter of a gourd fruit from its early stage as small ovary primordium until maturity, Sinnott (1960) provided a good example of the different interpretations that could emerge based on different graphical representation of growth. 2. Units of Measurement. In horticultural science and fruit growth studies in particular, evaluation of previous research is often convoluted by the plethora of units used in measurement and presentation of data. As in other aspects of scientific research, conversion by the end user of data from alternative systems of units relies on assumptions which may not be true. Although each system of units has advantages and disadvantages for different situations, expediency is often the basis for selection, because some systems require more computational effort than others (Banks et al. 1995). With recent advances in dynamic or continuous non-destructive measurement of fruit growth using computerbased systems (Wagenmakers 1996), it is now easier to obtain data in small increments, which may elucidate significant changes in growth pattern due to treatment, orchard management, or environmental

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effects. There is, therefore, a need to re-examine existing units for measuring and presenting fruit growth data with a view to rationalizing them. This rationalization may reflect fruit types (e.g. pome, berry, stone fruits) or be based on a standardized system that provides factors for converting alternative units that are frequently used in the literature. Such a system would eliminate the ongoing misinterpretation of growth data (Staudt et al. 1986; Magein 1989). For instance, Staudt et. al. (1986) reported that the course of berry growth in Vitis vinifera has been interpreted differently by various authors. Divisions into two, three, or four growth phases have been postulated, but Staudt et al. divided growth into just two phases. Researchers have also been inconsistent in using fruit growth terminology, and the choice of time during the day when measurement is made appears to be a matter of logistical convenience rather than targeting measurement time to account for the very well known diurnal growth patterns of most fruit. Fruit growth researchers must avoid the misuse and abuse of units; for instance, one measures the mass of fruit and not the weight! It is admissible that the relative effect of a treatment may be evident within an individual study; however, the adoption of standardized measuring and reporting protocols are essential if one wishes to compare results from various researchers and growing regions. 3. Symbols and Notations. As in whole plant growth analysis, notation in fruit growth evolved slowly until the first comprehensive and coherent system of notation was attempted (Evans 1972). However, the benefits of this system had been sacrificed considerably by researchers in the interest of preserving some link with other conventions (Hunt 1982a,b; Causton and Venus 1981). The use of consistent notation in fruit growth analysis is beneficial, especially in the classical and functional techniques where mathematical expressions and equations are common. The principles of notation followed in whole plant analysis that are applicable to fruit growth include (Hunt 1990): 1. 2. 3. 4. 5.

6.

Contractions of names: roman capitals (or small capitals); e.g. AGR, absolute growth rate; Measured quantities: italics; e.g. M, mass; Derived quantities: bold capitals; e.g. R, relative growth rate; Parameters of equations: italics lower case; e.g. constants a, b; Subscript defining time: suffix position; e.g. t l , M I , initial time and initial total dry mass; Conventional signs and mathematical symbols: roman or italic, upper or lower case as required; e.g. loge' natural logarithm.

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C. Future Research

The development and refinement of the techniques currently used in fruit analysis have benefited tremendously from earlier advances in whole plant growth analysis. There is, however, considerable scope for future research and development in fruit analysis. Many functional models exist for analyzing fruit growth data; however, no single model adequately explains the interactions between genetic, environmental, and cultural control of growth. Existing population growth models could be applied to fruit growth by treating the fruit as a population of cells and intercellular air spaces. The current revolution in computer and information technology provides opportunities for application of more advanced instrumentation to reduce drudgery and improve the precision of existing fruit measurement and analysis techniques. Geographical information systems (GIS) and global positioning systems (GPS) have significantly improved data acquisition and monitoring for many applications in agriculture and ecology. If current growth measurement techniques can be incorporated in GIS/GPS systems, the prospects for precision horticulture and yield mapping of orchards are real. This could provide other benefits in crop size forecasting, and better management of fruit variability. With the recent global interest in the commercialization of less known fruit crops which have potential as commercial crops, especially in lessdeveloped countries, there are opportunities to make contributions to the growing of these crops through better quantitative understanding of their growth attributes. Because of the relation between internal cellular structures and whole fruit growth, a combination of conventional fruit growth measurements (size and mass) with microscopic analysis of rates of cell growth (division and expansion) will provide a more complete understanding on the growth dynamics of fruits. As consumers demand better and consistent fruit quality, resulting in further segregation of fruit lines, in-depth knowledge of the developmental aspects of the fruit becomes more relevant to the control of growth and size (Webb et al. 1980). With recent advances in computer modeling, researchers may need to adopt a multi-disciplinary approach to integrate the existing models of fruit growth with models for predicting fruit quality at harvest and beyond. Synergy between preharvest (growth) and postharvest (quality) models could further improve our ability to optimize fruit quality. Since continued progress in fruit production depends on our ability to control growth, a challenge in growth studies is to exploit existing quantitative skills and technology to improve our understanding of the very important processes of fruit development and maturation.

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Warrington, I. J., T. A. Fulton, K A. Halligan, and H. N. de Silva. 1998. Temperature impacts on growth of apple. J. Am. Soc. Hort. Sci. (In press). Watada, A. K, R. C. Herner, A. A. Kader, R. J. Romani, and G. 1. Staby. 1984. Terminology for the description of developmental stages of horticultural crops. HortScience 19:20-21. Watson, D. J. 1947a. Comparative physiological studies on the growth offield crops. 1. Variation in net assimilation rate and leaf area between species and varieties, and within and between years. Ann. Bot (N.S.) 11:41-76. Watson, D. J. 1947b. Comparative physiological studies on the growth offield crops. 1. The effect of varying nutrient supply on net assimilation rate and leaf area. Ann. Bot. (N.S.) 11:375-407. Watson, D. J. 1952. The physiological basis of variation in yield. Adv. Agron. 4:101-144. Watson, D. J. 1956. Leaf growth in relation to crop yield. p. 178-191. In: F. 1. Milthorpe (ed.), The growth ofleaves. Butterworths, London. Watson, D. J. 1958. The dependence of the net assimilation rate on leaf area index. Ann. Bot. (N.S.) 22:37-54. Watson, M., and K. S. Gould. 1993. The development of fruit shape in kiwifruit: Growth characteristics and positional differences. J. Hort. Sci. 68(2):185-194. Waynick, D. D. 1927. Growth rates of Valencia oranges. California Citrograph 12:150, 164. Webb, R. A., J. V. Purves, and M. G. Beech. 1980. Size factors in apple fruit. Scientia Hort. 13:205-212. Wehner, T. c., and M. K Saltveit, Jr. 1983. Photographic analysis of cucumber fruit elongation. J. Am. Soc. Hort. Sci. 108:465-468. Weinberger, J. H. 1948. Influence of temperature following bloom on fruit development period of Elberta peach. Proc. Am. Soc. Hort. Sci. 51:175-178. Weiss, J. 1995. The biology of flowering, pollination and fruit development in nightblooming and day-blooming cacti. Ph.D. thesis, Ben-Gurion Univ. of the Negev, BeerSheva, Israel. Welte, H. F. 1990. Forecasting harvest fruit size during the season. Acta Hort. 276:275-282. West, c., G. K Briggs, and F. Kidd. 1920. Methods and significant relations in the quantitative analysis of plant growth. New Phytol. 19:200-207. Westwood, M. N. 1978. Temperate-zone pomology. W. H. Freeman, San Francisco. Westwood, M. N., and 1. T. Blaney. 1963. Non-climatic factors the shape of apple fruits. Nature 200:802-803. Whitehouse, W. K 1916. A study of variation in apples during the growing season. Oregon Agr. College Expt. Sta. 134:1-13. Worrell, D. B., C. M. S. Carrington, and D. J. Huber. 1994. Growth, maturation and ripening of soursop (Annona muricata 1.) fruit. Scientia Hort. 57:7-15. Worrell, D. B., C. M. S. Carrington, and D. J. Huber. 1998. Growth, maturation and ripening of breadfruit, Artocarpus altilis (Park.) Fosb. Scientia Hort. 76:17-28. Wright, S. T. C. 1956. Studies of fruit development in relation to plant hormones. III. J. Hort. Sci. 31:196-211. Yaffa,1. G., and T. M. DeJong. 1995a. Maximum fruit growth potential and seasonal patterns of resource dynamics during peach growth. Ann. Bot. 75:553-560. Yaffa,1. G., and T. M. DeJong. 1995b. Maximum fruit growth potential following resource limitation during peach growth. Ann. Bot. 75:561-567. Yamaguchi, H., Y. Kanayama, J. Soejima, and S. Yamaki. 1996. Changes in the amounts of the NAD-dependent sorbitol dehydrogenase and its involvement in the development of apple fruit. J. Am. Soc. Hort. Sci. 121:848-852.

8. FRillT GROWTH MEASUREMENT AND ANALYSIS

431

Zeger, S. L., and S. D. Harlow. 1987. Mathematical models from laws of growth to tools far biologic analysis: fifty years of growth. Growth 51:1-21. Zilkah, S., and 1. Klein. 1987. Growth kinetics and determination of shape and size oflarge avocado fruits cultivar Hass on the tree. Scientia Hart. 32:195-202. Zou, Q. 1998. CFD modeling of airflow and heat transfer in a ventilated fruit carton. M.Appl.Sc. thesis. Massey Univ., Palmerston North, New Zealand. Zucconi, F. 1981. Regulation of abscission in growing fruit. Acta Hart. 120:89-94.

Subject Index Volume 24 A

N

Asexual embryogenesis, 6-7

Nutrition, ecologically-based, 156-172

B

p

Bioreactor technology, 1-30

Pest control, ecologically-based, 172-201

c Citrus: Practices for young trees, 319-372 Viroid dwarfing, 277-317 D

Physiology: Floral scents, 31-53 Lettuce seed germination, 229-275

Triazoles, 55-138 Pollination, floral scents, 31-53 Propagation, bioreactor technology, 1-30

Dedication, Kamemoto, H., x-xiii

s

F

Floral scents, 31-53 Flower and flowering, scents, 31-53 Fruit, growth measurement, 373-431 Fruit crops: Citrus dwarfing by viroids, 277-317 Citrus, culture of young trees, 319-372

Seed, lettuce germination, 229-275 Stress, protectants (triazoles), 55-138 T

Tissue culture, 1-30 Triazoles, 55-138

G

v

Germination, seed, 229-275 Growth substances, 55-138

Vegetable crops: Ecologically-based practices,

I

In vitro, bioreactor technology, 1-30 L

Lettuce, seed germination, 229-275

139-228

Lettuce seed germination, 229-275

Tropical production, 139-228 Viroid, dwarfing for citrus, 277-317 Virus, dwarfing for citrus, 277-317 Volatiles, 31-53 433

Cumulative Subject Index (Volumes 1-24) A

Abscisic acid: chilling injury 15:78-79 cold hardiness, 11:65 dormancy, 7:275-277 genetic regulation, 16:9-14,20-21 mechanical stress, 17:20 rose senescence, 9:66 stress, 4:249-250 Abscission: anatomy and histochemistry, 1:172-203

citrus, 15:145-182, 163-166 flower and petals, 3:104-107 regulation, 7:415-416 rose, 9:63-64 Acclimatization: foliage plants, 6:119-154 herbaceous plants, 6:379-395 micropropagation, 9:278-281, 316-317

Actinidia, 6:4-12 Adzuki bean, genetics, 2:373 Agaricus, 6:85-118 Agrobacterium tumefaciens, 3:34 Air pollution, 8:1-42 Almond: bloom delay, 15:100-101 in vitro culture, 9:313 postharvest technology and utilization, 20:267-311 Alocasia, 8:46, 57: see also Aroids Alternate bearing: chemical thinning, 1:285-289 fruit crops, 4:128-173 pistachio, 3:387-388 434

Aluminum: deficiency and toxicity symptoms in fruits and nuts, 2:154 Ericaceae, 10:195-196 Amorphophallus, 8:46, 57, see also Aroids Anatomy and morphology: apple flower and fruit, 10:273-308 apple tree, 12:265-305 asparagus, 12:71 cassava, 13:106-112 citrus, abscission, 15:147-156 embryogenesis, 1:4-21, 35-40 fig, 12:420-424 fruit abscission, 1:172-203 fruit storage, 1:314 ginseng, 9:198-201 grape flower, 13:315-337 grape seedlessness, 11:160-164 heliconia, 14:5-13 kiwifruit, 6:13-50 magnetic resonance imaging, 20:78-86, 225-266

orchid, 5:281-283 navel orange, 8:132-133 pecan flower, 8:217-255 petal senescence, 1:212-216 pollution injury, 8:15 waxes, 23:1-68 Androgenesis, woody species, 10:171-173

Angiosperms, embryogenesis, 1:1-78 Anthurium: See also Aroids, ornamental fertilization, 5:334-335

Antitranspirants, 7:334 cold hardiness, 11 :65

CUMULATIVE SUBJECT INDEX

Apical meristem, cryopreservation,

435

Asexual embryogenesis, 1:1-78; 2:268-310; 3:214-314; 7:163168, 171-173,176-177, 184, 185-187, 187-188, 189, 10:153-181, 14:258-259, 337-339, 24:6-7

6:357-372

Apple: alternate bearing, 4:136-137 anatomy and morphology of flower and fruit, 10:273-309 bitter pit, 11:289-355 bioregulation, 10:309-401 bloom delay, 15:102-104 CA storage, 1:303-306 chemical thinning, 1:270-300 fertilization, 1:105 fire blight control, 1:423-474 flavor, 16:197-234 flower induction, 4:174-203 fruiting, 11:229-287 fruit cracking and splitting, 19:217-262

in vitro, 5:241-243; 9:319-321 light, 2:240-248 maturity indices, 13:407-432 mealiness, 20:200 nitrogen metabolism, 4:204-246 replant disease, 2:3 root distribution, 2:453-456 stock-scion relationships, 3:315-375 summer pruning, 9:351-375 tree morphology and anatomy, 12:265-305

vegetative growth, 11:229-287 watercore, 6:189-251 yield, 1:397-424 Apricot: bloom delay, 15:101-102 CA storage, 1:309 origin and dissemination, 22:225-266

Aroids: edible, 8:43-99; 12:166-170 ornamental, 10:1-33 Arsenic, deficiency and toxicity symptoms in fruits and nuts, 2:154

Artemisia, 19:319-371 Artemisinin, 19:346-359 Artichoke, CA storage, 1:349-350

Asparagus: CA storage, 1:350-351 fluid drilling of seed, 3:21 postharvest biology, 12:69-155 Auxin: abscission, citrus, 15:161, 168-176 bloom delay, 15:114-115 citrus abscission, 15:161, 168-176 dormancy, 7:273-274 flowering, 15:290-291, 315 genetic regulation 16:5-6,14,21-22 geotropism, 15:246-267 mechanical stress, 17:18-19 petal senescence, 11:31 Avocado: CA and MA, 135-141 flowering, 8:257-289 fruit development, 10:230-238 fruit ripening, 10:238-259 rootstocks, 17:381-429 Azalea, fertilization, 5:335-337 B

Babaco, in vitro culture, 7:178 Bacteria: diseases of fig, 12:447-451 ice nucleating, 7:210-212, 11:69-71

pathogens of bean, 3:28-58 tree short life, 2:46-47 wilt of bean, 3:46-47 Bacteriocides, fire blight, 1:450-459 Bacteriophage, fire blight control, 1:449-450

Banana: CA and MA, 22:141-146 CA storage, 1:311-312 fertilization, 1:105 in vitro culture, 7:178-180

CUMULATIVE SUBJECT INDEX

436

Banksia, 22:1-25 Bean: CA storage, 1:352-353 fluid drilling of seed, 3:21 resistance to bacterial pathogens, 3:28-58

Bedding plants, fertilization, 1:99-100; 5:337-341

Beet: CA storage, 1:353 fluid drilling of seed, 3:18-19 Begonia (Rieger), fertilization, 1:104 Biennial bearing. See Alternate bearing Biochemistry, petal senescence, 11:15-43

Bioreactor technology, 24:1-30 Bioregulation: See also Growth substances apple and pear, 10:309-401 Bird damage, 6:277-278 Bitter pit in apple, 11:289-355 Blackberry harvesting, 16:282-298 Black currant, bloom delay, 15:104 Bloom delay, deciduous fruits, 15:97

Blueberry: developmental physiology, 13:339-405

harvesting, 16:257-282 nutrition, 10:183-227 Botanic gardens, 15:1-62 Boron: deficiency and toxicity symptoms in fruits and nuts, 2:151-152 foliar application, 6:328 nutrition, 5:327-328 pine bark media, 9:119-122 Bramble, harvesting, 16:282-298 Branching, lateral: apple, 10:328-330 pear, 10:328-330 Brassicaceae, in vitro, 5:232-235 Breeding. See Genetics and breeding Broccoli, CA storage, 1:354-355 Brussels sprouts, CA storage, 1:355

Bulb crops: See Tulip genetics and breeding, 18:119-123 in vitro, 18:87-169 micropropagation, 18:89-113 root physiology, 14:57-88 virus elimination, 18:113-123

c CA storage. See Controlledatmosphere storage Cabbage: CA storage, 1:355-359 fertilization, 1:117-118 Cactus: crops, 18:291-320 reproductive biology, 18:321-346 Caladium. See Aroids, ornamental Calciole, nutrition, 10:183-227 Calcifuge, nutrition, 10:183-227 Calcium: bitter pit, 11:289-355 cell wall, 5:203-205 container growing, 9:84-85 deficiency and toxicity symptoms in fruits and nuts, 2:148-149

Ericaceae nutrition, 10:196-197 foliar application, 6:328-329 fruit softening, 10:107-152 nutrition, 5:322-323 pine bark media, 9:116-117 tipburn, disorder, 4:50-57 Calmodulin, 10:132-134, 137-138 Carbohydrate: fig, 12:436-437 kiwifruit partitioning, 12:318-324 metabolism, 7:69-108 partitioning, 7:69-108 petal senescence, 11:19-20 reserves in deciduous fruit trees, 10:403-430

Carbon dioxide, enrichment, 7:345-398,544-545

Carnation, fertilization, 1:100; 5:341-345

437

CUMULATIVE SUBJECT INDEX

Carrot: CA storage, 1:362-366 fluid drilling of seed, 3:13-14 Caryophyllaceae, in vitro, 5:237-239 Cassava, 12:158-166; 13:105-129 Cauliflower, CA storage, 1:359-362 Celeriac, CA storage, 1:366-367 Celery: CA storage, 1:366-367 fluid drilling of seed, 3:14 Cell culture, 3: 214-314 woody legumes, 14:265-332 Cell membrane: calcium, 10:126-140 petal senescence, 11:20-26 Cellular mechanisms, salt tolerance, 16:33-69

Cell wall: calcium, 10:109-122 hydrolases, 5:169-219 ice spread, 13:245-246 tomato, 13:70-71 Chelates, 9:169-171 Cherimoya, CA and MA, 22:146-147 Cherry: bloom delay, 15:105 CA storage, 1:308 origin, 19:263-317 Chestnut: blight, 8:281-336 in vitro culture, 9:311-312 Chicory, CA storage, 1:379 Chilling: injury, 4:260-261,15:63-95 injury, chlorophyll fluorescence, 23:79-84

pistachio, 3:388-389 Chlorine: deficiency and toxicity symptoms in fruits and nuts, 2:153 nutrition, 5:239 Chlorophyll fluorescence, 23:69-107 Chlorosis, iron deficiency induced, 9:133-186

Chrysanthemum fertilization, 1:100-101; 5:345-352

Citrus: abscission, 15:145-182 alternate bearing, 4:141-144 asexual embryogenesis, 7:163-168 CA storage, 1:312-313 chlorosis, 9:166-168 cold hardiness, 7:201-238 fertilization, 1:105 flowering, 12:349-408 honey bee pollination, 9:247-248 in vitro culture, 7:161-170 juice loss, 20:200-201 navel orange, 8:129-179 nitrogen metabolism, 8:181 practices for young trees, 24:319-372

rootstock, 1:237-269 viroid dwarfing, 24:277-317 Cloche (tunnel), 7:356-357 Coconut palm: asexual embryogenesis, 7:184 in vitro culture, 7:183-185 Cold hardiness, 2:33-34 apple and pear bioregulation, 10:374-375

citrus, 7:201-238 factors affecting, 11:55-56 herbaceous plants, 6:373-417 injury, 2:26-27 nutrition, 3:144-171 pruning, 8:356-357 Co10casia, 8:45, 55-56, see also Aroids Common blight of bean, 3 :45-46 Compositae, in vitro, 5:235-237 Container production, nursery crops, 9:75-101

Controlled environment agriculture, 7:534-545, see also Greenhouse and greenhouse crops; hydroponic culture; protected culture Controlled-atmosphere (CA) storage: asparagus, 12:76-77, 127-130 chilling injury, 15:74-77 flowers, 3:98, 10:52-55

CUMULATIVE SUBJECT INDEX

438

Controlled-atmosphere (CA) storage (cont'd) fruit quality, 8:101-127 fruits, 1:301-336; 4:259-260 pathogens, 3:412-461 seeds, 2:134-135 tropical fruit, 22:123-183 tulip, 5:105 vegetable quality, 8:101-127 vegetables, 1:337-394; 4:259-260 Copper: deficiency and toxicity symptoms in fruits and nuts, 2:153 foliar application, 6:329-330 nutrition, 5:326-327 pine bark media, 9:122-123 Corynebacterium flaccumfaciens, 3:33,46 Cowpea: genetics, 2:317-348 U.S. production, 12:197-222 Cranberry: botany and horticulture, 21:215-249 fertilization, 1:106 harvesting, 16:298-311 Cryphonectria parasitica. See Endothia parasitica Cryopreservation: apical meristems, 6:357-372 cold hardiness, 11:65-66 Crytosperma, 8:47, 58, see also Aroids Cucumber, CA storage, 1:367-368 Currant, harvesting, 16:311-327 Custard apple, CA and MA, 22:164 Cytokinin: cold hardiness, 11:65 dormancy, 7:272-273 floral promoter, 4:112-113 flowering, 15:294-295, 318 genetic regulation, 16:4-5, 14, 22-23 grape root, 5:150, 153-156 lettuce tipburn, 4:57-58 petal senescence, 11:30-31 rose senescence, 9:66

D

Date palm: asexual embryogenesis, 7:185-187 in vitro culture, 7:185-187 Daylength. See Photoperiod Dedication: Bailey, L.H., 1:v-viii Beach, S.A., 1:v-viii Bukovac, M.J., 6:x-xii Campbell, C.W., 19:xiii Cummins, J.N., 15:xii-xv Dennis, F.G., 22:xi-xii Faust, Miklos, 5:vi-x Hackett, W.P., 12:x-xiii Halevy, A.H., 8:x-xii Hess, C.E., 13:x-xii Kader, A.A., 16:xii-xv Kamemoto, H., 24:x-xiii Looney, N.K, 18:xiii Magness, J.R, 2:vi-viii Moore, J.N., 14:xii-xv Pratt, c., 20:ix-xi Proebsting, Jr., KL., 9:x-xiv Rick, Jr., C.M., 4:vi-ix Sansavini, S., 17:xii-xiv Sherman, W.B., 21:xi-xiii Smock, RM., 7:x-xiii Weiser, c.J., 11:x-xiii Whitaker, T.W., 3:vi-x Wittwer, S.H., 10:x-xiii Yang, S.F., 23:xi Deficit irrigation, 21:105-131 Deficiency symptoms, in fruit and nut crops, 2:145-154 Defoliation, apple and pear bioregulation, 10:326-328 'Delicious' apple, 1:397-424 Desiccation tolerance, 18:171-213 Dieffenbachia. See Aroids, ornamental Dioscorea. See Yam Disease: and air pollution, 8:25 aroids, 8:67-69; 10:18; 12:168-169 bacterial, of bean, 3:28-58 cassava, 12:163-164

439

CUMULATIVE SUBJECT INDEX

control by virus, 3:399-403 controlled-atmosphere storage,

controlled for agriculture,

3:412-461 cowpea, 12:210-213 fig, 12:447-479 flooding, 13:288-299 hydroponic crops, 7:530-534 lettuce, 2:187-197 mycorrhizal fungi, 3:182-185 ornamental aroids, 10:18 resistance, acquired, 18:247-289 root, 5:29-31 stress, 4:261-262 sweet potato, 12:173-175 tulip, 5:63, 92 turnip moasic virus, 14:199-238 waxes, 23:1-68 yam (Dioscorea), 12:181-183

controlled for energy efficiency,

Disorder: see also Postharvest physiology: bitterpit, 11:289-355 fig, 12:477-479 pear fruit, 11:357-411 watercore, 6:189-251; 11:385-387 Dormancy, 2:27-30 blueberry, 13:362-370 release in fruit trees, 7:239-300 tulip, 5:93 Drip irrigation, 4:1-48 Drought resistance, 4:250-251 cassava, 13:114-115 Durian, CA and MA, 22:147-148 Dwarfing: apple, 3:315-375 apple mutants, 12:297-298 by virus, 3:404-405

7:534-545 1:141-171,9:1-52

embryogenesis, 1:22,43-44 fruit set, 1:411-412 ginseng, 9:211-226 greenhouse management, 9:32-38 navel orange, 8:138-140 nutrient film technique, 5:13-26 Epipremnum. See Aroids, ornamental Eriobotrya japonica. See Loquat Erwinia: amylovora, 1:423-474 lathyri, 3 :34 Essential elements: foliar nutrition, 6:287-355 pine bark media, 9:103~131 plant nutrition 5:318-330 soil testing, 7:1-68 Ethylene: abscission, citrus, 15:158-161, 168-176

apple bioregulation, 10:366-369 avocado, 10:239-241 bloom delay, 15:107-111 CA storage, 1:317-319, 348 chilling injury, 15:80 citrus abscission, 15:158-161, 168-176

cut flower storage, 10:44-46 dormancy, 7:277-279 flowering, 15:295-296, 319 flower longevity, 3:66-75 genetic regulation, 16:6-7, 14-15, 19-20

kiwifruit respiration, 6:47-48 mechanical stress, 17:16-17 petal senescence, 11:16-19,

E

Easter lily, fertilization, 5:352-355 Embryogenesis. See Asexual embryogenesis Endothia parasitica, 8:291-336 Energy efficiency, in greenhouses,

27-30

rose senescence, 9:65-66

F

1:141-171; 9:1-52

Environment: air pollution, 8:20-22

Feed crops, cactus, 18:298-300 Feijoa, CA and MA, 22:148

440

Fertilization and fertilizer: anthurium, 5:334-335 azalea, 5:335-337 bedding plants, 5:337-341 blueberry, 10:183-227 carnation, 5:341-345 chrysanthemum, 5:345-352 controlled release, 1:79-139; 5:347-348 Easter lily, 5:352-355 Ericaceae, 10:183-227 foliage plants, 5:367-380 foliar, 6:287-355 geranium, 5:355-357 greenhouse crops, 5:317-403 lettuce, 2:175 nitrogen, 2:401-404 orchid, 5:357-358 poinsettia, 5:358-360 rose, 5:361-363 snapdragon, 5:363-364 soil testing, 7:1-68 trickle irrigation, 4:28-31 tulip, 5:364-366 Vaccinium, 10:183-227 zinc nutrition, 23:109-128 Fig: industry, 12:409-490 ripening, 4:258-259 Filbert, in vitro culture, 9:313-314 Fire blight, 1:423-474 Flooding: fruit crops, 13:257-313 Floral scents, 24:31-53 Floricultural crops: see also individual crops: Banksia, 22:1-25 fertilization, 1:98-1 04 growth regulation, 7:399-481 heliconia, 14:1-55 Leucospermum, 22:27-90 postharvest physiology and senescence, 1:204-236; 3:59-143; 10:35-62; 11:15-43 Florigen, 4:94-98 Flower and flowering: alternate bearing, 4:149

CUMULATIVE SUBJECT INDEX

apple anatomy and morphology, 10:277-283 apple bioregulation, 10:344-348 aroids, ornamental, 10:19-24 avocado, 8:257-289 Banksia, 22:1-25 blueberry development, 13:354-378 cactus, 18:325-335 citrus, 12:349-408 control, 4:159-160, 15:279-334 development (postpollination), 19:1-58 fig, 12:424-429 grape anatomy and morphology, 13:354-378 honey bee pollination, 9:239-243 induction, 4:174-203; 254-256 initiation, 4:152-153 in vitro, 4:106-127 kiwifruit, 6:21-35; 12:316-318 Leucospermum, 22:27-90 orchid,5:297-300 pear bioregulation, 10:344-348 pecan, 8:217-255 perennial fruit crops, 12:223-264 phase change, 7:109-155 photoperiod,4:66-105 pistachio, 3:378-387 postharvest physiology, 1:204-236; 3:59-143; 10:35-62; 11:15-43 postpollination development, 19:1-58 protea leaf blackening, 17:173-201 pruning, 8:359-362 raspberry, 11:187-188 regulation in floriculture, 7:416-424 rhododendron, 12:1-42 rose, 9:60-66 scents, 24:31-53 senescence, 1:204-236; 3:59-143; 10:35-62; 11:15-43; 18:1-85 sugars, 4:114 thin cell layer morphogenesis, 14:239-256

441

CUMULATIVE SUBJECT INDEX

tulip, 5:57-59 water relations, 18:1-85 Fluid drilling, 3:1-58 Foliage plants: acclimatization, 6: 11 9-154 fertilization, 1:102-103; 5:367-380 Foliar nutrition, 6:287-355 Freeze protection. See Frost protection Frost: apple fruit set, 1:407-408 citrus, 7:201-238 protection, 11 :45-109 Fruit: abscission, 1:172-203 abscission, citrus, 15:145-182 apple anatomy and morphology, 10:283-297

apple bioregulation, 10:348-374 apple bitter pit, 11:289-355 apple flavor, 16:197-234 apple maturity indices, 13:407-432

apple ripening and quality, 10:361-374

avocado development and ripening, 10:229-271 bloom delay, 15:97-144 blueberry development, 13:378-390

cactus physiology, 18:335-341 CA storage and quality, 8:101-127 chilling injury, 15:63-95 cracking, 19:217-262 diseases in CA storage, 3:412-461 drop, apple and pear, 10:359-361 fig, 12:424-429 growth measurement, 24:373-431 kiwifruit, 6:35-48; 12:316-318 loquat, 23:233-276 maturity indices, 13:407-432 navel orange, 8:129-179 nectarine, postharvest, 11:413-452 nondestructive postharvest quality evaluation, 20:1-119 peach, postharvest, 11:413-452 pear, bioregulation, 10:348-374

pear, fruit disorders, 11:357-411 pear maturity indices, 13:407-432 pear ripening and quality, 10:361-374

pistachio, 3:382-391 plum, 23:179-231 quality and pruning, 8:365-367 ripening, 5:190-205 set, 1:397-424; 4:153-154 set in navel oranges, 8:140-142 size and thinning, 1:293-294; 4:161

softening, 5:109-219, 10:107-152 splitting, 19:217-262 strawberry growth and ripening, 17:267-297

texture, 20:121-224 thinning, apple and pear, 10:353-359

tomato parthenocarpy, 6:65-84 tomato ripening, 13:67-103 Fruit crops: alternate bearing, 4:128-173 apple bitter pit, 11:289-355 apple flavor, 16:197-234 apple fruit splitting and cracking, 19:217-262

apple growth, 11:229-287 apple maturity indices, 13:407-432

apricot, origin and dissemination, 22:225-266

avocado flowering, 8:257-289 avocado rootstocks, 17:381-429 berry crop harvesting, 16:255-382 bloom delay, 15:97-144 blueberry developmental physiology, 13:339-405 blueberry harvesting, 16:257-282 blueberry nutrition, 10:183-227 bramble harvesting, 16:282-298 cactus, 18:302-309 carbohydrate reserves, 10:403-430 CA and MA for tropicals, 22:123-183

CA storage, 1:301-336 CA storage diseases, 3:412-461

CUMULATIVE SUBJECT INDEX

442

nectarine postharvest, 11:413-452 nondestructive postharvest quality evaluation 20:1-119 nutritional ranges, 2:143-164 olive salinity tolerance,

Fruit crops (cont'd) cherry origin, 19:263-317 chilling injury, 15:145-182 chlorosis, 9:161-165 citrus abscission, 15:145-182 citrus cold hardiness, 7:201-238 citrus, culture of young trees,

21:177-214

orange, navel, 8:129-179 orchard floor management,

24:319-372

citrus dwarfing by viroids,

9:377-430

peach origin, 17:331-379 peach postharvest, 11:413-452 pear fruit disorders, 11: 3 5 7-411 pear maturity indices, 13:407-432 pecan flowering, 8:217-255 photosynthesis, 11:111-157 Phytophthora control, 17:299-330 plum origin, 23:179-231 pruning, 8:339-380 rambutan, 16:143-196 raspberry, 11:185-228 roots, 2:453-457 sapindaceous fruits, 16:143-196 short life and replant problem,

24:277-317

citrus flowering, 12:349-408 cranberry, 21:215-249 cranberry harvesting, 16:298-311 currant harvesting, 16:311-327 deficit irrigation, 21:105-131 dormancy release, 7:239-300 Ericaceae nutrition, 10:183-227 fertilization, 1: 104-1 06 fig, industry, 12:409-490 fireblight, 11 :423-474 flowering, 12:223-264 foliar nutrition, 6:287-355 frost control, 11 :45-109 grape flower anatomy and morphology, 13:315-337 grape harvesting, 16:327-348 grape nitrogen metabolism, 14:407-452

grape purning, 16:235-254, 336-340

grape root, 5:127-168 grape seedlessness, 11:164-176 grapevine pruning, 16:235-254, 336-340

2:1-116

strawberry fruit growth, 17:267-297

strawberry harvesting, 16:348-365 summer pruning, 9:351-375 Vaccinium nutrition, 10:183-227 water status, 7:301-344 Fungi: fig, 12:451-474 mushroom, 6:85-118 mycorrhiza, 3:172-213;

honey bee pollination, 9:244-250, 254-256 jojoba, 17:233-266 in vitro culture, 7:157-200; 9:273-349 irrigation, deficit, 21:105-131 kiwifruit, 6:1-64; 12:307-347 longan, 16:143-196 loquat, 23:233-276 lychee, 16:143-196

muscadine grape breeding, 14:357-405

navel orange, 8:129-179

10:211-212

pathogens in postharvest storage, 3:412-461

truffle cultivation, 16:71-107 Fungicide, and apple fruit set, 1:416 G

Garlic, CA storage, 1:375 Genetic variation: alternate bearing, 4:146-150 photoperiodic response, 4:82 pollution injury, 8:16-19

443

CUMULATIVE SUBJECT INDEX

ternperature-photoperiod interaction, 17:73-123 Genetics and breeding: aroids (edible), 8:72-75; 12:169 aroids (ornamental), 10:18-25 bean, bacterial resistance, 3:28-58 bloom delay in fruits, 15:98-107 bulbs, flowering, 18:119-123 cassava, 12:164 chestnut blight resistance, 8:313-321

citrus cold hardiness, 7:221-223 cranberry, 21:236-239 embryogenesis, 1:23 fig, 12:432-433 fire blight resistance, 1:435-436 flowering, 15:287-290, 303-305, 306-309,314-315

flower longevity, 1:208-209 ginseng, 9:197-198 in vitro techniques, 9:318-324; 18:119-123

lettuce, 2:185-187 loquat, 23:252-257 muscadine grapes, 14:357-405 mushroom, 6:100-111 navel orange, 8:150-156 nitrogen nutrition, 2:410-411 pineapple, 21:138~164 plant regeneration, 3:278-283 pollution insensitivity, 8:18-19 potato tuberization, 14:121-124 rhododendron, 12:54-59 sweet potato, 12:175 sweet sorghum, 21:87-90 tomato parthenocarpy, 6:69-70 tomato ripening, 13:77-98 tree short life, 2:66-70 Vigna, 2:311-394 waxes, 23:50-53 woody legume tissue and cell culture, 14:311-314 yam (Dioscorea), 12:183 Geophyte. See Bulb, tuber Geranium, fertilization, 5:355-357 Germination, seed, 2:117-141, 173-174,24:229-275

Germplasm preservation: cryopreservation, 6:357-372 in vitro, 5:261-264; 9:324-325 pineapple, 21:164-168 Germplasm resources: pineapple, 21:133-175 Gibberellin: abscission, citrus, 15:166-167 bloom delay, 15:111-114 citrus, abscission, 15:166-167 cold hardiness, 11:63 dormancy, 7:270-271 floral promoter, 4:114 flowering, 15:219-293, 315-318 genetic regulation, 16:15 grape root, 5:150-151 mechanical stress, 17:19-20 Ginseng, 9:187-236 Girdling, 4:251-252 Glucosinolates, 19:99-215 Graft and grafting: incompatibility, 15:183-232 phase change, 7:136-137, 141-142 rose, 9:56-57 Grape: CA storage, 1:308 chlorosis, 9:165-166 flower anatomy and morphology, 13:315-337

harvesting, 16:327-348 muscadine breeding, 14:357-405 nitrogen metabolism, 14:407-452 pollen morphology, 13:331-332 pruning, 16:235-254, 336-340 root, 5:127-168 seedlessness, 11:159-187 sex determination, 13:329-331 Gravitropism, 15:233-278 Greenhouse and greenhouse crops: carbon dioxide, 7:357-360, 544-545

energy efficiency, 1:141-171; 9:1-52 growth substances, 7:399-481 nutrition and fertilization, 5:317-403

pest management, 13:1-66 vegetables, 21:1-39

CUMULATIVE SUBJECT INDEX

444

Growth regulators. See Growth substances Growth substances, 2:60-66, 24:55-138 see also Abscisic acid, Auxin, Cytokinins, Ethylene, Gibberellins abscission, citrus, 15:157-176 apple bioregulation, 10:309-401 apple dwarfing, 3:315-375 apple fruit set, 1:417 apple thinning, 1:270-300 aroids, ornamental, 10:14-18 avocado fruit development, 10:229-243 bloom delay, 15:107-119

H

Halo blight of beans, 3 :44-45 Hardiness, 4:250-251 Harvest: flower stage, 1:211-212 index, 7:72-74 lettuce, 2:176-181 mechanical of berry crops, 16:255-382

Hazelnut. See Filbert Heat treatment (postharvest), 22:91-121

Heliconia, 14:1-55 Herbaceous plants, subzero stress,

CA storage in vegetables, 1:346-348 cell cultures, 3:214-314 chilling injury, 15:77-83 citrus abscission, 15:157-176 cold hardiness 7:223-225; 11:58-66 dormancy, 7:270-279 embryogenesis, 1:41-43; 2:277-281 floriculture, 7:399-481 flower induction, 4:190-195 flowering, 15:290-296 flower storage, 10:46-51 genetic regulation, 16:1-32 ginseng, 9:226 grape seedlessness, 11:177-180 in vitro flowering, 4:112-115 mechanical stress, 17:16-21

meristem and shoot-tip culture, 5:221-227

navel oranges, 8:146-147 pear bioregulation, 10:309-401 petal senescence, 3:76-78 phase change, 7:137-138, 142-143

raspberry, 11:196-197 regulation, 11:1-14 rose, 9:53-73 seedlessness in grape, 11:177-180 triazole, 10:63-105

6:373-417

Herbicide-resistant crops, 15:371-412 Histochemistry: flower induction, 4:177-179 fruit abscission, 1:172-203 Histology, flower induction, 4:179-184, see also Anatomy and morphology Honey bee, 9:237-272 Horseradish, CA storage, 1:368 Hydrolases, 5:169-219 Hydroponic culture, 5:1-44; 7:483-558

Hypovirulence, in Endothia parasitica, 8:299-310 I

Ice, formation and spread in tissues, 13:215-255

Ice-nucleating bacteria, 7:210-212; 13:230-235

Industrial crops, cactus, 18:309-312 Insects and mites: aroids, 8:65-66 avocado pollination, 8:275-277 fig, 12:442-447 hydroponic crops, 7:530-534 integrated pest management, 13:1-66

lettuce, 2:197-198

445

CUMULATIVE SUBJECT INDEX

ornamental aroids, 10:18 tree short life, 2: 5 2 tulip, 5:63, 92 waxes, 23:1-68 Integrated pest management: greenhouse crops, 13:1-66 In vitro: abscission, 15:156-157 apple propagation, 10:325-326 artemisia, 19:342-345 aroids, ornamental, 10:13-14 bioreactor technology, 24:1-30 bulbs, flowering, 18:87-169 cassava propagation, 13:121-123 cellular salinity tolerance,

fruit trees, 7:331-332 grape root growth, 5:140-141 lettuce industry, 2:175 navel orange, 8:161-162 root growth, 2:464-465

J Jojoba, 17:233-266 Juvenility, 4:111-112 pecan, 8:245-247 tulip, 5:62-63 woody plants, 7:109-155 K

16:33-69

cold acclimation, 6:382 cryopreservation, 6:357-372 embryogenesis, 1:1-78; 2:268-310; 7:157-200; 10:153-181

Kale, fluid drilling of seed, 3:21 Kiwifruit: botany, 6:1-64 vine growth, 12:307-347

environmental control, 17:123-170

flowering bulbs, 18:87-169 flowering, 4:106-127 pear propagation, 10:325-326 phase change, 7:144-145 propagation, 3:214-314; 5:221-277; 7:157-200; 9:57-58,273-349; 17:125-172

thin cell layer morphogenesis, 14:239-264

woody legume culture, 14:265-332

Iron: deficiency and toxicity symptoms in fruits and nuts, 2:150 deficiency chlorosis, 9:133-186 Ericaceae nutrition, 10:193-195 foliar application, 6:330 nutrition, 5:324-325 pine bark media, 9:123 Irrigation: deficit, deciduous orchards, 21:105-131

drip or trickle, 4:1-48 frost control, 11: 76-82

L

Lamps, for plant growth, 2:514-531 Lanzon, CA and MA, 22:149 Leaves: apple morphology, 12:283-288 flower induction, 4:188-189 Leek: CA storage, 1:375 fertilization, 1:118 Leguminosae, in vitro, 5:227-229; 14:265-332

Lemon, rootstock, 1:244-246, see also Citrus Lettuce: CA storage, 1:369-371 fertilization, 1:118 fluid drilling of seed, 3:14-17 industry, 2:164-207 seed germination, 24:229-275 tipburn, 4:49-65 Leucospermum, 22:27-90 Light: fertilization, greenhouse crops, 5:330-331

446 Light (cont'd) flowering, 15:282-287,310-312 fruit set, 1:412-413 lamps, 2:514-531 nitrogen nutrition, 2:406-407 orchards, 2:208-267 ornamental aroids, 10:4-6 photoperiod,4:66-105 photosynthesis, 11:117-121 plant growth, 2:491-537 tolerance, 18:215-246 Longan. See Sapindaceous fruits CA and MA, 22:150 Loquat: botany and horticulture, 23:233-276 CA and MA, 22:149-150 Lychee. See Sapindaceous fruits CA and MA, 22:150

M Magnesium: container growing, 9:84-85 deficiency and toxicity symptoms in fruits and nuts, 2:148 Ericaceae nutrition, 10:196-198 foliar application, 6:331 nutrition, 5:323 pine bark media, 9:117-119 Magnetic resonance imaging, 20:78-86, 225-266 Male sterility, temperaturephotoperiod induction, 17:103-106 Mandarin, rootstock, 1:250-252 Manganese: deficiency and toxicity symptoms in fruits and nuts, 2:150-151 Ericaceae nutrition, 10:189-193 foliar application, 6:331 nutrition, 5:235-326 pine bark media, 9:123-124 Mango: alternate bearing, 4:145-146 asexual embryogenesis, 7:171-173 CA and MA, 22:151-157

CUMULATIVE SUBJECT INDEX

CA storage, 1:313 in vitro culture, 7:171-173 Mangosteen, CA and MA, 22:157 Mechanical harvest, berry crops, 16:255-382 Mechanical stress regulation, 17:1-42 Media: fertilization, greenhouse crops, 5:333 pine bark, 9:103-131 Medicinal crops artemisia, 19:319-371 poppy, 19:373-408 Meristem culture, 5:221-277 Metabolism: flower, 1:219-223 nitrogen in citrus, 8:181-215 seed,2:117-141 Micronutrients: container growing, 9:85-87 pine bark media, 9:119-124 Micropropagation, see also In vitro; propagation: bulbs, flowering, 18:89-113 environmental control, 17:125-172 nuts, 9:273-349 rose, 9:57-58 temperate fruits, 9:273-349 tropical fruits and palms, 7:157-200 Microtus. See Vole Modified atmosphere (MA) for tropical fruits, 22:123-183 Moisture, and seed storage, 2:125-132 Molybdenum nutrition, 5:328-329 Monocot, in vitro, 5:253-257 Monstera. See Aroids, ornamental Morphology: navel orange, 8:132-133 orchid, 5:283-286 pecan flowering, 8:217-243 Moth bean, genetics, 2:373-374 Mung bean, genetics, 2:348-364 Mushroom: CA storage, 1:371-372

CUMULATIVE SUBJECT INDEX

cultivation, 19:59-97 spawn, 6:85-118 Muskmelon, fertilization, 1:118-119 Mycoplasma-like organisms, tree short life, 2:50-51 Mycorrhizae: container growing, 9:93 Ericaceae, 10:211-212 fungi,3:172-213 grape root, 5:145-146 N

Navel orange, 8:129-179 Nectarine: bloom delay, 15:105-106 CA storage, 1:309-310 postharvest physiology, 11:413-452 Nematodes: aroids, 8:66 fig, 12:475-477 lettuce, 2:197-198 tree short life, 2:49-50 NFT (nutrient film technique), 5:1-44 Nitrogen: CA storage, 8:116-117 container growing, 9:80-82 deficiency and toxicity symptoms in fruits and nuts, 2:146 Ericaceae nutrition, 10:198-202 fixation in woody legumes, 14:322-323 foliar application, 6:332 in embryogenesis, 2:273-275 metabolism in apple, 4:204-246 metabolism in citrus, 8:181-215 metabolism in grapevine, 14:407-452 nutrition, 2:395, 423; 5:319-320 pine bark media, 9:108-112 trickle irrigation, 4:29-30 vegetable crops, 22:185-223 Nondestructive quality evaluation of fruits and vegetables, 20:1-119 Nursery crops: fertilization, 1:106-112 nutrition, 9:75-101

447

Nut crops: almond postharvest technology and utilization, 20:267-311 chestnut blight, 8:291-336 fertilization, 1:106 honey bee pollination, 9:250-251 in vitro culture, 9:273-349 nutritional ranges, 2:143-164 pistachio culture, 3:376-396 Nutrient: concentration in fruit and nut crops, 2:154-162 film technique, 5:1-44 foliar-applied, 6:287-355 media, for asexual embryogenesis, 2:273-281 media, for organogenesis, 3:214-314 plant and tissue analysis, 7:30-56 solutions, 7:524-530 uptake, in trickle irrigation, 4:30-31 Nutrition (human): aroids, 8:79-84 CA storage, 8:101-127 Nutrition (plant): air pollution, 8:22-23, 26 blueberry, 10:183-227 calcifuge, 10:183-227 cold hardiness, 3:144-171 container nursery crops, 9:75-101 cranberry, 21:234-235 ecologically based, 24:156-172 embryogenesis, 1:40-41 Ericaceae, 10:183-227 fire blight, 1:438-441 foliar, 6:287-355 fruit and nut crops, 2:143-164 ginseng, 9:209-211 greenhouse crops, 5:317-403 kiwifruit, 12:325-332 mycorrhizal fungi, 3:185-191 navel orange, 8:162-166 nitrogen in apple, 4:204-246 nitrogen in vegetable crops, 22:185-223 nutrient film techniques, 5:18-21, 31-53

CUMULATIVE SUBJECT INDEX

448

Nutrition (plant) (cont'd) ornamental aroids, 10:7-14 pine bark media, 9:103-131 raspberry, 11:194-195 slow-release fertilizers, 1:79-139

o

Leucospermum, 22:27-90 orchid pollination regulation, 19:28-38

poppy, 19:373-408 protea leaf blackening, 17:173-201 rhododendron, 12:1-42 p

Oil palm: asexual embryogenesis, 7:187-188 in vitro culture, 7:187-188 Okra: botany and horticulture, 21:41-72

CA storage, 1:372-373 Olive: alternate bearing, 4:140-141 salinity tolerance, 21:177-214 Onion: CA storage, 1:373-375 fluid drilling of seed, 3:17-18 Opium poppy, 19:373-408 Orange: see also Citrus alternate bearing, 4:143-144 sour, rootstock, 1:242-244 sweet, rootstock, 1:252-253 trifoliate, rootstock, 1:247-250 Orchard and orchard systems: floor management, 9:377-430 light, 2:208-267 root growth, 2:469-470 water, 7:301-344 Orchid: fertilization, 5:357-358 pollination regulation of flower development, 19:28-38 physiology, 5:279-315 Organogenesis, 3:214-314, see also In vitro; tissue culture Ornamental plants: Banksia, 22:1-25 chlorosis, 9:168-169 fertilization, 1:98-104, 106-116 flowering bulb roots, 14:57-88 flowering bulbs in vitro, 18:87-169 foliage acclimatization, 6:119-154 heliconia, 14:1-55

Paclobutrazol. See Triazole Papaya: asexual embryogenesis, 7:176-177 CA and MA, 22:157-160 CA storage, 1:314 in vitro culture, 7:175-178 Parsley: CA storage, 1:375 drilling of seed, 3:13-14 Parsnip, fluid drilling of seed, 3:13-14

Parthenocarpy, tomato, 6:65-84 Passion fruit: in vitro culture, 7:180-181 CA and MA, 22:160-161 Pathogen elimination, in vitro, 5:257-261

Peach: bloom delay, 15:105-106 CA storage, 1:309-310 origin, 17:333-379 postharvest physiology, 11:413-452 short life, 2:4 summer pruning, 9:351-375 wooliness, 20:198-199 Peach palm (Pejibaye): in vitro culture, 7:187-188 Pear: bioregulation, 10:309-401 bloom delay, 15:106-107 CA storage, 1 :306-308 decline, 2:11 fruit disorders, 11:357-411 fire blight control, 1:423-474 in vitro, 9:321 maturity indices, 13:407-432 root distribution, 2 :456 short life, 2:6

449

CUMULATIVE SUBJECT INDEX

Pecan: alternate bearing, 4:139-140 fertilization, 1:106 flowering, 8:217-255 in vitro culture, 9:314-315 Pejibaye, in vitro culture, 7:189 Pepper (Capsicum): CA storage, 1:375-376 fertilization, 1:119 fluid drilling in seed, 3:20 Persimmon: CA storage, 1:314 quality, 4:259 Pest control: aroids (edible), 12:168-169 aroids (ornamental), 10:18 cassava, 12:163-164 cowpea, 12:210-213 ecologically based, 24:172-201 fig, 12:442-477 fire blight, 1:423-474 ginseng, 9:227-229 greenhouse management, 13:1-66 hydroponics, 7:530-534 sweet potato, 12:173-175 vertebrate, 6:253-285 yam (Dioscorea), 12:181-183 Petal senescence, 11:15-43 pH: container growing, 9:87-88 fertilization greenhouse crops, 5:332-333

pine bark media, 9:114-117 soil testing, 7:8-12, 19-23 Phase change, 7:109-155 Phenology: apple, 11:231-237 raspberry, 11:186-190 Philodendron. See Aroids, ornamental Phosphonates, Phytophthora control, 17:299-330 Phosphorus: container growing, 9:82-84 deficiency and toxicity symptoms in fruits and nuts, 2:146-147 nutrition, 5:320-321

pine bark media, 9:112-113 trickle irrigation, 4:30 Photoautotrophic micropropagation, 17:125-172

Photoperiod, 4:66-105,116-117; 17:73-123

flowering, 15:282-284, 310-312 Photosynthesis: cassava, 13:112-114 efficiency, 7:71-72; 10:378 fruit crops, 11:111-157 ginseng, 9:223-226 light, 2:237-238 Physiology: see also Postharvest physiology bitter pit, 11:289-355 blueberry development, 13:339-405

cactus reproductive biology, 18:321-346

calcium, 10:107-152 carbohydrate metabolism, 7:69-108

cassava, 13:105-129 citrus cold hardiness, 7:201-238 conditioning 13:131-181 cut flower, 1:204-236; 3:59-143; 10:35-62

desiccation tolerance, 18:171-213 disease resistance, 18:247-289 dormancy, 7:239-300 embryogenesis, 1:21-23; 2:268-310

floral scents, 24:31-53 flower development, 19:1-58 flowering, 4:106-127 fruit ripening, 13:67-103 fruit softening, 10:107-152 ginseng, 9:211-213 glucosinolates, 19:99-215 heliconia, 14:5-13 juvenility, 7:109-155 lettuce seed germination, 24:229-275

light tolerance, 18:215-246 loquat, 23:242-252 male sterility, 17:103-106

450

flower development (coni'd) mechanical stress, 17:1-42 nitrogen metabolism in grapevine, 14:407-452 nutritional quality and CA storage, 8:118-120 olive salinity tolerance, 21:177-214 orchid,5:279-315 petal senescence, 11:15-43 photoperiodism, 17:73-123 pollution injury, 8:12-16 polyamines, 14:333-356 potato tuberization, 14:89-188 pruning, 8:339-380 raspberry, 11:190-199 regulation, 11:1-14 root pruning, 6:158-171 roots of flowering bulbs, 14:57-88 rose, 9:3-53 salinity hormone action, 16:1-32 salinity tolerance, 16:33-69 seed,2:117-141 seed priming, 16:109-141 subzero stress, 6:373-417 summer pruning, 9:351-375 sweet potato, 23:277-338 thin cell layer morphogenesis, 14:239-264 tomato fruit ripening, 13:67-103 tomato parthenocarpy, 6:71-74 triazoles, 10:63-105,24:55-138 tulip, 5:45-125 vernalization, 17:73-123 volatiles, 17:43-72 watercore, 6:189-251 water relations cut flowers, 18:1-85 waxes, 23:1-68 Phytohormones. See Growth substances Phytophthora control, 17:299-330 Phytotoxins, 2:53-56 Pigmentation: flower, 1:216-219 rose, 9:64-65 Pinching, by chemicals, 7:453-461

CUMULATIVE SUBJECT INDEX

Pineapple: CA and MA, 22:161-162 CA storage, 1:314 genetic resources, 21:138-141 in vitro culture, 7:181-182 Pine bark, potting media, 9:103-131 Pistachio: alternate bearing, 4:137-139 culture, 3:376-393 in vitro culture, 9:315 Plantain: CA and MA, 22:141-146 in vitro culture, 7:178-180 Plant protection, short life, 2:79-84 Plum: CA storage, 1:309 origin, 23:179-231 Poinsettia, fertilization, 1:103-104; 5:358-360 Pollen, desiccation tolerance, 18:195 Pollination: apple, 1:402-404 avocado, 8:272-283 cactus, 18:331-335 embryogenesis, 1:21-22 floral scents, 24:31-53 fig, 12:426-429 flower regulation, 19:1-58 fruit crops, 12:223-264 fruit set, 4:153-154 ginseng, 9:201-202 grape, 13:331-332 heliconia, 14:13-15 honey bee, 9:237-272 kiwifruit, 6:32-35 navel orange, 8:145-146 orchid, 5:300-302 petal senescence, 11:33-35 protection, 7:463-464 rhododendron, 12:1-67 Pollution, 8:1-42 Polyamines, 14:333-356 chilling injury, 15:80 Polygalacturonase, 13:67-103 Postharvest physiology: almond,20:267-311 apple bitter pit, 11:289-355

451

CUMULATIVE SUBJECT INDEX

apple maturity indices, 13:407-432 aroids, 8:84-86 asparagus, 12:69-155 CA for tropical fruit, 22:123-183 CA storage and quality, 8:101-127 chlorophyll fluorescence, 23:69-107 cut flower, 1:204-236; 3:59-143; 10:35-62 foliage plants, 6:119-154 fruit, 1:301-336 fruit softening, 10:107-152 heat treatment, 22:91-121 lettuce, 2:181-185 low-temperature sweetening, 17:203-231 MA for tropical fruit, 22:123-183 navel orange, 8:166-172 nectarine, 11:413-452 nondestructive quality evaluation, 20:1-119 pathogens, 3:412-461 peach,11:413-452 pear disorders, 11:357-411 pear maturity indices, 13:407-432 petal senescence, 11:15-43 protea leaf blackening, 17:173-201 quality evaluation, 20:1-119 seed,2:117-141 texture in fresh fruit, 20:121-244 tomato fruit ripening, 13:67-103 vegetables, 1:337-394 watercore, 6:189-251; 11:385-387 Potassium: container growing, 9:84 deficiency and toxicity symptoms in fruits and nuts, 2:147-148 foliar application, 6:331-332 nutrition, 5:321-322 pine bark media, 9:113-114 trickle irrigation, 4:29 Potato: CA storage, 1:376-378 fertilization, 1:120-121 low temperature sweetening, 17:203-231 tuberization, 14:89-198

Propagation: see also In vitro apple, 10:324-326; 12:288-295 aroids, ornamental, 10:12-13 bioreactor technology, 24:1-30 cassava, 13:120-123 floricultural crops, 7:461-462 ginseng, 9:206-209 orchid,5:291-297 pear, 10:324-326 rose, 9:54-58 tropical fruit, palms 7:157-200 woody legumes in vitro, 14:265-332 Protaceous flower crop: see also Protea Banksia, 22:1-25

Leucospermum, 22:27-90 Protea, leaf blackening, 17:173-201 Protected crops, carbon dioxide, 7:345-398 Protoplast culture, woody species, 10:173-201 Pruning, 4:161, 8:339-380 apple, 9:351-375 apple training, 1:414 chemical, 7:453-461 cold hardiness, 11:56 fire blight, 1:441-442 grapevines, 16:235-254 light interception, 2:250-251 peach,9:351-375 phase change, 7:143-144 root, 6:155-188 Prunus: see also Almond; Cherry; Nectarine; Peach; Plum in vitro, 5:243-244; 9:322 root distribution, 2:456

Pseudomonas: phaseolico1a, 3:32-33, 39,44-45 solanacearum, 3:33 syringae, 3:33,40; 7:210-212 Q Quality evaluation: fruits and vegetables, 20:1-119, 121-224 nondestructive, 20:1-119 texture in fresh fruit, 20:121-224

CUMULATIVE SUBJECT INDEX

452

R

Rabbit, 6:275-276 Radish, fertilization, 1:121 Rambutan. See Sapindaceous fruits Rambutan, CA and MA, 22:163 Raspberry: harvesting, 16:282-298 productivity, 11:185-228 Rejuvenation: rose, 9:59-60 woody plants, 7:109-155 Replant problem, deciduous fruit trees, 2:1-116 Respiration: asparagus postharvest, 12:72-77 fruit in CA storage, 1:315-316 kiwifruit, 6:47-48 vegetables in CA storage, 1:341-346 Rhizobium, 3:34,41 Rhododendron, 12:1-67 Rice bean, genetics, 2:375-376 Root: apple, 12:269-272 cactus, 18:297-298 diseases, 5:29-31 environment, nutrient film technique, 5:13-26 Ericaceae, 10:202-209 grape, 5:127-168 kiwifruit, 12:310-313 physiology of bulbs, 14:57-88 pruning, 6:155-188 raspberry, 11:190 rose, 9:57 tree crops, 2:424-490 Root and tuber crops: aroids, 8:43-99; 12:166-170 cassava, 12:158-166 low-temperature sweetening, 17:203-231

minor crops, 12:184-188 potato tuberization, 14:89-188 sweet potato, 12:170-176 sweet potato physiology, 23:277-338

yam (Dioscorea), 12:177-184

Rootstocks: alternate bearing, 4:148 apple, 1:405-407; 12:295-297 avocado, 17:381-429 citrus, 1:237-269 cold hardiness, 11:57-58 fire blight, 1:432-435 light interception, 2:249-250 navel orange, 8:156-161 root systems, 2:471-474 stress, 4:253-254 tree short life, 2:70-75 Rosaceae, in vitro, 5:239-248 Rose: fertilization, 1:104; 5:361-363 growth substances, 9:3-53 in vitro, 5:244-248

s Salinity: air pollution, 8:25-26 olive, 21:177-214 soils, 4:22-27 tolerance, 16:33-69 Sapindaceous fruits, 16:143-196 Sapodilla, CA and MA, 22:164 Scoring, and fruit set, 1:416-417 Seed: abortion, 1:293-294 apple anatomy and morphology, 10:285-286

conditioning, 13:131-181 desiccation tolerance, 18:196-203 environmental influences on size and composition, 13:183-213

flower induction, 4:190-195 fluid drilling, 3:1-58 grape seedlessness, 11:159-184 kiwifruit, 6:48-50 lettuce, 2:166-174 lettuce germination, 24:229-275 priming, 16:109-141 rose propagation, 9:54-55 vegetable, 3:1-58 viability and storage, 2:117-141

453

CUMULATIVE SUBJECT INDEX

Secondary metabolites, woody legumes, 14:314-322 Senescence: chlorophyll senescence, 23:88-93 cut flower, 1:204-236; 3:59-143; 10:35-62; 18:1-85 petal, 11:15-43 pollination-induced, 19:4-25 rose, 9:65-66 whole plant, 15:335-370 Sensory quality: CA storage, 8:101-127 Shoot-tip culture, 5:221-277, see also Micropropagation Short life problem, fruit crops, 2:1-116 Small fruit, CA storage, 1:308 Snapdragon fertilization, 5:363-364 Sodium, deficiency and toxicity symptoms in fruits and nuts, 2:153-154 Soil: grape root growth, 5:141-144 management and root growth, 2:465-469 orchard floor management, 9:377-430 plant relations, trickle irrigation, 4:18-21 stress, 4:151-152 testing, 7:1-68; 9:88-90 zinc, 23:109-178 Soilless culture, 5:1-44 Solanaceae, in vitro, 5:229-232 Somatic embryogenesis. See Asexual embryogenesis Sorghum, sweet, 21:73-104 Spathiphyllum. See Aroids, ornamental Stem, apple morphology, 12:272-283 Storage: see also Postharvest physiology, Controlledatmosphere (CA) storage cut flower, 3:96-100; 10:35-62 rose plants, 9:58-59 seed,2:117-141

Strawberry: fertilization, 1:106 fruit growth and ripening, 17:267-297 harvesting, 16:348-365 in vitro, 5:239-241 Stress: benefits of, 4:247-271 chlorophyll fluorescence, 23:69-107 climatic, 4:150-151 flooding, 13:257-313 mechanical, 17:1-42 petal, 11:32-33 plant, 2:34-37 protectants (triazoles), 24:55-138 protection, 7:463-466 salinity tolerance in olive, 21:177-214 subzero temperature, 6:373-417 waxes, 23:1-68 Sugar: see also Carbohydrate allocation, 7:74-94 flowering, 4:114 Sugar apple, CA and MA, 22:164 Sugar beet, fluid drilling of seed, 3:18-19 Sulfur: deficiency and toxicity symptoms in fruits and nuts, 2:154 nutrition, 5:323-324 Sweet potato: culture, 12:170-176 fertilization, 1:121 physiology, 23:277-338 Sweet sop, CA and MA, 22:164 Symptoms, deficiency and toxicity symptoms in fruits and nuts, 2:145-154 Syngonium. See Aroids, ornamental T

Taro. See Aroids, edible Tea, botany and horticulture, 22:267-295

CUMULATIVE SUBJECT INDEX

454

Temperature: apple fruit set, 1 :408-411 bloom delay, 15:119-128 CA storage of vegetables, 1:340-341

chilling injury, 15:67-74 cut flower storage, 10:40-43 cryopreservation, 6:357-372 fertilization, greenhouse crops, 5:331-332

fire blight forecasting, 1:456-459 flowering, 15:284-287, 312-313 interaction with photoperiod, 4:80-81

low temperature sweetening, 17:203-231

navel orange, 8:142 nutrient film technique, 5:21-24 photoperiod interaction, 17:73-123 photosynthesis, 11:121-124 plant growth, 2:36-37 seed storage, 2:132-133 subzero stress, 6:373-417 Texture in fresh fruit, 20:121-224 Thinning, apple, 1:270-300 Tipburn, in lettuce, 4:49-65 Tissue: see also In vitro culture, 1:1-78; 2:268-310; 3:214-314; 4:106-127; 5:221-277; 6:357-372; 7:157-200; 8:75-78; 9:273-349; 10:153-181, 24:1-30 dwarfing, 3:347-348 nutrient analysis, 7:52-56; 9:90

Tomato: CA storage, 1:380-386 chilling injury, 20:199-200 fertilization, 1:121-123 fluid drilling of seed, 3:19-20 fruit ripening, 13:67-103 galacturonase, 13:67-103 parthenocarpy, 6:65-84 Toxicity symptoms in fruit and nut crops, 2:145-154 Transport, cut flowers, 3:100-104 Tree decline, 2:1-116

Triazoles, 10:63-105, 24:55-138 chilling injury, 15:79-80 Trickle irrigation, 4:1-48 Truffle cultivation, 16:71-107 Tuber, potato, 14:89-188 Tuber and root crops. See Root and tuber crops Tulip: See Bulb fertilization, 5:364-366 in vitro, 18:144-145 physiology, 5:45-125 Tunnel (cloche), 7:356-357 Turfgrass, fertilization, 1:112-117 Turnip, fertilization, 1:123-124 Turnip Mosaic Virus, 14:199-238

u Urd bean, genetics, 2:364-373 Urea, foliar application, 6:332

v Vaccinium, 10:185-187, see also Blueberry; Cranberry Vase solutions, 3:82-95; 10:46-51 Vegetable crops: aroids, 8:43-99; 12:166-170 asparagus postharvest, 12:69-155 cactus, 18:300-302 cassava, 12:158-166; 13:105-129 CA storage, 1:337-394 CA storage and quality, 8:101-127 CA storage diseases, 3:412-461 chilling injury, 15:63-95 ecologically based, 24:139-228 fertilization, 1:117-124 fluid drilling of seeds, 3:1-58 greenhouse management, 21:1-39 greenhouse pest management, 13:1-66

honey bee pollination, 9:251-254 hydroponics, 7:483-558 lettuce seed germination, 24:229-275

low-temperature sweetening, 17:203-231

CUMULATIVE SUBJECT INDEX

minor root and tubers, 12:184-188 mushroom cultivation, 19:59-97 mushroom spawn, 6:85-118 N nutrition, 22:185-223 nondestructive postharvest quality evaluation, 20:1-119 okra, 21:41-72 potato tuberization, 14:89-188 seed conditioning, 13:131-181 seed priming, 16:109-141 sweet potato, 12:170-176 sweet potato physiology, 23:277-338

tomato fruit ripening, 13:67-103 tomato parthenocarpy, 6:65-84 tropical production, 24:139-228 truffle cultivation, 16:71-107 yam (Dioscorea), 12:177-184 Vegetative tissue, desiccation tolerance, 18:176-195 Vernalization, 4:117,15:284-287; 17:73-123

Vertebrate pests, 6:253-285 Vigna: see also Cowpea genetics, 2:311-394 U.S. production, 12:197-222 Viroid, dwarfing for citrus, 24:277-317

Virus: benefits in horticulture, 3:394411

dwarfing for citrus, 24:277-317 elimination, 7:157-200; 9:318; 18:113-123

fig, 12:474-475 tree short life, 2:50-51 turnip mosaic, 14:199-238 Volatiles, 17:43-72,24:31-53 Vole, 6:254-274

w Walnut, in vitro culture, 9:312 Water relations: cut flower, 3:61-66; 18:1-85 deciduous orchards, 21:105-131 desiccation tolerance, 18:171-213

455

fertilization, greenhouse crops, 5:332

fruit trees, 7:301-344 kiwifruit, 12:332-339 light in orchards, 2:248-249 photosynthesis, 11:124-131 trickle irrigation, 4:1-48 Watercore, 6:189-251 pear, 11:385-387 Watermelon, fertilization, 1:124 Wax apple, CA and MA, 22;164 Waxes, 23:1-68 Weed control, ginseng, 9:228229

Weeds: lettuce research, 2:198 virus, 3:403 Woodchuck, 6:276-277 Woody species, somatic embryogenesis, 10:153-181

x Xanthomonas phaseoli, 3:29-32,41, 45-46

Xanthophyll cycle, 18:226-239 Xanthosoma, 8:45-46, 56-57, see also Aroids y Yam (Dioscorea), 12:177-184 Yield: determinants, 7:70-74; 97-99 limiting factors, 15:413-452

z Zantedeschia. See Aroids, ornamental Zinc: deficiency and toxicity symptoms in fruits and nuts, 2:151 foliar application, 6:332, 336 nutrition, 5:326; 23:109-178 pine bark media, 9:124

Cumulative Contributor Index (Volumes 1-24) Abbott, J.A., 20:1 Adams III, W.W., 18:215 Aldwinckle, H.S., 1:423; 15:xiii Anderson, I.e., 21:73 Anderson, J.L., 15:97 Anderson, P.e., 13:257 Andrews, P.K., 15:183 Ashworth, KN., 13:215; 23:1 Asokan, M.P., 8:43 Atkinson, D., 2:424 Aung, L.H., 5:45 Bailey, W.G., 9:187 Baird, L.A.M., 1:172 Banks, N.H., 19:217 Barden, J.A., 9:351 Barker, A.V., 2:411 Bass, L.N., 2:117 Becker, J.S., 18:247 Beer, S.V., 1:423 Behboudian, M.H., 21:105 Bennett, A.B., 13:67 Benschop, M., 5:45 Ben-Ya'acov, A., 17:381 Benzioni, A., 17:233 Bevington, K.B., 24:277 Bewley, J.D., 18:171 Binzel, M.L., 16:33 Blanpied, G.D., 7:xi Bliss, F.A., 16:xiii Borochov, A., 11:15 Bower, J.P., 10:229 Bradley, G.A., 14:xiii Brennan, R, 16:255 Broadbent, P., 24:277 Broschat, T.K., 14:1 456

Brown, S. 15:xiii Buban, T., 4:174 Bukovac, M.J., 11:1 Burke, M.J., 11:xiii Buwalda, J.G., 12:307 Byers, RK, 6:253 Caldas, L.S., 2:568 Campbell, L.K, 2:524 Cantliffe, D.J., 16:109, 17:43; 24:229 Carter, G., 20:121 Carter, J.V., 3:144 Cathey, H.M., 2:524 Chambers, RJ., 13:1 Charron, e.S., 17:43 Chin, C.K., 5:221 Clarke, N.D., 21:1 Cohen, M., 3:394 Collier, G.F., 4:49 Collins, W.L., 7:483 Compton, M.K, 14:239 Conover, e.A., 5:317; 6:119 Coppens d'Eeckenbrugge, G., 21:133 Coyne, D.P., 3:28 Crane, J.e., 3:376 Criley, RA., 14:1; 22:27; 24:x Crowly, W., 15:1 Cutting, J.G., 10:229 Daie, J., 7:69 Dale, A., 11:185; 16:255 Darnell, RL., 13:339 Davenport, T.L., 8:257; 12:349 Davies, F.S., 8:129; 24:319 Davies, P.J., 15:335 Davis, T.D., 10:63; 24:55

457

CUMULATIVE CONTRIBUTOR INDEX

DeEll, J.R, 23:69 DeGrandi-Hoffrnan, G., 9:237 De Hertogh, A.A., 5:45; 14:57; 18:87 Deikman, J., 16:1 DellaPenna, D., 13:67 Demmig-Adams, B., 18:215 Dennis, F.G., Jr., 1:395 Doud, S.L., 2:1 Dudareva, N., 24:31 Duke, 5.0., 15:371 Dunavent, M.G., 9:103 Duval, M.-F., 21:133 Diizyaman, K, 21:41 Dyer, W.K, 15:371 Early, J.D., 13:339 Elfving, D.e., 4:1; 11:229 El-Goorani, M.A., 3:412 Esan, KB., 1:1 Evans, D.A., 3:214 Ewing, KK, 14:89 Faust, M., 2:vii, 142; 4:174; 6:287; 14:333; 17:331; 19:263; 22:225; 23:179 Fenner, M., 13:183 Fenwick, G.R, 19:99 Ferguson, A.R, 6:1 Ferguson, LB., 11:289 Ferguson, J.J., 24:277 Ferguson, L., 12:409 Ferree, D.e., 6:155 Ferreira, J.F.S., 19:319 Fery, RL., 2:311; 12:157 Fischer, RL., 13:67 Fletcher, RA., 24:53 Flick, e.K, 3:214 Flore, J.A., 11:111 Forshey, e.G., 11:229 Fujiwara, K., 17:125 Geisler, D., 6:155 Geneve, RL., 14:265 George, W.L., Jr., 6:25 Gerrath, J.M., 13:315 Gilley, A., 24:55

Giovannetti, G., 16:71 Giovannoni, rJ., 13:67 Glenn, G.M., 10:107 Goffinet, M.e., 20:ix Goldschmidt, KK, 4:128 Goldy, RG., 14:357 Goren, R, 15:145 Goszczynska, D.M., 10:35 Grace, S.e., 18:215 Graves, e.J., 5:1 Gray, D., 3:1 Grierson, W., 4:247 Griffen, G.J., 8:291 Grodzinski, B., 7:345 Gucci, R, 21:177 Guest, D.L, 17:299 Guiltinan, M.J., 16:1 Hackett, W.P., 7:109 Hallett, I.e., 20:121 Halevy, A.H., 1:204; 3:59 Hammerschmidt, R, 18:247 Hanson, KJ., 16:255 Harker, F.R, 20:121 Heaney, RK., 19:99 Heath, RR, 17:43 Helzer, N.L., 13:1 Hendrix, J.W., 3:172 Henny, RJ., 10:1 Hergert, G.B., 16:255 Hess, F.D., 15:371 Heywood, V., 15:1 Hogue, KJ., 9:377 Holt, J.S., 15:371 Huber, D.J., 5:169 Hunter, KL., 21:73 Hutchinson, J.F., 9:273 Hutton, RJ., 24:277 Indira, P., 23:277 Isenberg, F.M.R, 1;337 Iwakiri, B.T., 3:376 Jackson, J.K, 2:208 Janick, J., l:ix; 8:xi; 17:xiii; 19:319; 21:xi; 23:233

458

Jarvis, W.R, 21:1 Jenks, M.A., 23:1 Jensen, M.H., 7:483 Jeong, B.R, 17:125 Jewett, T.J., 21:1 Joiner, J.N., 5:317 Jones, H.G., 7:301 Jones, J.B., Jr., 7:1 Jones, RB., 17:173 Kagan-Zur, V., 16:71 Kang, S.-M., 4:204 Kato, T., 8:181 Kawa, L., 14:57 Kawada, K., 4:247 Kelly, J.F., 10:ix; 22:xi Khan, A.A., 13:131 Kierman, J., 3:172 Kim, K.-W., 18:87 Kinet, J.-M., 15:279 King, G.A., 11:413 Kingston, C.M., 13:407-432 Kliewer, W.M., 14:407 Knight, RJ., 19:xiii Knox, RB., 12:1 Kofranek, A.M., 8:xi Korcak, RF., 9:133; 10:183 Kozai, T., 17:125 Krezdarn, A.H., l:vii Lakso, A.N., 7:301; 11:111 Lamb, RC., 15:xiii Lang, G.A., 13:339 Larsen, RP., 9:xi Larson, RA., 7:399 Leal, F., 21:133 Ledbetter, C.A., 11:159 Li, P.H., 6:373 Lill, RK, 11:413 Lin,S., 23:233 Lipton, W.J., 12:69 Litz, RK, 7:157 Lockard, RG., 3:315 Loescher, W.H., 6:198 Lorenz, O.A., 1:79 Lu, R, 20:1

CUMULATIVE CONTRIBUTOR INDEX

Lurie,S., 22:91-121 Lyrene, P., 21:xi Manivel, L., 22:267 Maraffa, S.B., 2:268 Marangoni, A.G., 17:203 Marini, RP., 9:351 Marlow, G.c., 6:189 Maronek, D.M., 3:172 Martin, G.G., 13:339 Mayak, 5., 1:204; 3:59 Maynard, D.N., 1:79 McConchie, R, 17:173 McNicol, RJ., 16:255 Merkle, S.A., 14:265 Michailides, T.J., 12:409 Michelson, K, 17:381 Mika, A., 8:339 Miller, 5.5., 10:309 Mills, H.A., 2:411; 9:103 Mills, T.M., 21:105 Mitchell, C.A., 17:1 Mizrahi, Y., 18:291, 321 Molnar, J.M., 9:1 Monk, G.J., 9:1 Monselise, S.P., 4:128 Moore, G.A., 7:157 Mar, Y., 9:53 Morris, J.R, 16:255 Murashige, T., 1:1 Murr, D.P., 23:69 Murray, S.H., 20:121 Myers, P.N., 17:1 Nadeau, J.A., 19:1 Nascimento, W.M., 24:229 Neilsen, G.H., 9:377 Nerd, A., 18:291, 321 Niemiera, A.X., 9:75 Nobel, P.S., 18:291 Nyujto, F., 22:225 O'Donoghue, KM., 11:413 Ogden, RJ., 9:103 O'Hair, S.K., 8:43; 12:157 Oliveira, C.M., 10:403

CUMULATIVE CONTRlBUTOR INDEX

Oliver, M.J., 18:171 O'Neill, S.D., 19:1 Opara, L.U., 19:217; 24:373 Ormrod, D.P., 8:1 Palser, B.F., 12:1 Papadopoulos, A.P., 21:1 Pararajasingham, S., 21:1 Parera, CA., 16:109 Pegg, K.G., 17:299 Pellett, H.M., 3:144 Perkins-Veazil, P., 17:267 Pichersky, E., 24:31 Piechulla, B., 24:31 Ploetz, RC, 13:257 Pokorny, F.A., 9:103 Poole, RT., 5:317;6:119 Poovaiah, B.W., 10:107 Portas, CA.M., 19:99 Porter, M.A., 7:345 Possingham, J.V., 16:235 Prange, RK., 23:69 Pratt, C, 10:273; 12:265 Preece, J.E., 14:265 Priestley, CA., 10:403 Proctor, J.T.A., 9:187 Quamme, H., 18:xiii Raese, J.T., 11:357 Ramming, D.W., 11:159 Ravi, V., 23:277 Reddy, A.S.N., 10:107 Redgwell, RJ., 20:121 Reid, M., 12:xiii, 17:123 Reuveni, M., 16:33 Richards, n, 5:127 Rieger, M., 11:45 Roper, T.R, 21:215 Rosa, E.A.S., 19:99 Roth-Bejerano, N., 16:71 Roubelakis-Angelakis, K.A., 14:407 Rouse, J.L., 12:1 Royse, D.J., 19:59 Rudnicki, RM., 10:35 Ryder, E.J., 2:164; 3:vii

459

Sachs, R, 12:xiii Sakai, A., 6:357 Salisbury, F.B., 4:66; 15:233 Saltveit, M.E., 23:x San Antonio, J.P., 6:85 Sankhla, N., 10:63; 24:55 Saure, M.C, 7:239 Schaffer, B., 13:257 Schenk, M.K., 22:185 Schneider, G.W., 3;315 Schuster, M.L., 3:28 Scorza, R, 4:106 Scott, J.W., 6:25 Sedgley, M., 12:223; 22:1 Seeley, S.S., 15:97 Serrano Marquez, C, 15:183 Sharpe, RH., 23:233 Sharp, W.R, 2:268; 3:214 Shattuck, V.I., 14:199 Shear, CB., 2:142 Sheehan, T.J., 5:279 Shipp, J.L., 21:1 Shirra, M., 20:267 Shorey, H.H., 12:409 Simon, J.E., 19:319 Sklensky, D.E., 15:335 Smith, G.S., 12:307 Smock, RM., 1:301 Sommer, N.F., 3:412 Sondahl, M.R, 2:268 Sopp, P.L, 13:1 Soule, J., 4:247 Sparks, D., 8:217 Splittstoesser, W.E., 6:25; 13:105 Srinivasan, C, 7:157 Stang, E.J., 16:255 Steffens, G.L., 10:63 Stevens, M.A., 4:vii Stroshine, RL., 20:1 Struik, P.C, 14:89 Studman, CJ., 19:217 Stutte, G.W., 13:339 Styer, D.J., 5;221 Sunderland, K.D., 13:1 Sung, Y., 24:229 Suranyi, D., 19:263; 22:225; 23:179

460

Swanson, B., 12:xiii Swietlik, D., 6:287; 23:109 Syvertsen, J.P., 7:301 Tattini, M., 21:177 Tetenyi, P., 19:373 Tibbitts, T.W., 4:49 Timon, B., 17:331 Tindall, H.D., 16:143 Tisserat, B., 1:1 Titus, J.S., 4:204 Trigiano, RN., 14:265 Tunya, G.O., 13:105 Upchurch, B.L., 20:1 Valenzuela, H.R, 24:139 van Doorn, W.G., 17:173; 18:1 van Kooten, 0., 23:69 Veilleux, RK, 14:239 Vorsa, N., 21:215 Wallace, A., 15:413 Wallace, D.H., 17:73 Wallace, G.A., 15:413 Wang, c.Y., 15:63 Wang, S.Y., 14:333 Wann, S.R, 10:153

CUMULATIVE CONTRIBUTOR INDEX

Watkins, c.B., 11:289 Watson, G.W., 15:1 Webster, B.D., 1:172; 13:xi Weichmann, J., 8:101 Wetzstein, H.Y., 8:217 Whiley, A.W., 17:299 Whitaker, T.W., 2:164 White, J.W., 1:141 Williams, KG., 12:1 Williams, M.W., 1:270 Wismer, W.V., 17:203 Wittwer, S.H., 6:xi Woodson, W.R, 11:15 Wright, RD., 9:75 Wutscher, H.K., 1:237 Yada, RY., 17:203 Yadava, U.L., 2:1 Yahia, KM., 16:197; 22:123 Yan, W., 17:73 Yarborough, D.K, 16:255 Yelenosky, G., 7:201 Zanini, K, 16:71 Zieslin, N., 9:53 Zimmerman, RH., 5:vii; 9:273 Ziv, M., 24:1 Zucconi, F., 11:1