Advances in Agronomy, Volume 54

DVANCES IN

Lgronomy

V O L U M5 E4

Advisory Board Martin Alexander

Eugene J. Kamprath

Cornell University

North Carolina State University

Kenneth J. Frey

Larry P. Wilding

Iowa State University

Texas A&M University

Prepared in cooperation with the American Society of Agronomy Monographs Committee P. S. Baenziger J. Bartels J. N. Bigham L. P. Bush

M. A. Tabatabai, Chairnuan R. N. Carrow W. T. Frankenberger, J . D. M. Kral S. E. Lingle

G. A. Peterson D. E. Rolston

D. E. Stott J. W. Stuck

Edited by

Donald L. Sparks Department of Plant and Soil Sciences University of Delaware Newark, Delaware

ACADEMIC PRESS, INC. Harcourt Brace & Company San Diego New York Boston

London Sydney Tokyo Toronto

This book is printed on acid-free paper.

@

Copyright 0 1995 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road. London NWl 7DX International Standard Serial Number: 0065-2 1 13 International Standard Book Number: 0- 12-000754- 1 PRINTED IN THE UNITED STATES OF AMERICA 95 96 9 7 9 8 99 O O Q W 9 8 7 6

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4

3 2 1

Contents CONTRIBUTORS ...................................................... PREFACE ..............................................................

ix

xi

IMPACTS OF AGRICULTURAL PRACTICES ON SUBSURFACE MICROBIAL ECOLOGY

Eugene L . Madsen I. Introduction and Scope ......................................... I1. Subsurface Microbial Ecology ................................... 111. Agricultural Practices and Their Impact on Subsurface Habitats .... Iv. Impact of Agricultural Practices on Subsurface Microbial Ecology . . V. Concluding Remarks ............................................ References .....................................................

HERBICIDE-RESISTANT

FIELD

CROPS

Jack Dekker and Stephen 0. Duke ...................................................

I . Introduction I1. Mechanisms of Herbicide Resistance

i

5 35 46 56 57

.............................

111. Selection for Herbicide-Resistant Variants ........................ rv. Herbicide-Resistant Crops by the Herbicide Chemical Family ..... V. Summary ...................................................... References .....................................................

69 71

77 80 100 101

ACIDSOIL TOLERANCE IN WHEAT

Brett F. Carver and James D . Ownby I . T h e Problem: Causes. Symptomatology. and Severity ............. I1. Physiology of Aluminum and Manganese ‘Tolerance in Wheat .....

I11. Genetic Mechanisms of Tolerance to Acid Soils ................... Iv. Breeding for Acid Soil Tolerance ................................ v. Sustainable Production in Acid Soils ............................. VI. Conclusions .................................................... References .....................................................

V

117 124 136 146 161 162 164

vi

CONTENTS

MICROBIAL REDUCTIONOF IRON. MANGANESE. AND OTHER METALS

Derek R . Lovley I . Introduction ................................................... Fe(II1) and Mn(rV) Reduction ................................... Uranium Reduction ............................................. Selenium Reduction ............................................ Chromate Reduction ............................................ VI . Microbial Reduction of Other Metals ............................ VII . Conclusions .................................................... References ..................................................... 11. 111. IV. V.

176 176 202 205 210 216 216 217

NITRIFICATION INHIBITORSFOR AGRICULTURE. HEALTH. AND THE ENVIRONMENT I. I1. 111. I v. V. VI . VII .

Rajendra Prasad and J . F. Power Introduction ................................................... Nitrification Inhibitors .......................................... N l s . NI I;/NO; Ratios. and Plant Growth ....................... NIs and Crop Yields ............................................ Phytotoxicity of NIs ............................................ Health and Nitrates ............................................. NIs and Environnient ........................................... References ..................................................... PRODUCTION AND

234 235 243 246 252 254 262 269

BREEDINGOF LENTIL

F.J . Muehlbauer. W.J. Kaiser. S . L . Clement. and R .J . Summerfield 284 1. Introduction ................................................... 285 I1. Background .................................................... 286 I11. Origin. Taxonomy. Cytology. and Plant Description .............. 291 IV. Production of Lentil ............................................ V. Fertilization and Weed Control .................................. 296 297 VI . Principal Uses .................................................. 298 VII. Major Constraints to Production ................................ ......................................... 303 Hybridization Methods VIII . .............................................. 307 Genetic Resources IX . Genetics ....................................................... 308 X. 317 XI . Interspecific Hybridization ...................................... VIII . Methods Used for Lentil Breeding ............................... 318

CONTENTS

ix.

Breeding Objectives ............................................ X. Summary ...................................................... References .....................................................

vii 321 326 327

USE OF APOMIXIS IN CULTIVAR DEVELOPMENT I. I1. I11.

rv. v.

VI.

Wayne W. Hanna Introduction ................................................... T h e Gene(s) Controlling Apomixis .............................. Breeding ....................................................... impact on Seed Industry ........................................ International Impact ............................................ Evaluation ..................................................... References

.....................................................

INDEX ............................................................

333 334 337 345 346 347 347 351

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Contributors Numbers in parentheses indicate the pagcs on which the authors’ contributions begin.

BRETT F. CARVER (I 17), Department OfAgronomy, Oklahoma State University, Stillwater, Oklahoma 74078 S. L. CLEMENT ( 2 8 3 ) , United States Department of Agriculture, Agriailtural Research Service, Regional Plant Introduction Station, WashingtonState University, Pidlman, Washington 991 64 JACK DEKKER (69), Agronomy Department, Iowa State University, Ames, Iowa 5001I STEPHEN 0. DUKE (69), United States Department of Agrinclture, Agricultural Research Service, Southern Weed Science Laboratory, Stoneville, Mississippi 38776 WAYNE W. HANNA ( 3 3 3 ) , United States Department of Agriculture, Ap’ailtziral Research Service, Coastal Plain Experiment Station, Tifon, Georgia 31 793 W. J. KAISER ( 2 8 3 ) , United States Department of Agriculture, Agricultural Research Service, Regional Plant Introduction Station, Washington State University, Pullman, Washington 991 64 DEREK R. LOVLEY (17 S), Water Resozcrces Division, United States Geological Survey, Reston, Virginia 22092 EUGENE L. MADSEN (l), Division of Biological Sciences, Section of Mimobiology, Cornell University,Ithaca, New York 14853 F. J. MUEHLBAUER ( 2 8 3 ) , United States Department of Agriailtzire, Agricultural Research Service, Grain Legiime Genetics and Physiology Research Unit, Washington State University, Piillman, Washington 991 64 JAMES D. OWNBY (1 17), Department of Botany, Oklahoma State University, Stillwater, Oklahoma 74078 J. F. POWER ( 2 3 3), United States Department of Agricziltiire, Agricultural Research Service, Universityof Nebraska, Lincoln, Nebraska 68583 RAJENDRA PRASAD ( 2 3 3 ) , Division of Agronomy, Indian Agrikziltural Research Institute, New Dehli, India R. J. SUMMERFIELD, ( 2 8 3 ) Department of Agriculture, Plant Environment Laboratoi-y, University of Reading, Berkshire RG2 9AD, United Kingdom

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Preface This book is the 54th volume of Advances in Agronomy. Under the excellent editorships of A. G. Norman and N. C. Brady, this venerable serial publication has included state-of-the-art and classic reviews over the years. The excellent quality of Advances in Agronomy and its recognition by scientists as a first-rate reference source continues. I am pleased to report that in a recent Science Citation Index Journal Citation Report, Advunces in Agronomy was ranked Number 1 in Agriculture. In addition, we are publishing at least two volumes per year, which means that reviews are published on a timely basis. Volume 54 contains seven excellent reviews that cover some important and contemporary topics in the crop and soil sciences. Chapter 1 is a comprehensive and timely review on the impacts of agricultural practices on subsurface microbial ecology. Chapter 2 also addresses a topic of much interest in the area of the environment, herbicide-resistant crops. Chapter 3 provides a thorough review on acid tolerance of wheat. Chapter 4 addresses microbial reduction of iron, manganese, and other metals, a topic that is of much interest to scientists. Chapter 5 covers nitrification inhibitors with particular emphasis on their impacts on health and the environment. Chapter 6 is a review of production and breeding of lentil, an important crop in many parts of the world. Chapter 7 addresses an important subject in plant improvement and production, use of apomixis in cultivar development. I appreciate the excellent contributions from the authors. DONALD L. SPARKS

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m A C T S OF AGRICULTURAL PRACTICES ON SUBSURFACE MCROBIAL ECOLOGY Eugene L. Madsen Division of Biological Sciences Section of Microbiology Cornell University Ithaca, New York 14853

I. Introduction and Scope Background Definitions 11. Subsurface Microbial Ecology A. Structure of the Habitat B. Function 111. Agricultural Practices and Their Impact on Subsurface Habitats Types of Agricultural Practices IV.Impact of Agricultural Practices on Subsurface Microbial Ecology A. A Historical Perspective for Inquiry into Subsurface versus Surface Habitats B. How Can the Impacts of Agricultural Practices on Subsurface Microbial Ecology Be Measured? C. Measures of the Impact of Agricultural Practices on Subsurface Microorganisms V. Concluding Remarks References

I. INTRODUCTION AND SCOPE Impetus for writing this chapter arises from the convergence of two major concerns of society. The first is an awareness, developing most prominently during the 1980s. that groundwater systems, including subsurface aquifer sediments and vadose zone formations, are precious yet vulnerable resources worthy of both protection and scientific investigation. The second is a realization that agricultural practices may carry with them unforeseen negative consequences such as erosion (Morgan, 1979; Rose, 1985), biomagnification of pesticides (Carson, 1962; Moriarty, 1977), and nitrate pollution of groundwater (Spalding and Exner, 1993). 1 Advonces in Agmnoriq. Vor’ume 14 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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E. L. MADSEN

This chapter’s purpose is to reveal what is and what is not presently understood about how agricultural practices influence microorganisms in the groundwater habitats. Of primary importance to this purpose is recognition that physical manipulations of the Earth’s surface that are practiced by humans in order to produce food (i.e., agricultural practices) are likely to influence subsurface microbial communities as they carry on their normal ecosystem processes beneath the Earth’s surface (i.e., subsurface microbial ecology). Furthermore, the relationship between agricultural practices and subsurface microbial ecology is one of the “impacts.” Implicit in this latter term is the fact that microorganisms are responsive to changes in their surroundings and that the responses may take many forms. As is evident from the Table of Contents for this chapter and Fig. 1, both subsurface microbial ecology (depicted as the land surface in Fig. 1) and agricultural practices (depicted as a bounding sphere in Fig. 1) have their own independent characteristics. Furthermore, the domains of subsurface microbial ecology and agricultural practices are discontinuous in time and space. Nonetheless, as will become evident in the course of this chapter, points of contact between agricultural practices and subsurface microbial ecology (marked by cross-hatched craters in Fig. 1) have the potential to be very significant. To date, however, very little scientific research has directly addressed this important interdisciplinary subject. This chapter develops the scheme shown in Fig. 1 by defining subsurface microbial ecology (emphasizing its unperturbed status and responsive capabilities), then by defining agricultural practices (emphasizing mechanisms for influencing the subsurface habitats beneath), and finally by addressing documented or yet-to-be documented interactions between agricultural practices and subsurface microbial ecology. In order to achieve these goals, several pertinent review articles and/or books are frequently referenced both implicitly and explicitly. In the area of subsurface microbiology, these include Chapelle (1993); Madsen and Ghiorse ( 1 993); Matthess et al. (1992); and Pederson ( 1 993). In the area of agricultural ecology,

Figure 1. Dynamic bounding sphere metaphor for the impacts of agricultural practices on subsurface microbial ecology (see text for explanation).

SUBSURFACE MICROBIAL ECOLOGY

3

these include Briggs and Courtney (1985); Carroll et a/. (1990); Soule er al. (1990); and Tivy (1990). In the area of hydrogeology, these include Davis and Dewiest (1966); Domenico and Schwartz (1990); Freeze and Cherry (1979); and Nachtnebel and Kovar (1991).

BACKGROUND DEFINITIONS The terrestrial subsurface habitat and its synonym, the groundwater habitat, reside directly beneath all continental portions of the globe. To access the terrestrial subsurface, one must excavate or drill through surface materials comprised of soil or rock (in upland areas) or freshwater and sediments (in aquatic areas). It is this vertical stratification of the Earth’s surface and its implicit gradation of exposure to climatic and biological influences that makes surface and subsurface habitats distinctive. In descending from the surface of the earth through soil, one typically encounters materials in the following vertical sequence: the A and B soil horizons; the C soil horizon, from which the other soil horizons may have been derived (Brady, 1990); an unsaturated (or vadose) zone (that begins with the C soil horizon and ends at the water table); and a capillary fringe zone residing directly above a saturated zone which may extend through many different geologic strata (Fig. 2; Madsen and Ghiorse, 1993). Where does the surface habitat end and the subsurface begin? For the purposes of this chapter, the groundwater habitat begins immediately below the B soil horizon where soil scientists traditionally have felt that major biological activity ceases (Alexander, 1977; Brady, 1990; Madsen and Ghiorse, 1993). However, the transition between soil and groundwater habitats is not delineated by soil horizons per se because the demarcation is gradual. But regardless of the type of overlying material (be it soil, rock, or freshwater bodies and sediment), the subsurface occurs where the influences of climate, animals, and plant roots diminish and these are replaced by predominantly hydrological, geochemical, and microbiological influences. Although freshwater habitats represent a relatively small proportion of the continental surface area [<2% of global surface area (Wetzel, 1983) vs 24% of global surface area for forested or cultivated soil (Ehrlich er al., 1977)], free passage of water from the subsurface to lakes and stream and vice versa may have significant hydrological and biological impact. This may be especially true for agricultural lands because these components of the terrestrial habitat are frequently adjacent to fresh waters (Blum e f al., 1993; Fernandezalvarez et al., 1991; Gilbert et a/., 1990; Tremolieres et a/., 1993). In a hydrogeologic sense, groundwater refers to water that is easily extractable from saturated, highly permeable geologic strata known as aquifers (Davis and Dewiest, 1966; Domenico and Schwartz, 1990; Freeze and Cherry, 1979). Be-

4

E. L. MADSEN

cause the water in these high yielding formations are major sources of drinking and irrigation water, aquifers are of principal concern in water management and conservation programs. Most groundwater is interstitial-it resides within a matrix of sediments and/or minerals with variable porosity, chemistry, and degree of saturation. Although water may be readily obtained only from aquifers, the vertical profile in a typical landscape reveals a continuum of water tensions and availabilities. Water may exist in many forms: as part of the crystal matrix of minerals in rocks; in unconnected pores of solid rock; in connected pores in solid rock; in the saturated portions of aquifers; and in unsaturated strata as liquid gaseous and solid phases (the latter only under extremely cold climatic conditions) in capillaries and pores. To microorganisms which, by definition, live in microhabitats, all available forms of water, except those in chemical combination with minerals, may be important. Therefore, this chapter uses a broad definition of groundwater which includes capillary water, water vapor, and water within aquifers (Madsen and Ghiorse, 1993). As a habitat for microorganisms, the subsurface includes the unsaturated zone because it may contain significant amounts of biologically available water. Also, unsaturated zones may be transiently saturated during recharge events and they may influence both the chemistry and microbiology of the saturated zone. In summary, throughout this chapter, groundwater refers to all subsurface water found beneath the soil A and B horizons that is available to sustain and influence microbial life in the terrestrial subsurface (Ghiorse and Wilson, 1988; Madsen and Ghiorse, 1993). Agriculture is derived from Latin “agri cu1tura”-meaning “cultivation of the land.” Agriculture is the science, art, and business of cultivating the soil, producing crops, and raising livestock useful to humankind. As stated by Tivy (1990), the principal resource base for agriculture is the physical environment. The primary process in agriculture is photosynthesis-a process that has not yet been replicated outside living chloroplast-containing cells of green plants. Thus, the cultivated crop plant is the basic “production unit” of agriculture because of its ability to manufacture complex organic compounds from inorganic materials supplied by the atmosphere and soil. The challenge of agriculture is to efficiently manage the physical environment to provide for the biological demands of the crop plant. Tivy (1990) insightfully has written that this linkage between crop and environment is established through the management practices which: (1) select the desired crop to match the prevailing climatic and edaphic conditions; ( 2 ) propagate the crop via tillage practices that favor proper crop germination and growth; and (3) protect the crop from competition with weeds for growth-related resources (light, COz, water, nutrients) and from yield reduction by animal pests and plant pathogens.

SUBSURFACEMICROBIAL ECOLOGY

5

11. SUBSURFACE MICROBIAL ECOLOGY OF THE HABITAT A. STRUCTURE

1. Hydrology and Geology

The terrestrial subsurface is an important component of the landscape through which water passes as it cycles among the atmosphere, soil, lakes, streams, and oceans (Fig. 2, Section LA). Once water has infiltrated below the surface layer of soil, it has several possible fates. It may (i) return to soil via capillary, gaseous, or saturated transport; (ii) be intercepted by plant roots; (iii) reach streams, lakes, or ponds via saturated flow; (iv) reverse its saturated flow direction from streams or lakes back into subsurface strata when levels of surface waters are high; (v) directly reach the ocean via saturated flow; (vi) become mixed with seawater when groundwater withdrawal in coastal areas causes seawater to intrude inland; or (vii) enter a closed deep continental basin (Fig. 2 ; Domenico and Schwartz, 1990). Regardless of the flow path taken through the subsurface, groundwater remains in the biosphere. However, the residence time before water exits the subsurface is highly variable. Return of subsurface water to the soil may occur within a few days or weeks, though return from a deep continental basin may require thousands of years (Madsen and Ghiorse, 1993; Freeze and Cherry, 1979). In conceptualizing the routes taken by water through the terrestrial segment of the hydrologic cycle, Chapelle’s presentation (Chapelle, 1993) of local, intermediate, and regional flow systems is insightful. Chapelle provides the following definitions for these three flow systems based on relationships among surface topography, large-scale geological structures, and the depth of water penetration along its path from recharge to discharge areas: ( 1 ) A local system has its recharge area at topographic high and its discharge area at a topographic low that are located adjacent to each other; (2) an intermediate system occurs when recharge and discharge areas are separated by one or more topographic highs; and (3) in a regional system, the recharge area occupies the regional water divide and the discharge area occurs at the bottom of the basin. Figure 2, which incorporates Chapelle’s flow systems (Chapelle, 1993), illustrates the spatial and functional relationships between the geological setting of the subsurface and its most dynamic component, water. Beneath the soil which, by definition, is the zone of pedogenesis, lie the unsaturated and saturated subsurface zones. This view of the subsurface habitat as being delineated in terms of the degree to which water occupies voids in a porous matrix (if air has been completely displaced by water, the system is “saturated”; if not, the system is “unsaturated”) is satisfying, but it is also simplistic. For superim-

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E. L. MADSEN

Preclpitatlon Evaporation

Recharge area

Infiltration

Stream

Ocean

-

Soil A horizon B horizon

unsaturated zone capillary water

Flow path taken by groundwater

V

topography determined by

-

adjacent eievational

saturated zone

water in unconnected pores

water in chemical combination with minerals

Destination: soil, vegetation and both surface and subsurface water bodies

extremes

m: recharge and discharge

areas are separated by one or more elevational maxima

Path 01 water Is deep beneath other flow systems; flow path connects highest elevation of regional recharge area to lowest discharge point 01 regional basin.

Figure 2. Conceptual Row system for understanding the role of the soil and the subsurface habitat in the hydrologic cycle. (Figure from Madsen and Ghiorse (1993) modified according to flow system categories by Chapelle (1993) and groundwater categories by Domenico and Schwartz (1990)l.

posed upon the degree of water saturation are the geological, geographic, and climatic characteristics. At a given location on the Earth’s surface, the stratigraphy beneath reflects a unique and complex history of geological, hydrological, and chemical events (e.g., sedimentation, erosion, volcanism, tectonic activity, dissolution, precipitation, and biogeochemical activity). The result often is a heterogeneous geologic profile whose complexity may be compounded by variations in pore water chemistry that may stem from localized aberrations in mineral phases

SUBSURFACE MICROBIAL ECOLOGY

7

or inorganic or organic solute concentrations. The large surface area provided by rocks and sediments in the porous matrix may strongly influence the physical and chemical conditions of the groundwater habitat by altering concentrations of dissolved aqueous constituents at the surfaces and by sorbing microbial cells (Madsen and Ghiorse, 1993; van Loosdrecht et al., 1990). Sorption and aqueous equilibrium reactions are most likely to be influential in the saturated zone. But many subsurface habitats are dominated by unsaturated zones as well. In arid climates, the unsaturated zone may be hundreds of meters deep. Rainfall in such desert climates may be insufficient to allow saturated infiltration of soil to reach to the water table, except in restricted low-lying areas (Davis and Dewiest, 1966). Therefore, rather large area portions of deserts may have unsaturated zones beneath them with little or no saturated water flux. Under such circumstances, vapor phase reactions may be the prevalent form of geochemical change. Such conditions have important implications for agricultural irrigation practices as well as both microbial physiology and activity (see Sections II.A.2, II.B, 111, and 1V.C). Freeze and Cherry (1979) have presented the idea of “chemical evolution” of groundwater as it passes from the atmosphere in recharge zones along the variety of flow paths such as those depicted in Fig. 2 and described in Section 1I.A. I. As precipitation, water begins as pure distillate containing only atmospheric gaseous and atmospheric particulate materials. After contact with soil and deeper subsurface sediments, the chemical composition of the water changes Substantially. Not only do components in surface and subsurface matrixes dissolve, volatilize, and precipitate, but, as the water reaches zones that are more remote from the atmosphere, complexation and oxidation/reduction reactions also occur. Many of the reactions are strictly geochemical (Chapelle, 1993; Domenico and Schwartz, 1990; Stumm and Morgan, 1981; Morel and Hering, 1993; Schwarzenbach et al., 19931, but many are also microbiologically mediated (see Sections 1I.B and 1V.C). The chemical composition of a given sample of groundwater reflects the integrated history of chemical and biochemical reactions that occur along a given flow path through soil and geologic strata. Because of the diversity of flow paths and biogeochemical reactions, the composition of groundwater is quite variable. Nonetheless, some generalizations can be made. In aquifers used for drinking water supplies that are not influenced significantly by human activity, major chemical constituents (>5 mg/liter) typically include calcium, magnesium, silica, sodium, bicarbonate, chloride, and sulfate while minor constituents (0.01- 10 mgniter) include iron, potassium, boron, fluoride, and nitrate; with trace amounts (<0.1 mg/ liter) of many inorganics and organics (including humic acids, fulvic acids, carbohydrates, amino acids, tannins, lignins, hydrocarbons, acetate, and propionate (Domenico and Schwartz, 1990). However, as discussed in Sections III and IV, human activities (including septic systems, landfills, other types of waste disposal, and agricultural practices) may alter the chemistry of groundwater substantially by adding high concentrations of solutes such as both toxic and nontoxic organic

8

E. L. MADSEN

carbon compounds and nutrients. Detailed descriptions of geological and geochemical principles and the hydrogeologic properties of the Earth’s crust are beyond the scope of this chapter. For these, readers are referred to Domenico and Schwartz (1990), Freeze and Cherry (1979). Larson and Birkeland (1982), Morel and Hering (1993), Stumm and Morgan (1981), and Strahler (1984).

2. Organisms The subsurface biological community is unique in that it consists primarily of unicellular bacteria, fungi, and protozoa. Though algae may be present under some circumstances (Madsen and Ghiorse, 1993), absence of sunlight severely limits the activity and significance of any photosynthetic microorganisms that may be transported into aquifers from adjacent habitats. Larger organisms (such as fish) occur only rarely in subterranean caves or cavernous aquifers that are connected to the surface by suitably large channels or fissures (Ghiorse and Wilson, 1988; Hynes, 1983; Longley, 1981). Other aquatic fauna, such as amphipods, may inhabit ecotone habitats such as riverbank or lake sediments that are transitional to the subsurface (Danielpool er al., 1991; Gilbert et al., 1990). The accumulating evidence indicates that most of the known physiological types of bacteria that we have come to expect in moderate (as opposed to extreme) marine and freshwater habitats on the surface of Earth are also present in moderate subsurface habitats. Ghiorse and Wilson (1988) and later Madsen and Ghiorse ( 1993) compiled extensive lists of reports examining microorganisms and their potential metabolic activities in subsurface habitats. A variety of experimental techniques performed on samples from many sites have revealed wide-ranging metabolic capabilities of both aerobic and anaerabic microorganisms. KolbelBoelke et al. (1988) listed physiological properties of 2700 aerobic heterotrophic bacteria isolated from a Pleistocene sand aquifer in Northern Germany. Also, Balkwill ( 1 990) tabulated 27 metabolic groups or physiological types of aerobic and anaerobic bacteria isolated from a single deep aquifer drilling site in South Carolina. Similarly,Haldeman et al. ( 1 993) isolated and characterized 2 10 aerobic bacteria from freshly exposed rock faces in a vadose zone tunnel system 400 m beneath the Nevada test site. Additionally, Kampfer er al. (1993) isolated and characterized 3446 aerobic and anaerobic bacteria from a shallow contaminated aquifer where a large-scale bioremediation project was underway. To further illustrate the breadth of metabolic diversity found among subsurface microorganisms in shallow and deep aquifer systems, results of recent studies (published since 1991) in which the physiological types or activities of subsurface microorganisms were determined have been summarized (Table I). In evaluating the meaning of these results, it is important to recognize the distinction between metabolic potential, determined by incubating field samples in the laboratory, and in siru microbial activity. This distinction between what can be measured in laboratory-incubated

SUBSURFACEMICROBIAL ECOLOGY

9

experimental samples and the actual expression of metabolic activity in field sites has been extensively discussed by Madsen et al. (1991), Madsen (1991), Madsen and Ghiorse ( 1993), the National Research Council ( 1993), and Madsen ( 1995). Despite this caveat, it is clear from Table I that the metabolic diversity of subsurface microbial communities, like those of all other moderate habitats in the biosphere, is substantial. This diversity has major implications for all aspects of groundwater microbiology, especially for the responses of subsurface microorganisms to agriculture-induced change (see Sections 111 and IV). Surface soils in temperate regions typically contain lo* to lo9 bacteria per gram. The major chemical and physical influences which govern bacterial abundances in soil [available organic carbon, nitrogen, phosphorus, sulfur, moisture, pH, electron acceptors, grazing by predators, immigration of microorganisms from other habitats, etc. (Alexander, 1977)] are modified in the subsurface along the hydrologic flow paths. The nature of the geologic stratum (mineral type, particle size distribution, texture, hydraulic conductivity, etc.) also may determine the abundance and distribution of bacteria in a given subsurface zone. Madsen and Ghiorse (1993) have presented a generalized scheme for the vertical distribution and potential metabolic activity of subsurface microorganisms. In descending from the A to B soil horizons into the C horizon, a decline in nutrient levels is accompanied by a drastic decline in bacterial abundance. Indeed, many reports in the older soil microbiology literature suggested that few, if any bacteria, existed in the C horizon. The C soil horizon often marks the beginning of the unsaturated subsurface zone, which supports far fewer bacteria than the B soil horizon. However, the numbers of bacteria usually do not continue to diminish with depth. Instead, microbial abundance typically increases substantially at the water table and just above it in the capillary water zone (Fig. 2 ) . It is possible that these interface zones between the unsaturated and saturated zones may be the site of relatively dynamic mixing of oxygen and recently recharged nutrients in shallow unconfined aquifers. As one continues deeper through the water table into the saturated zone, the abundance and potential activities of microorganisms generally remain high relative to the unsaturated zone. In a given locale, both the moisture regime and the type of geologic strata below the water table may be highly varied (e.g., sediments high in clay of low transmissivity, beds of crystalline or porous rock with varying degrees of fracturing, and zones of highly transmissive sand and gravel may be present). Highly transmissive saturated zones (high-yield aquifers containing water that was recharged relatively recently) typically show microbial abundances and metabolic activity potentials that are two to four orders of magnitude greater than those of hydrologically nontransmissive zones, whose waters and nutrients may be relatively old and depleted. Thus, depth per se does not govern the abundance and activity of bacteria in the saturated zone; rather, the hydrological, physical, and geochemical properties of each stratum appear to govern the population density and degree of metabolic activity of its own community.

-

Table I Summary of Microbiological Groups Detected in Subsurface Habitats"

0

Microbiological group

Aerobic cbemoheterotrophic bacteria

Sample type

Method of detection

Field site underlain by glacio-fluvial sand

Injection of herbicides, MCPP, and atrazine in siru into groundwater; microcosms amended with herbicides Microscopy, viable counts on agar media, '*CO, production from radiolabeled acetate and phenol; [ 'Hlthymidine incorporation into cells Viable counts on agar media; characterization of 47 isolated bacteria using measures such as metal resistance, phospholipid fatty acid profiles, and carbon source utilization [ 'JC]Glucose and toluene mineralization; 14C-labeledamino acid incorporation; MPN, and direct microscopic counts Six bacteria were stored in media for 100 days; cellular and physiological changes were monitored Random selection and isolation of 63 bacteria; these were challenged to grow under various exposures to UV radiation and hydrogen peroxide I4CO, production from '"C-labeled chelating agents (DTPA, EDTA, NTA) Enrichment and isolation of 25 methanotrophic bacterial strains; phospholipid fatty acid analyses; GC analysis of culture fluids for TCE and PCE; T O z production from [ '"C-ITCE; DNA hybridization assays

4 to 3 1-m depths of sediments

One water sample and three vadose zone deep rock samples from the Nevada test site

Well water from shallow aquifer at hazardous waste site

Deep rock vadose samples from the Nevada test site Also, UV radiation and hydrogen peroxide-resistant bacteria

Methanotrophs capable of cometabolizing chlorinated alphatic hydrocarbons

Deep coastal plain acquifer sediments from depths of 150-500 m

Deep coastal plain acquifer sedimenls from 0- to 376-m depths Trichloroethylene- and perchloroethylenecontaminated well water from the Atlantic coastal plain

Reference Agertved er uI. ( 1992)

Albrechtson and Winding (1992)

Amy ef a/. (1992)

Armstrong

el a/. ( 1991 )

Amy et al. ( 1993)

Bolton et al. 1993)

Bolton e t a / . 1993) Bowman et a/. (1 993)

Also, actinomycetes

Vadose zone paleosols at depths ranging from 53.6 to 63.7 m; also vertical cliff faces Carbonate sands beneath a pentachlorophenol (PCP) wood-treating facility Deep Atlantic coastal plain acquifer

Deep Atlantic coastal plain acquifer

Fresh rock faces in a deep subsurface vadose zone tunnel system, 400 m in depth (Nevada)

Sediments and ground waters to depth of 550 m in southeastern Atlantic coastal plain Sand and gravel acquifer sediments and water from I to 2-m depth

Also, TCE-cometabolizing phenol degraders

Shallow confined sandy acquifer 5-m depth interval (central California)

Microscopy/respiratory activity stain; viable counts on agar media; ATP concentration; mineralization of [ “C-Iglucose; ecological diversity indices G U M S determination of PCP and metabolites in field samples, determination of sediment/PCP sorption coefficients Microscopy; growth on various compounds; “CO2evolution from toluene; naphthalene; hybridization with pWW0 and NAH7 plasmids Agar media, 108 physiological tests applied to 198 isolated bacteria Microscopy, agar media, isolation and testing of 210 bacterial strains; growth on agar media; analysis of fatty acid methyl esters; microbial diversity analyses; Euclidean distance cluster analyses Microscopy; growth on agar media; screening of isolated bacteria for physiological capabilities Microscopy; ATP analysis; laboratory and field ( i n situ) incubation of sediment and water contained within a steel cylinder and amended with ’ H z Oand 23 organic contaminants; fluids were periodically removed from vessels and analyzed to demonstrate contaminant losses Injection and circulation of phenol into aerobic groundwater at a field site stimulated metabolism of TCE

Brockman et crl. ( I 992)

Davis et crl. ( I994a)

Fredrickson el al. (1991a)

Fredrickson er al. ( I991 b) Haldeman et a/. ( 1993)

Hazen et ui. ( I99 I )

Holm et al. ( 1992)

Hopkins et al. ( I 993a)

(continues)

e

Table I-Continued Microbiological group Also. TCE-cometabolizing phenol degraders

Sample type

Method of detection

Shallow confined sandy aquifer 5-m depth interval (central California)

Field and laboratory metabolism trials demonstrated that phenol stimulates high efficiency cometabolism of chlorinated ethenes by indigenous subsurface microorganisms Microscopy; growth on agar media, production of “CO, from [ ‘‘C]glucose and -acetate; water potential measurements performed on field samples and on growing cultures Isolation of bacterium able to grow on methylanilines and aniline; oxygen uptake by cells in the presence of organic compounds, enzyme assays; GC determination of anilines in culture media ‘“COzevolution and “ C incorporation into biomass from [ “Clglucose, -phenol, and -aniline; microscopy, agar media; 32Pincorporation into phospholipid Isolation of two bacterial strains; examined influence of nutrient status on distribution of bacteria between solids and solution using a continuous flow system I4CO2production from [ I4C]p-hydroxybenzoate, -naphthalene, and phenanthrene; enumeration of microorganisms, including protozoan predators, inside and outside a plume of contaminated ground water

Deep vadose zone (2- to 450-m depths) in the intermountain zone of the western United States

Shallow unsaturated zone sediments to 5-m depth

Shallow unsaturated zone

7- to 8-m depth saturated zone in sedimentary strata

Shallow unconfined sandy acquifer at depths ranging from 1 to 6 m

Reference Hopkins et al. (1993b)

Kieft el a/.( 1993)

Konopka ( 1993)

Konopka and Turco (1991)

Lindqvist and Bengtsson (1991)

Madsen et a/.( 1991 )

Two different sand and gravel aquifers contaminated with coal tar waste

Microscopy: growth on agar media: enumeration of bacteria, fungi, actinomycetes; ‘ T O 2production from 1 “ C l p hydroxybenzoate, -naphtahlene, and -phenanthrene

Madsen ef a / . ( 1992)

Propane oxidizers capable of cometabolizing chlorinated aliphatic hydrocarbons

Four strains isolated from TCEcontaminated Atlantic coastal plain sediments

Aerobic incubations fed with propane: “CO, production from [ “CITCE: scintillation counting: resting cell assays; GC analyses of a variety of aromatic and chlorinated aliphatic compounds

Malachowsky et (11. (1994)

Also, pesticide-metabolizing

Glacial outwash aquifer 5.2- to 18.8-m depths

“ C 0 2 production from I“C]glucose and [ ‘AC]ethylatrazine

McMahon et a/.(1992)

microorganisms Methanotrophs capable of cometabolizing chlorinated aliphatic hydrocarbons

Unconfined fine sand aquifer 15-32 m in depth (Minnesota)

Column microcosms amended with 0,. methane, and chlorinated ethenes; subsequent measurements of all amendments eluting from columns; computer modeling of site hydrology: simulation modeling of methanotrophic bioremediation of site Shake flask assays demonstrating biodegradation potential of creosote compounds; toxicity assays Growth on heterotrophic media: bacterial sorption and transport through continuous flow columns

McCarty et 01. ( 1 99 1)

Injection of methane and chlorinated ethenes into highly instrumented field site; periodic field measurements of the chlorinated ethenes, metabolites, oxygen, and methane

Semprini and McCarty (1991)

Well water from 7-m depth at a pentachlorophenol- and creosote-contaminated field site Well water I 1 .8 and 20 m in depth

Methanotrophs capable of cometabolizing chlorinated aliphatic hydrocarbons

Shallow confined sandy aquifer 5-m depth interval (central California)

Mueller et a / . ( 1991 )

Scholl and Harvey ( 1992)

(continues)

-* Table

I-Conrinued Microbiological group

Sample type

Method of detection

Methanotrophs capable of cometabolizing chlorinated aliphatic hydrocarbons

Shallow confined sandy aquifer 5-m depth interval (central California)

Also, protozoa

Sandy sediments in field site contaminated with aviation fuel (0-5 m depth)

Details similar to Semprini (1991a). but methane was found to competitively inhibit the metabolism of chlorinated ethenes Viable enrichment counts of predatory protozoa; viable bacterial counts on agar media, determination of 0 z, CO 2 , and fuel concentrations in sediments Microscopy; fused glass columns of varying pore geometries and gas saturations; cell transpon and breakthrough curves in flow-through systems Continuous cultures of bacteria able to metabolize toluene and chlorobenzoates; DNA extraction, chlorobenzoate gene amplification by PCR Anaerobic laboratory incubations of sediment amended with nitrate, glucose, and aromatic hydrocarbons; in situ incubation of chambers filled with aquifer sediments and amended with acetate, lactate, or yeast extract; and nitrate or sulfate; and toluene, ethylbenzene, xylenes, cumene, and trimethylbenzene “CO, production from 1 I4C]benzenein laboratory incubated aquifer slurries; assays for benzene sorption onto sediments; computer simulations of benzene transport at the field site; analysis of field samples for inorganic nutrients

Two bacterial strains from Atlantic coastal plain sediments (203- and 324-m depths). One additional strain from a shallow groundwater aquifer Wellwater receiving groundwater leachate

Aerobic and anaerobic chemoheterotrophic bacteria Also, methanogens and nitrate reducers

Fine-sand aquifer sediments from 4.6 to 6. I m below the water table; well water from 5.0-m depth

Sand and gravel aquifer to depths of 30 m

Reference Semprini and McCarty (1992)

Sinclair et al. (1993)

Wan er ( I / . ( 1994)

Wyndham

YI

nl. (1994)

Acton and Barker ( 1992)

Davis ef a/. (1994b)

Also, methanogens, denitrifiers

Quartz sand aquifer sediments from 6.1 -m depth

MPN enumeration; microscopy; GClMS analyses and ratios of metabolizable to nonmetabolizable chemicals; and laboratory incubation of slurries demonstrate microbial metabolism of creosote

Godsy et al. (1992)

Also, actinomycetes, fungi, dentrifiers

Seven rock samples 50-450 m deep beneath the Nevada test site, western United States

Haldeman and Amy (1993)

Nitrate reducers

2- to 6-m depth of glacial aquifer

Also, methylotrophs, fermenters, denitrifiers, sulfate reducers, and hydrocarbon degraders

Mixed textured sediments from 2- to 10-m depth adjacent to river

Microscopy, extraction of microbial phospholipids, enrichments for nitrifying, sulfur oxidizing, sulfur reducing microorganisms; diversity indices Bioremediation field demonstration in which water and nitrates and other nutrients were ciculated as declining concentrations of toluene, xylenes, and ethylbenzene in water and sediment cores were measured Isolation of 3446 bacterial strains; growth on agar media: protein determination; fluoresceine diacetate hydrolysis; diversity indices; assays performed on field plots and samples from large scale bioremediation project Measurements of adenylate energy charge, ATF', and bacterial DNA in field and laboratory-incubated samples of groundwater, and on laboratoryincubated cultures '"CO, production from [ ''C]glucose and pyridine

Well water from anaerobic portions of a sandy shallow sewage-impacted Cape Cod aquifer

Also, methanogens and sulfate reducers

Deep coastal plain aquifer sediments from 0.1- to 526-111depths

Hutchins er al. ( I 99 1 )

Kampfer et al. ( I 993)

Metge er al. ( I 993)

Shankar er al. ( 1991 )

(continues)

Table I-Continued Microbiological group

Also, chemoheterotrophs

Other anaerobic microorganisms denitrifiers

Sample type

Sandy and peat subsoils 0.3-4 m in depth

Mixed sand and clay sediments I .5 to 8.2 m in depth

Iron-reducing bacteria

Atlantic coastal plain sediments, sampled from depths of 25 to 200 m

Acetogens

Deep coastal plain acquifer sediments from depths of 0. I lo 526 m

Method of detection

Reference

Microscopy; concentration of cells in sand traps; enrichment on 20 different combinations of electron donors and acceptors

Stevens e f al. (1993)

' T O 2production from [ I4C]acetateand -glucose; fluorescence microscopy; addition of chloride, pentachlorophenol, Cd, Zn, and acid of varying doses to inhibit mineralization activity Anaerobic laboratory incubations of sediment slurries amended with acetylene to allow N,O production to indicate denitrification; gas chromatographic determinations of H2 and C 0 2 ;ion chromatographic determination of nitrate and nitrite Analysis of waters and sediments for organic acids, iron, and sulfur species and other inorganic species; measurements of isotopic ratios of inorganic "C and "C; enrichment of Fe- and SO,=-reducing bacteria; conversion of [ IiC]acetate to 1 4 C 0 , Depletion of model lignin compounds from anaerobic aquifer slurries and from culture media inoculated with an isolated acetogenic bacterium; chemical analysis of the growth media

VanBeelen et al. ( 199I )

Bradley et al. ( 1992)

Chapelle and Lovley ( 1992)

Liu and Suflita (1993)

Anaerobic autotrophs

Water pumped from bedrock at 830- to 1078-m depths

Native bacteria colonizing glass slides in flow-through system; metabolic potential assessed using scintillation counting and autoradiography to assess assimilation of ‘ T O ? , ‘“C]formate. [ HIacetate, I C 1lactate, [ ‘“C1glucose, [ 3H]leucine

Pederson and Ekendahl ( 1992)

Methanogens that utilize aromatic hydrocarbons

Aquifer sediment from 6-m depth

Edwards and Grbic-Galic ( 1994)

Sulfate reducers that utilize aromatic hydrocarbons

Sandy silt 0.25 m above water table

Anaerobic incubation of slurries amended with rn-xylene, p-xylene, benzene, ethylbenzene, naphthalene, o-xylene, toluene; chemical analyses demonstrating microbial metabolism; electron microscopy Anaerobic laboratory incubation of slurries amended with benzene, toluene, and xylenes; loss of xylenes and toluene occurred only in the presence of sulfate; ‘“CO, production [ “Cltoluene and o-xylene

Perchloroethylene (PCE)-dechlorinating anaerobes

Well water and mixed-textured sediments from a 12-m deep aquifer contaminated with PCE

Denitrifiers, sulfate reducers, and carbon tetrachloride degraders

Shallow confined sandy aquifer 5-m depth interval (central California)

Field mapping of concentration contours for PCE and metabolites at industrial site; anaerobic laboratory incubations of ground water samples periodically monitored for loss of PCE and production of metabolites; phospholid fatty acid analyses of sediment samples Injection of acetate into field site groundwater to foster conditions for in siru carbon tetrachloride metabolism

Edwards erul. (1991)

Major el ul. ( I99 I )

Semprini et ul. ( 1992)

(continues)

Table I-Continued Microbiological group

Sample type

Method of detection

Reference

~ ~ ~ i hsulfate f i reducers, ~ ~ ~ and , carbon tetrachloride degraders

Shallow confined sandy aquifer 5-m depth interval (central California)

Under controlled hydrologic field conditions, inject acetate, nitrate, and carbon tetrachloride into an aquifer; monitor concentrations of these and other compounds; conclude in siru metabolism of carbon tetrachloride is not directly linked to denitrification

Semprini el al. ( 199Ib)

Denitrifiers

Sandy sediments to a depth of 15 m

Geochemical analyses of field samples; field and laboratory incubations of microcosms amended with acetylene to block completion of denitrification reactions; loss of NO3 and production of N Z Ometabolite in laboratory and in situ; stimulation of denitrification by glucose addition Placement of silicate minerals in situ in anaerobic wells; chemical analyses and electron microscopy

Starr and Gillham (1993)

~~~

Other metabolicallyactive microorganisms Silica-dissolving microorganisms

Wells, 8 m deep

‘This compilation updates that of Madsen and Ghiorse (1993), showing articles published since 1991

Hiebert and Bennett (1992)

SUBSURFACE MICROBIAL ECOLOGY

19

Four major trends in subsurface microbiological investigations have developed since 1991 (Table 1). These are: 1. New knowledge of deep vadose-zone microbiology. Sponsored largely by the U.S. Department of Energy Subsurface Science Program, a broad group of investigators (e.g., Brockman et al., 1992; Fredrickson et al., 1991a,b;Haldeman and Amy, 1993; Haldeman et al., 1993; Kieft e l al., 1993) have documented the presence, abundance, ecological diversity and potential aerobic and anaerobic metabolic activities of microorganisms in rock, sediment, and paleosols beneath the western arid intermountain zone of the United States. In many deep subsurface samples, desiccation-tolerant viable bacteria were detected (Kieft et al., 1993). But another major finding of Kieft et al. (1993) was that many rock samples contained microscopically visible cells capable of respiring [ ''C]glucose and -acetate; yet, viable counts on agar media were below detection. This supports the concept that subsurface habitats may support viable but nonculturable microorganisms (see Section II.B.2). In subsurface desert samples from the northwestern United States, Kieft et al. (1 995) have also recently discovered unsaturated sediments with high numbers of bacteria and saturated zones with low numbers. This trend seems to be contrary to that described above, but probably merely reflects the fact that in desert areas the depositional history of geologic strata may be more influential on microbial numbers than site hydrology. 2. Documentation of the microorganisms and/or their in situ biodegradation activity at bioremediation field sites that have been intentionally or inadvertently contaminated with organic chemicals. These types of investigations (e.g., Acton and Barker, 1992; Agertved et al., 1992; Davis et al., 1994a,b; Dobbins et al., 1992; Godsy et al., 1992; Holm et al., 1992; Hopkins et al., 1993a; Hutchins et al., 1991; Kampfer et al., 1993; Madsen et al., 1991, 1992; Semprini et al., 1991a,b) have often had objectives of exploiting the metabolic capabilities of subsurface microorganisms as a means to cleaning up contaminated groundwaters. 3 . New knowledge of microbial communities inhabiting saturated deep granitic environments.Sponsored largely by the Swedish Natural Science Research Council and the Swedish Nuclear Fuel and Waste Management Co., participating researchers [summarized by Pederson ( I993)] have characterizedphysiological and other properties of microorganismsin crystalline rock and water in the Stripa mine nuclear waste storage area. 4. The response of microorganisms in shallow aquifers to exposure to agricultural chemicals. This research has been sponsored by a variety of both European and North American agencies. Reports (e.g., Agertved et al., 1992; Dippel et al., 1991; Konopka and Turco, 1991; McMahon et al., 1992; Sinclair and Lee, 1990) often demonstrate the physiological potential for subsurface microorganisms to metabolize pesticides.

Both actinomycetes (gram-positive, filamentousbacteria) and fungi (predominantly filamentous eukaryotes) are spore-forming microorganisms that are well

20

E. L. MADSEN

adapted to environments subjected to wide variations in water availability and low nutrient conditions (Alexander, 1977; Brock et al., 1994; Henis, 1987). Despite their metabolic versatility, actinomycetes and fungi have been reported in minor abundances in the majority of subsurface microbiological studies reported to date. Approximately 4% of 1200 bacteria isolated from Atlantic coastal plain sediments were morphologically similar to actinomycetes; these were restricted largely to transmissive, sandy strata (Balkwill, 1989). In examining a shallow sandy aquifer system in the northeastern United States, Madsen et al. (1991) found that actinomycetes and fungi were restricted to the surface soil and unsaturated zone. Other investigations examining a range of microbiological types in subsurface sediments from both humid (Sinclair et al., 1990) and arid (Colwell, 1989) climates have specifically searched for actinomycetes and failed to find them. Low numbers of fungi (<50 CFU/g) were detected in many subsurface samples from depths down to 216 m in the Atlantic coastal plain system (Sinclair and Ghiorse, 1989). Similarly, in another sandy aquifer, Federle et al. (1990) identified characteristic fungal phospholipid fatty acids whose abundance varied in both unsaturated and saturated zones of the subsurface profiles to a depth of 19 m. Numerous microbiological investigations completed beneath the arid intermountain zone of the western United States (e.g., Amy et al., 1992; Haldeman er al., 1993; Kieft et al., 1993) have concluded that fungi are rare in arid-zone subsurface environments. One slight departure from this complete absence of fungi in deep vadose zone samples is the report from Haldeman and Amy (1993) which detected two different fungal colonies on agar plates from one of seven rock samples beneath the Nevada test site. Exceptions to the rule of actinomycetes being absent from the subsurface also have been reported by Brockman et d. ( 1992) stated that the microflora in one sample from a freshly exposed desiccated cliff face was dominated by actinomycetes. This finding contrasted with the dominant nonspore-forming gram-negative microflora that predominated in a comparable cliff face that was subject to periodic moisture recharge events (Brockman et al., 1992). In another notable recent report, Haldeman and Amy (1993) found that the microflora in two Nevada test site subsurface samples was completely dominated (93 and 92%) by actinomycetes. These results suggest that only seldom do the key traits of actinomycetes (such as filamentous morphology, tolerance to desiccation, spore formation, and varied heterotrophic metabolism) confer a selective advantage which leads to numerical dominance of the subsurface microflora. However, it should be recognized that both fungal and actinomycete metabolism would be included in the radiorespirometric metabolic potential assays listed in Table I. Most metabolic potential studies do not attempt to identify the contribution of any particular group within the community. Fungi and actinomycetes could be very active in aerobic heterotrophic metabolism within the larger subsurface microbial community. Thus, the contributions of these organisms to potential aerobic metabolic activity may be significant, even when they are initially present at low numbers.

SUBSURFACE MICROBIAL. ECOLOGY

21

Protozoa are unicellular eukaryotic microorganisms with diverse morphological and physiological characteristics(Fenchel, 1987; Finlay, 1990; Foissner, 1987; Sleigh, 1989; Stanier et al., 1986). Many protozoa are able to survive nutrient starvation and other environmental stresses by producing cysts. Photosynthetic and parasitic protozoa are unlikely to have any significance except as transients in the subsurface habitat; however, the importance of the remaining types of freeliving phagotrophic protozoa should not be underestimated. Ingestion of bacterial prey is the dominant mode of nutrition for free-living protozoa in aquatic and terrestrial habitats (Fenchel, 1987), and it is in this predatory capacity that protozoa may accelerate the cycling of carbon and other nutrients (Fenchel, 1987; Stout, 1980). Because protozoan biomass usually reflects the rate at which they are able to graze on their bacterial prey (Fenchel, 1987; Foissner, 1987; Stout, 1980), the population density of protozoa may serve as an indirect measure of in situ bacterial growth rate. To the degree that bacterial growth rate can be tied to specific types of metabolism (i.e., denitrification, methane oxidation, biodegradation of pesticides or organic pollutants), protozoan biomass becomes an indirect means of determining in situ microbial processes (Madsen et al., 1991). In this regard, the presence of protozoa in the subsurface may be very important in documenting the expression of metabolic potential in groundwater habitats. Early studies of the abundance and distribution of protozoa in deep soil horizons (Sandon, 1927; Waksman, 1916) indicated that, like bacterial numbers, protozoan numbers decline drasticallywith depth in horizons beneath the soil surface. These observationswere confirmed by more recent studies of Sinclair and Ghiorse (l987), Beloin et al. (1988), and Sinclair et al. (1990) which showed that in shallow aquifer systems in the south central United States, the abundance of cystforming protozoa declined to below the detection limit (0.2 protozoa/g) in the unsaturated zone. However, protozoa reappeared in samples from the capillary water zone. Cores from 7-8 m deep in the aquifer contained 50 to 100 protozoan cysts/g of sediment. Protozoa also were detected in other samples from various subsurface depths. Numbers were lowest in clay-confining layers and highest in the coarse-textured sands and gravels in which bacterial abundance also was elevated (Beloin et al., 1988; Sinclair and Ghiorse, 1987; Sinclair et al., 1990). Sinclair and Ghiorse (1987) noted that nearly all of the protozoa in their samples were in the cyst form instead of the vegetative form. The dominant populations of subsurface protozoa were flagellates and amoebae. Ciliates were never found in sediment samples from depths greater than 0.5 m. Data on protozoa in subsurface samples in the south central United States were supported by studies completed in other humid locations. Working in New York state, Madsen et al. (1991) found as many as 19,000 protozoa per gram in water table samples at a sandy forested coal tar-contaminated site where indigenous subsurface bacteria were actively metabolizing polycyclic aromatic hydrocarbons in the groundwater. Also, Sinclair and Ghiorse (1989) examined 47 different deep subsurface samples from the Atlantic coastal plain and found that protozoa abundance was positively correlated with

22

E. L. MADSEN

the coarseness of sediment texture. At a Michigan study site, Sinclair et al. (1993) reported development of dense protozoa populations (as many as 6.5 X los per gram of sediment) in a shallow aquifer contaminated with aviation fuel. The abundance of protozoa was considered high enough to significantly affect the density of bacteria (Sinclair et al., 1993). All of these investigations enumerated protozoa with a most-probable number (MPN) technique that relies on predation of bacterial prey. Additional evidence for protozoa in the subsurface has been obtained by analyzing phospholipid fatty acids extracted directly from sediments. Federle et al. (1986, 1990) found fatty acids characteristic of protozoa in subsurface-saturated zones in Alabama and Wisconsin. Madsen and Ghiorse (1993) concluded their discussion of subsurface protozoa by stating that three common themes have emerged in the protozoology of subsurface sediments in humid climates: (i) a modest population density of 1 to 100 cyst-forming protozoa/g may occur in shallow pristine aquifer sediments and in some deep aquifer sediments containing bacterial population densities of I Oh107/g;(ii) the abundance of protozoa is related to sediment texture-high numbers of protozoa are restricted to sandy transmissive zones containing abundant bacteria; and (iii) cyst-forming flagellates and amoebae appear to be the principal types of protozoa in subsurface ecosystems. In arid climates or subsurface systems where crystalline bedrock is dominant, the role of protozoa may be substantially diminished compared to sediments in humid climates. None of the recent studies examining deep vadose-zone (e.g., Haldeman et al., 1993; Kieft et al., 1993) and granitic (e.g., Pedersen, 1993) environments have reported that protozoans have been prominent members of the subsurface microbial community.

B. FUNCTION 1. Responsiveness of Microorganisms to Environmental Changes The success of microorganisms, particularly bacteria, in colonizing all available habitats on Earth (including those in the most extreme environments) and their ability to carry out key biogeochemical processes attests to the ability of extant microorganisms to adapt and respond to environmental change. Micropaleontological evidence indicates that bacteria-like cells were the earliest life forms on Earth (see Brock et al., 1994). The propensity for responsiveness to environmental change is highly evolved among microorganisms. Unlike multicellular organisms, the five major groups of microorganisms [bacteria, fungi, protozoa, algae, and viruses; which by definition comprise all microscopic unicellular or colonial life forms (Brock et al., 1994)] have evolved few, if any, cellular structures for ensuring homeostatic living conditions. Instead, microorganisms have

SUBSURFACE MICROBIAL ECOLOGY

23

developed mechanisms for coping, as individual cells or colonies, with adversities found in often widely fluctuating environmental conditions (temperature, pH, radiation, water potential, carbon and energy sources, toxins, oxygen concentration, concentrations of other final electron acceptors, etc.) that occur in the virtually unlimited variety of microhabitats that comprise the biosphere. Given the innate ability of microorganisms, in general, to adapt and respond to environmental change, one would expect subsurface microorganisms to exhibit the same responsiveness when challenged by environmental perturbations stemming from agricultural practices. Because bacteria dominate in most subsurface habitats (see Section 11.A.2), this section focuses primarily on bacterial responses to environmental change. Table 11 provides an overview of the types of responses to environmental change that have been studied in bacteria. To provide a broad perspective, the information in Table I1 is presented according to traditions of hierarchical organization in both ecology [from cell to biosphere (Allen and Hoekstra, 1992; Hobbie and Ford, 1993)] and microbiology [from cell to gene (Brock et al., 1994)]. As is characteristic of multivariable and complex open systems (such as the biosphere, biomes, landscapes, and ecosystems), simple cause-and-effect linkages between particular stimuli and subsequent responses are difficult to decipher (Table 111). Indeed, the complex microscale interactions between biotic and abiotic processes and intricate food webs that make up these large-scale systems greatly inhibit our ability to understand and predict how the microbial components of the systems will behave when the larger scale system is modified intentionally or unintentionally by human activities (Hobbie and Ford, 1993). For example, the burning of fossil fuels, the release of chlorofluorocarbons, and the practice of modern day agriculture all have had effects at the global scale. However, the responsiveness of microbiological components of ecosystems has yet to be fully investigated upward from microscale to global scale. Perusal of pertinent recent literature (e.g., Groffman, 1993) leaves an impression that the large-scale factors controlling microbial processes at the microscale are largely unknown. At the community level of organization (Table 11), prospects for understanding bacterial responses to environmental change are somewhat more tractable. While factors controlling ecosystem, landscape, biome, and the biosphere microbial processes are vague and ill defined, community-level processes may be understood by analogy with pure cultures. After all, microbiologists routinely grow, measure, and manipulate large populations of microbial cells in pure culture. Populations are the basic units of bacterial communities. But by no means are the responses of microbial communities in open, natural systems simple to assess. In most natural environments (including subsurface environments), individual or groups of microorganisms share their microhabitats with one another. Under these mixed population conditions, a wide variety of interactions between populations can occur. These interactions vary over space and time with changes in habitat charac-

24

E. L. MADSEN Table I1 Responses of Bacteria to Environmental Changes

Ecological level of response

Change eliciting response (examples)

Biosphere, biome, landscape, ecosystem"

Global warming; increasing atmospheric concentrations of chloro-fluorocarbons, and carbon dioxide; deforestation, wild fires, radiation Changes in moisture status and oxidation reduction potential; starvation; addition of carbon compounds, nutrients, pesticides, heavy metals, etc. Changes in moisture status and oxidation reduction potential; starvation; addition of carbon compounds, nutrients, pesticides, heavy metals, etc. Heat or cold

Community

Population

Cell

Heat or cold Starvation Nutritional status

Nutrient-dependent growth rate

Decline in nutritional status

Type of response Microbiological response to these global change issues are unresolved. These changes are likely to disrupt biogeochemical cycling of nutrients through a variety of complex mechanismsb Selective growth and/or survival of populations capable of exploiting new resources and/or resisting adverse effects of changes.' Selective growth and/or survival of advantaged members of each population."

Alteration in cellular protein distributions.e Alteration in fatty acid composition of cellular phospholipidsf Bacillus cells convert from vegetative to sporulated form.' Adenylate energy charge: proportion of adenine nucleotides bearing high energy phosphate bonds. This ratio (approximately 0.8 for rapidly growing cells and 0.5 for dead cells) provides an index for metabolic state and regulates many enzyme activities.e Alteration in cell size and macromolecular composition (RNA, protein, DNA, cell mass) as governed by cell diameter.' Shift-down response: RNA synthesis and cell enlargement immediately cease while DNA and cell numbers increase at preshift levels until new growth ratedependent composition is reached; then all synthetic-, cell size- and cell division-related processes resume.'

SUBSURFACEMICROBIAL ECOLOGY

25

Table 11-Continued Ecological level of response

Change eliciting response (examples) Improvement in nutritional status

Protein

Heat (also extreme pH and toxins)

Nutrient-dependent growth rate

Phosphate limitation

Availability of preferred carbon growth substrate

Deprivation of essential growth factor (e.g., amino acid, carbon source, nitrogen source) or addition of toxic agent which may interfere with energy transduction and/or amino acid synthesis Ultraviolet light and other DNA damage

Gene

Heat (also extreme pH and toxins)

Type of response Shift-up response: rate of RNA synthesis (especially tRNA and rRNA) increases, cell size increases; protein and DNA systhesis lag behind cell size; therefore, proportion of DNA and protein per cell diminishes.' Heat shock response: translation of sigma factor protein allows binding of RNA polymerase holo-enzyme to DNA promoter. This confers specificity on RNA polymerase so that only heat-induced genes are translated and transcribed into protein.' Growth rate regulates the synthesis of 52 distinct proteins required in ribosome structure and function. Phosphate starvation induces elevated production of approximately 100 proteins.' Catabolite repression: inhibition of synthesis of enzymes required for metabolism of nonpreferred carbon and growth substrates.' Stringent response: total inhibition of ribosome and tRNA synthesis so that cell can conserve resources and redirect them toward adaptation to new growth conditions.'

SOS response: DNA damage activates the RecA protein which facilitates proteolytic cleavage of the LexA protein which represses expression of approximately 20 genes: when LexA is elminated, these genes are expressed.' Heat shock response: htpR gene (in E. coli) encodes for u protein factor which favors transcription of over 80 heat-induced genes.' (continues)

26

E. L. MADSEN

Table 11-Continued Ecological level of response Gene (continued)

Change eliciting response (examples) Availability of preferred carbon growth substrate

Starvation

Deprivation of essential growth factors or addition of toxic agents

Phosphate limitation

Ultraviolet light and other DNA damage

Type of response a encodes Catabolite repression: c ~ gene adenylate cyclase, the cyclic adenosine monophosphate (CAMP)-regulating enzyme, in response to membrane sensed concentrations of the preferred substrate (glucose). crp gene encodes catabolite gene activator protein which, in combination with cyclic adenosine monophosphate, binds to promoters; this initiates transcription and translation of genes which allow growth on nonpreferred carbon compounds.'' SpoOa and SpoOF genes (in Bacillus) encode activator and modulator proteins, respectively, required for sporulation.' Stringent response: relA and spoT genes are transcribed and the translational products of these genes regulate intracellular levels ofguanosine pyrophosphate nucleotides which are essential for ribosome function.' A variety of genes in the Pho system of Enteric bacteria encode activation, modulation, and sensor molecules that code for the alkaline phosphatase enzyme and others? SOS response: upon damage of DNA, recA modulator gene is activated to code for the RecA protein which eliminates repression of the lexA gene; the l e d gene is then transcribed and translated to induce expression of more than 20 DNA repair genes.'

"The scientific basis for measuring the impact and understanding the importance of microscale phenomena at the ecosystem, landscape, biome, and biosphere scales is still in development. However, progess can be made through interdisciplinary cooperation of specialists who can identify the appropriate controlling factors and scale-related phenomena to measure (Groffman, I99 I , 1993; Groffman et ul., 1992, 1993). "Rogers and Whittinan (1991 ); Likens (1985); Woodwell and Houghton (1990); and Mayer e/ a/.. ( 1992). ' Atlas and Bartha ( 1993). "Stolp ( 1988). "Neidhardt e t a / . ,( 1990).

SUBSURFACEMICROBIAL ECOLOGY

27

teristics, types of microbial inhabitants, etc. Successional events and interactions such as competition, commensalism, parasitism, and predation, among others (Atlas and Bartha, 19931, may occur within microbial communities. Under many situations, especially anaerobic conditions, entire food webs are based on the interactions of different microbial populations (Zinder, 1993). The essence of community-level responses can be efficiently grasped by understanding that microbial communities are often composed of a large number of populations with the potential for a wide variety of interactions. When environmental changes (such as carbon and energy source or nutrient addition) are imposed on webs of interacting populations, the community will probably respond to the new conditions so that the resources made available are maximally exploited. If the environmental change is brought about by the introduction of toxicants, desiccation, starvation, or other physiological stresses, then resistant members of the microbial community may become dominant. These kinds of shifts in community structure can be observed utilizing a variety of cultural (Kampfer et al., 1993; Wollum, 1982), biochemical (Findlay and Dobbs, 1993; Karl, 1986, 1993a,b; White et al., 1983), and molecular (Atlas ef al., 1991; Paul, 1993; Torsvik et a/., 1990) methods and then transformed into diversity indices (Atlas eta/., 1991; Atlas and Bartha, 1993; Brockman et al., 1992; Haldeman etal., 1993). Resiliency, a key trait of microbial communities (Alexander, 197 1 ), is derived from shifting proportions and relationships of diverse populations. A discussion of community level response to environmental change is presented in Section IV. At the population level of organization (Table 11), microbial responses to environmental change are perhaps the best understood because of the rich traditions in microbial physiology established by studying the growth and behavior of populations of genetically similar cells in axenic cultures. Such populations reflect the integrated behavior of component cells whose life processes are frequently referred to in terms of growth, reproduction, mutation, regulation, and death. The microbiologist studies a variety of physiological, biochemical, and genetic processes at the population level and interprets them at the level of the individual cell (Brock et a/., 1994). As environmental changes are imposed on microbial populations, individuals contend with adversities and exploit resources to the best of their abilities. There is a constant selection process at the level of the individual cell which ultimately selects for “laboratory-adapted’’ cultures and may favor shifts in the abundance of rare or mutant genotypes within each population. This selective culture phenomenon can give rise to cultural artifacts which preclude understanding of microbial behavior at higher levels of organization depicted in Table 2. At the cellular and subcellular levels of response to environmental change, the richly varied and reductionistic approaches that have been successful in cell biology, enzymology, genetics, and molecular biology show their influence (Table 11). Each bacterial cell can be modeled as a highly organized, tightly regulated entity

28

E. L. MADSEN

whose macromolecular architecture includes (in diminishing order, based on the known composition of a few well studied bacteria such as Escherichiu coli) proteins, RNA, DNA, lipid, lipopolysaccharide, murein, and glycogen, among others (Neidhardt et al., 1990; Nanninga, 1985). Cell growth, division, as well as longterm survival, and dormancy are regulated by specific genes. Yet, after many decades of intense genetic and physiological research on the genome of E. Cali, the function of only about one-half of the entire genetic complement of this wellknown organism has been identified or mapped (Riley, 1993). By deleting large portions of the chromosome, it further has been shown that the majority of genes in E. coli are nonessential for survival under enriched laboratory growth conditions (Neidhardt et al., 1990). Although the specific physiological and genetic roles of the “nonessential” DNA are unknown, it is certain that a large part of the genome of E. coli and other laboratory-adapted microorganisms is dedicated to coping with environmental stresses (such as obtaining nutrients at low concentrations, long-term survival and dormancy, maintenance of position in microhabitats, and resisting effects of noxious chemicals) which are rarely encountered by laboratory cultures (Neidhardt et al., 1990). Neidhardt et al. (1990) present an excellent overview of how bacterial cells (especially E. coli) coordinate their overall cell metabolism, protein, and genetic responses to environmental change under laboratory culture conditions. Gene expression and its regulation are the fundamental mechanisms involved in processes aimed at efficient management of the limited carbon, energy, and nutrient resources available to microbial cells. In order to interpret some of the statements in Table 11, it is important for the reader to recall the cellular processes leading from DNA to RNA (transcription) to protein (translation) (Brock et al., 1994). Proteins serve essential structural, catalytic, and regulatory roles in cell function. Furthermore, proteins are the major macromolecular component of microbial cells, thus the synthesis of protein (and other macromolecules, especially DNA and RNA) is very tightly regulated in microorganisms. Enzyme levels and activities are controlled in a cascade of coordinated cellular mechanisms that include the initiation of transcription, transcription maintenance and termination, translation initiation, and translation efficiency. Transcription initiation may be positively or negatively controlled by DNA-binding activator or repressor proteins, respectively, which regulate RNA polymerase activity. Transcription attenuation can provide a negative feedback mechanism to achieve early termination of mRNA synthesis from DNA when the supply of the gene’s protein product is sufficient. Furthermore, translational repression may occur when the binding of a regulatory protein to mRNA prevents that transcript from directing protein synthesis. Numerous feedback and control mechanisms between products and substrates, proteins, RNA, and genes are involved in cellular growth and metabolism. To foster understanding of the complex details of metabolic regulation, Neidhardt et

SUBSURFACE: MICROBIAL ECOLOGY

29

al. (1990) provide a simple conceptualization (Fig. 3 ) for the stimulus-response network that bacterial cells utilize in adjusting to environmental changes. An environmental stimulus (e.g.. pH change or reduced levels of nutrients) affects a target, usually located at the surface of the cell, the sensor (e.g., a membranebound transport protein or change in membrane potential). In response to the status of the sensor, a signal is transmitted (such as the intracellular concentration of the transport protein's substrate or a modified form of the substrate). The signal is transduced by a cellular component such as an enzyme that undergoes conformational (hence functional) change when bound to the signal molecule. The transducer protein may then create a product which binds to DNA, thereby regulating gene expression. The expressed gene is transcribed and translated through a series of proteins that may regulate expression of a network of related genes. Ultimately, the protein products of the coregulated network of genes may be tied together via a negative feedback loop which returns to the signal-transducer-regulator complex (Fig. 3). This E. coli-based model provides a framework for understanding

Stimulus

t t

Sensor

r

?I

TransducerW

Regulator

Operon A

Operon B

Operon n

DNA in

Network proteins

Response

Figure 3. Mechanisms of microbial responses to environmental changes, a stimulus-response pathway (modified from Neidhardt e t a / . , 1990).

30

E. L. MADSEN

the type of molecular strategies that all bacteria may utilize in responding to environmental change.

2. Metabolic Status of Subsurface Microorganisms Regardless of the microbiological habitat examined, counts of viable cells on agar media usually are one to three orders of magnitude lower than microscopic (total) counts performed on the same sample (Ferguson et al., 1984: Lee and Fuhrman, 1990; Mor6 et al., 1994; Roszak and Colwell, 1987). This discrepancy is cause for an important conceptual and operational distinction between culturable and nonculturable microorganisms. The distinction reflects both the physiological requirements and metabolic status of microorganisms in nature. Each culture medium and set of incubation conditions is selective to some degree and therefore able to meet the physiological requirements of only part of the culturable component of a given microbial community. For a given culture medium, organisms that fail to grow either have different physiological needs than those presented or are nonculturable. Nonculturability may have two distinct physiological bases: cells can be dormant (i.e., they may require special resuscitative events prior to regaining an ability to grow) or they may be nonviable, (i.e., intact and, therefore, detectable microscopically; but unable to reproduce regardless of the growth conditions applied by the microbiologist) (Fuhrman and Azam, 1982; Henis, 1987; Hoppe, 1978; Roszak and Colwell, 1987). It is important to note here that this definition of nonviability (and by default, the definition of viability) is based on microbiologists’ inability to provide proper conditions for growth in laboratory media. In nature, the proper conditions may be provided by changes in any number of unknown environmental factors that can support growth. Also, this definition of nonviability does not take into account the ability of bacterial cells to maintain some of their enzymatic activities and physiological functions but lose the ability to reproduce. Thus, the concept of nonviability and death among bacterial cells in any natural setting is somewhat arbitrary [see Henis (1 987) for further discussion of the arbitrary nature of the terms: survival. dormancy, and death as applied to bacteria]. Understanding the dichotomies of culturable versus nonculturable and dormant versus nonviable microorganisms is important in assessing and predicting the responsiveness of subsurface microbial communities to perturbations, such as agriculturally related pollution events (see Sections 111 and IV), to groundwater habitats. Groundwater nutrient concentrations usually are very low and flow velocities can be very slow [hydraulic conductivities sometimes on the order of centimeters to meters per year (Freeze and Cherry, 1979)l. Thus, it is reasonable to assume correspondingly slow rates of metabolism and growth (or dormancy, under extremely low nutrient conditions) as the usual physiological status of subsurface microorganisms. However, accurate information on in situ metabolic rates and

SUBSURFACEMICROBIAL ECOLOGY

31

other direct measures of the physiological status of microorganisms in groundwater habitats are extremely difficult to obtain because of disturbance artifacts and uncertainties of interpreting field data (see Brockman et al., 1992; Madsen, 199 I; Madsen and Ghiorse, 1993; Madsen, 1995). Criteria for evaluating the physiological status of microorganisms in their natural settings (Findlay and Dobbs, 1993; Karl, 1986, 1993a,b; Tunlid and White, 1992) have been applied to subsurface samples. These include a wide variety of laboratory-orientedprocedures: cell size (Ghiorse and Balkwill, 1983; Hirsch and Rades-Rohkohl, 1983), cell structures indicative of active growth or dormancy (Ghiorse and Balkwill 1983), accumulation of storage polymers such as poly-phydroxyalkanoates (Balkwill et al., 1988; Bengtsson, 1989;Ghiorse and Balkwill, 19831, ATP levels (Balkwill etal., 1988; Beloin etal., 1988; Jensen, 1989; Metge et al., 1993; Wilson et al., 1983), various stains for viability (King and Parker, 1988; Marxsen 1988), extraction of polar lipid fatty acids (Federle et al., 1990; Haldeman et al., 1993; White et al., 1983), incorporation of [3H]thymidine into cell constituents (Albrechtsen and Winding, 1992; Phelps et al., 1989; Thorn and Ventullo, 1988), and use of time courses of substrate mineralization or other activity measures to infer both substrate turnover times (Armstrong’et al., 199 I ; Ventullo and Larson, 1985) and a state of metabolic readiness (Francis et al., 1989; Hutchins et al., 1991; Kuhn et al., 1989). (For additional references and details see Table I and Section IV.) Taken in total, there are no clear trends in the existing data to indicate the overall physiological status of subsurface microbial communities. Albeit for pristine aquifers, most of the evidence suggests that metabolic activity is very low. A portion of the studies cited in Table I were designed to reveal only the presence of or potential activity of microorganisms in subsurface samples. The results suggest that subsurface bacteria possess the ability to adapt quickly from nutrient-limited to nutrient-sufficientconditions. This nutritional adaptability may be a consistent trait of many of the culturable microorganisms in aquifers (Balkwill et al., 1988; Bengtsson, 1989; Kampfer et al., 1993; Thorn and Ventullo, 1988; Wilson et a/., 1983). Only occasionally have adaptive time lags indicative of metabolic dormancy (Armstrong et al., 1991; Madsen et al., 1991; Swindoll et al., 1988) been observed. To the contrary, many authors indicate that aquifer organisms appear to be poised for rapid utilization of added substrates (Federle and Pastwa, 1988; Madsen and Bollag, 1989; Phelps et a/., 1989). The large and rapidly growing body of evidence derived from bioremediation-directedresearch (e.g., Davis et al., 1994a,b; Edwards and Grbic-Galic, 1994; Godsy et al., 1992; Hopkins et al., 1993a,b; Hutchins et al., 1991; Major ef al., 1991; Madsen et al., 1991, 1992; McCarty et al., 1991; Semprini and McCarty, 1991; 1992; Semprini et al., 1991a,b, 1992) (see Table I) makes a compelling case for the capability of subsurface microorganisms to express metabolic activity, especially biodegradation activity, in situ. Broad conclusions about the ability of subsurface microorganisms

32

E. L. MADSEN

to catalyze agriculturally relevant subsurface processes can be inferred from bioremediation studies only if the subsurface physiological conditions required for biodegradation activity mimic the conditions required for other more ecologically relevant activity (i,e., pesticide metabolism and the cycling of carbon, nitrogen, and phosphorous). The fate of agriculture-related groundwater contaminants is discussed in Section IV. In all probability, as is true in other habitats, the physiological status of microorganisms in the terrestrial subsurface is highly variable over time and space, reflecting site-specific environmental conditions. As indicated earlier, microbial abundance and metabolic activity in subsurface zones vary depending on environmental properties such as the hydrolosic regime, pore water chemistry, mineral texture and type, and the availability of nutrients. The physiological status of the subsurface microbial community follows the dictates of the physical and chemical properties of the subsurface sediments. 3. Nutrient Cycling Because sunlight is precluded from groundwater environments, photosynthesis is impossible. Thus, the food chain in aquifers is primarily heterotrophic, reliant either on an influx of dissolved organic carbon along a given hydrologic flow path or on organic materials of sedimentary origin that subsequently may have been metamorphosed (Barker, 1979; vanbevelen, 1984). The potential for microbial oxidation of reduced inorganic energy sources has been demonstrated in some subsurface sediments [e.g., sulfide, hydrogen, ammonium (Fredrickson et al., 1989; Pedersen and Ekendahl, 1992)l. These energy sources may be utilized for autotrophic COz fixation or other microbial metabolic functions (Kelly, 1992; Kuenen and Bos, 1989), as has been demonstrated in deep sea thermal vent regions (Jannasch, 1989). But to our knowledge, aerobic chemosynthetic food chains have not been described in the terrestrial subsurface. The possibility for such food chains does exist though, especially in areas of geothermal activity and in ultradeep boreholes where reduced inorganic energy sources encounter oxygenated water. Because primary production is unlikely to occur in groundwater ecosystems, autochthonous carbon compounds for fueling ecosystem function are restricted to those which comprise the microorganisms themselves or are contained in the subsurface rocks and sediments. Much of the autochthonous carbon may be unavailable for metabolism. Thus, allochthonous dissolved organic carbon [the majority of which may consist of the recalcitrant fraction of organic substances derived from surface plant material (Madsen and Ghiorse, 1993)], delivered through hydrologic recharge and groundwater flow (see Sections I and 11), may govern subsurface microbial metabolism. Bormann and Likens ( 1967, 1979) have presented a comprehensive definition of nutrient cycling in forested ecosystems which links

33

SUBSURFACEMICROBIAL ECOLOGY

the physical transport of nutrient elements to their temporal exchange with biomass and other pools within the system. Figure 4 (taken from Madsen and Ghiorse, 1993) is based on figures presented by Bormann and Likens (1967, 1979) but modified to emphasize the importance of hydrologic flow and microorganisms in the groundwater habitat. The key role played by microorganisms in nutrient cycling is the enzymatic conversion of complex, high molecular weight carbon

Meteroioglc Input

Water I

(gases, solids, solutes) Subsurface Ecosystem

-

__________----

-1

System Atmosphere

living and dead microbial blomass: high molecular weight C, N, P, S. etc.

Microbial Decomposltlon

Microblal Uptake

I I I I

1

I I I I I * -

low molecular welght,

---- _ _

I

lntrasydem Cycle

-----

7Z-F MeterologicOutput

------

I

(gases, solids,

Figure 4. Nutrient cycling in subsurface ecosystems. Water flow through the subsurface is the dominant mechanism by which materials are transported within and exchanged between subsurface and adjacent ecosystems. Microorganisms are the principal inhabitants of the groundwater habitat. Delivery of organic substances to subsurface sediments, their microbiological utilization, and removal of by-products of microbial metabolism depend on hydrologic flow (from Madsen and Ghiorse, 1993).

34

E. L. MADSEN

compounds (e.g., polysaccharides, lipids, protein, lignin, or humic material) either to simple low molecular weight components (e.g., sugars, fatty acids or amino acids or phenolic compounds) or to inorganic compounds (e.g., COz, NH3, SO4?-, Pod3-) in the available nutrient pool. This catalysis is driven largely by the physiological need of heterotrophic microorganisms for carbon and energy. The other nutrient elements (N, P, S, etc.) accompany carbonaceous materials during their transformation. By definition, nutrient cycling describes the passage of an element through a series of steps that return it to its original state. Evidence arguing for completion of the simplest (two-step) case of a nutrient cycle must document both the decomposition and uptake shown in Fig. 4. In soil habitats, the most obvious evidence for microbiological cycling of nutrient elements is a balanced steady state between pools of soil organic matter, crop residues, leaf litter, and living vegetation. The linkages in soil between microbiological decomposition of crop residues, humification processes, and the growth of crop plants are obvious. But the same types of linkages are far from obvious in subsurface systems. Based on chemical analyses of groundwater samples, Baedecker and Back (1979) assembled a plausible case for multiple transformations of carbon and nitrogen in sediments beneath a landfill. Similarly, Chapelle (1993) has provided a comprehensive overview of the contributions of microbial metabolism to the changing geochemistry of groundwater. These results not withstanding, studies which unequivocally document complete nutrient cycles in groundwater habitats are very rare. This stems partially from the fact that the subsurface habitat is inaccessible, opaque, spatially and temporarily complex, and resistant to the rigorous documentation of the role of microorganisms in catalyzing even single geochemical reactions, let alone a series of reactions that constitute a true nutrient cycle (Madsen and Ghiorse, 1993; see also Section IV). In the simplest case, each microbial activity listed in Table I represents one-half of the nutrient cycling process (generalized as uptake or decomposition in Fig. 4). The task of measuring two complementary processes becomes extremely challenging, because the steps may occur simultaneously or may be separated by unknown scales of time and distance. A contributing reason for incomplete information on nutrient cycling in the subsurface is that the steps may often be unavoidably “truncated.” Chapelle ( 1993) has advanced the hypothesis that truncated cycles are implicit in subsurface systems because primary productivity (e.g., photosynthesis) cannot occur; thus, both materials (such as oxygen) and energy, required to drive complete biogeochemical cycles, may be lacking. Advancements in determining cyclic metabolic processes in subsurface ecosystems and testing Chapelle’s ( 1993) “truncation hypothesis” will depend on improvements in techniques for defining and measuring both hydrologic flow paths and microbiologically catalyzed fluxes between nutrient pools. One approach that has promise would apply the mass balance concept of Bormann and Likens (1967, 1979) to the subsurface. It is theo-

SUBSURFACEMICROBIAL ECOLOGY

35

retically possible to estimate the fraction of total annual photosynthesis that escapes the soil and is processed in the groundwater habitat by measuring pore water chemistry at different distances along defined hydrologic flow paths.

111. AGRICULTURAL PRACTICES AND THEIR IMPACT ON SUBSURFACE HABITATS By using analytical tools from ecology, agricultural activities can be conceptualized as managed systems that interact with other ecosystems in the landscapes. Figure 5 (from Briggs and Courtney, 1985) depicts the essential components of agriculture. The physical system consists of soil, crops, and livestock. Climatic and other inputs (e.g., water, fertilizers, pesticides, and a variety of organizational and logistical manipulations such as plowing, tillage, and animal husbandry) are directed through the physical system. As agricultural products are removed, materials (i.e., water, manures, crop residues) and influences (i.e., chemical, climatic, biological, and ecological changes) flow within the system and are exported to adjacent ecosystems. Each individual agricultural system has its own set of input, influence, flow, harvest, and external system parameters. The discussion that fol-

EXTERNAL SYSTEMS

&$= -

LIVESTOCK

-I

STREAMS GROUNDWATERS VEGETATION FAUNA, etc,

MANAGEMENT SYSTEM

INFLUENCES

mws

Figure 5. Conceptual structure of an agricultural system and its relationship with external systems (modifed from Briggs and Courtney 1985).

E. L. MADSEN

36

lows describes agricultural practices that occur within the “physical system” of Fig. 5. Emphasis is given to the influences and flows as they impinge on one particular external system, groundwater.

TYPES OF AGRICULTURAL PRACTICES This section discusses the mechanism(s) by which agricultural practices influence physical, chemical, and hydrologic conditions in the subsurface habitat (Fig. I). Agricultural practices change in time and space, while the groundwater habitat passively receives inputs from above (see Fig. 6). As was clear from Section 1I.A and Figs. 2 and 4, the subsurface habitat represents an unusual union between terrestrial and aquatic environments with often discontinuous and intermittent water flow from recharge areas. Entrainment of solutes (applied to or generated within surface soils) is virtually the only mechanism by which agricultural practices impact the subsurface environment and its microbial inhabitants. Even deep plowing activities such as moling and chiseling (see this section and Section III.A.2; Briggs and Courtney, 1985; Loomis and Connor, 1992) fail to penetrate through the soil habitat to directly transfer energy or materials to the underlying subsurface. Thus, water management practices emerge as the principal means by which agriculture influences the subsurface (Fig. 6). However, all of the other management practices (i.e., those involving soils, crops, pests, and livestock) may have significant indirect effects both by altering rates of water infil-

Soil Habitat Key agricultural practices: I

I

Hydrologic llnk between soil and subsurface

I

’

1. Water management(espec~aiiy

Irrigation, drainage, and tillage) may modifv the amount. extent. depth, location, temperature,and timing of Infiltrating water 2. Soil, crop, pest, and animal management may modify the compositonof intiitratingwaters, especially, salts, nutrients, organlc carbon, metals, pesticides, and other chemlcal components

SubsurfaceHabitat

Figure 6. The hydrologic link between the soil habitat, where agricultural practices occur, and the subsurface habitat which supports a responsive biotic community that is almost exclusively microbial.

SUBSURFACE MICROBIAL ECOLOGY

37

trating through soil and by altering the chemical composition of waterborne materials reaching the subsurface habitat.

1. Water Management Water availability represents one of the main controls on crop growth. Plants require water for two main reasons: to maintain cell turgor pressure and to supply nutrients (Briggs and Courtney, 1985). Water deficiencies lead, under extreme conditions, to loss of turgidity and wilting of the plant. Under less extreme water deficiency conditions, growth may be inhibited by lack of nutrients and by diminished photosynthetic efficiency. Excesses of water may also occur, however. Crop plants obtain most of their water from the soil, a complex, porous matrix in which both air and water reside in the same pores. When water is present in abundance, air may be excluded from the pores and plants may suffer from lack of oxygen. With the exception of wetland rice plants (Tivy, 1990), physiological problems induced by anaerobic conditions may ensue for the crop (Briggs and Courtney, 1985). In many parts of the world a soil water surplus, for part or all of the year, limits the amount of time available for crop growth. Some form of drainage is, therefore, necessary for successful crop growth. The basic aim of all drainage systems is to remove soil water so that soil aeration can be achieved. This, in turn, alleviates toxic anaerobic conditions and promotes deeper, more extensive root development (Tivy, 1990). Essentially three strategies exist for draining soil: (1) improve the vertical movement of water; (2) improve the lateral water movement; and (3) lower the regional water table (Briggs and Courtney, 1985). These drainage strategies are pursued by carrying out one or several of the following procedures: “moling” (formation of an unlined subsurface cylindrical channel, approximately 0.5 m deep that is created by drawing a bullet-shaped implement through the soil); “subsoiling” (which uses techniques much like moling to shatter impermeable pan layers beneath the normal plowing depth); pipe drains (which convey excess water away from subsoils; pipe drains are installed by digging trenches and lining them with tiles or top-perforated plastic pipes prior to being covered over); and open ditches (Briggs and Courtney, 1985; Tivy, 1990). In other parts of the world there is a deficiency of soil water for crop production for part or all of the year. Agriculture in these arid and semiarid regions relies on irrigation, the transport of water from a supply source to land whose atmospheric input of water fails to keep pace with evapotransporational losses. Three general methods of irrigation may be identified: surface methods, overhead methods, and subsurface methods (Briggs and Courtney, 1985). By far, the most widely used are surface methods of application for, in general, these involve relatively lowcost technology (i.e., gravity flow of water) and are easy to apply over a wide area.

38

E. L. MADSEN

Surface flooding may be achieved by damming and diverting main streams (uncommon in North America) and also by distributing water over the surface via furrows. The water seepage occurs through the base and sides of furrows and, via capillary action, reaches ridges within which the crops are planted (Briggs and Courtney, 1985). Overhead methods of irrigation are varied. Water is normally sprinkled onto the soil surface from nozzles. The nozzles may be mounted on sprinkler units that are static, rotating, or traveling. Overhead irrigation often has the advantage of being independent of surface topography (Briggs and Courtney, 1985). Trickle (or drip) irrigation involves the discharge of small amounts of water from small orifices located on or immediately below the soil surface near the crop roots. Trickle irrigation strives for highly effective use of water while minimizing water loss and soil disturbance. Finally, subirrigation is an expensive, specialized irrigation practice that uses damming procedures and drains to manage the water table level so as to bring the capillary fringe (see Sections I and 11) within the root zone of the crop (Tivy, 1990; Briggs and Courtney, 1985). There are four key concerns in managing water for agricultural purposes: the mechanics and hydraulics of distributing water; the timing of these water fluxes; the volume of these water fluxes; and the quality of the water (Briggs and Courtney, 1985). These four concerns apply to both drainage and irrigation practices and they raise several fundamental issues about the environmental impact of water management on the subsurface habitat. 1. Water (like all matter) is neither created nor destroyed-its mass is conserved. Whenever water is moved, the source necessarily becomes initially drier and the receiving area becomes wetter. This simple truism means that assessments of the environmental or ecological impacts of irrigation practices necessarily must consider both the habitat that provides the water and the one that receives it. 2. No water used for irrigation is free of dissolved or suspended materials. Thus, whenever water is conveyed, so also are conveyed its accompanying chemical constituents. Of particular concern is the salinization of soils that are under irrigation in arid areas. As irrigation waters evaporate, salts (commonly of sodium, calcium, magnesium, chloride, sulfate, bicarbonate, as well as a variety of trace elements) may accumulate over time. High salt concentrations in soil may contribute to the development of impermeable soil layers (pans) and may be deleterious to both soil structure and crop growth. As just discussed and shown in Fig. 6, water management practices are the single most influential means by which agriculture affects subsurface microorganisms. Rows 2-5 of Table I11 (summarized from Loomis and Connor, 1992; Tivy, 1990; Briggs and Courtney, 1985; Soule ef al., 1990) provide an overview of water management practices and their impact on the subsurface habitat.

SUBSURFACEMICROBIAL ECOLOGY

39

2. Soil Management Physical modification of the soil resource, via tillage practices, is designed to create favorable conditions for crop growth and development. Tillage is carried out for three main reasons: (1) physical preparation of the seedbed (i.e., by plowing or harrowing) to break up soil aggregates; ( 2 ) removal of crop residue and weeds by reducing their size and burying them to promote decay and nutrient cycling; and (3) improvement of rooting and drainage conditions (i.e., by deep plowing) (Briggs and Courtney, 1985). A variety of tillage systems have been developed, each of which may be implemented according to the prevailing agricultural conditions (geographic location, crop planted, climate, soil type, and a variety of socioeconomic factors). According to Loomis and Connor ( I 992), “primary (or conventional) tillage” utilizes a mold board plow, often in combination with a disk plow to dig into and invert top soil. “Secondary tillage” is the preparation of seed beds with field cultivators, light disks, and spring- or spike-toothed harrows. These implements reduce the size of soil peds and leave the soil smoother and firmer for improved contact among seed, soil, and its moisture. Conventional tillage approaches contrast with “reduced till” and “no till” systems in which the degree of plowing is diminished and reliance on herbicides for weed control is increased. Rows 6-9 of Table 111 provide an overview of soil management practices and their impact on the subsurface habitat. The principal means by which soil management methods impact ground water is by influencing the chemical composition and flow rates of infiltrating water.

3. Crop Management Crop management begins with the sowing of seeds, continues with crop maintenance during growth and development, and ends with crop harvest, storage, and distribution (Tivy, 1990). During seed sowing, a mechanized planter often opens a furrow in the prepared soil seed bed, places the seed in the exposed moist soil, covers the planted seed, and then often packs the soil down to assure firm seed-soil contact. In no-till systems, the crop is planted (“drilled”) directly into the soil through residue from the previous crop. Soil fertilization is an essential component of crop management to assure nutritional sufficiency for plant growth. The selection of type, amount, timing, and method of fertilizer application is determined by a variety of considerations including the crop type, the nature of the fertilizer, soil conditions, and weather. A generalized listing of common fertilizer applications follows (after Briggs and Courtney, 1985): ( I ) broadcast [application of fertilizer (often pelletized) to the soil surface before the crop emerges]; ( 2 ) plowing in (application of fertilizer to

P 0

Table I11 Agricultural Practices“and Their Impact on Conditionsin the Subsurface Habitat

Type of agricultural practice Water management (exerts a direct” influence on the hydrologic link between soil and the subsurface)

Key references documenting impact of agricultural practices on soil or groundwater

Practice

Impact on groundwater flux and types of solutes entrained

None (only climate and unmodified soil and vegetation govern water flux)

No change in ambient fluxes of water from surface to subsurface; naturally occurring inorganic and organic solutes are transported in recharge water

Land drainage

Decrease in flux of water and accompanying inorganic and organic solutes from soil to the subsurface

Danielpool et al. ( 1 99 I ); Schot and Molenaar ( 1992); Utermann et al.

Surface flood irrigation

May increase flux of water to the subsurface; intermittent flood irrigation may cause salinization of soil; high-volume flooding may transport salts to the subsurface Salinization of soils may occur; subsequent flooding may transport salts to subsurface May physically influence the subsurface by lowering the water table, increasing the extent of vadose zone, and (in some situations) causing land subsidence

Frenkel and Meiri (1985); Magaritz and Nadler

( 1990)

Soil management (exerts an indirecth influence on the hydrologic link between soil and the subsurface)

Overhead, drip, and subsurface imgation Withdrawal of water from subsurface and surface reservoirs None

No change in ambient fluxes of water and naturally occurring inorganic and organic solutes

(1993); McTerman and Mize (1992); Umali (1993) Bouwer (1981); Carbognin ( 1985)

Plowing: Chisel Moldboard Disk

Field cultivation Cultivator Harrows Tillers Hoes Reduced till and no till systems

Crop management (exerts and indirecth influence on the hydrologic link between soil and the subsurface)

Sowing, cultivation, mulching, manure application, fertilizer application, green manuring, harvesting

Soil compaction and reduction of soil porosity caused by tractor tires may decrease water infiltration and flux to subsurface. Enhanced infiltration may increase water flux to subsurface. Mixing of soils and crop residues may enhance their decay, hence accelerated nutrient release to soil solution and perhaps to groundwater. Increased soil porosity may reduce soil thermal conductivity hence raise soil temperatures and the temperature of water infiltrating to the subsurface. Under certain circumstances, repeated plowing may cause impermeable layers (pans) beneath the soil surface to form, hence decrease flux of water to subsurface. Soil compaction and reduction of soil porosity caused by tractor tires may decrease water infiltration and flux to subsurface. Altered seedbed aspect may increase radiation received and decrease radiation losses; resultant increased soil temperatures may warm the waters infiltrating to the subsurface. Decrease in water and wind erosion of soil may enhance or diminish water flux to the subsurface. Soil temperatures may increase because thermal turbulent transfer may decrease, this may warm the water infiltrating to the subsurface. However, reflection of sunlight may reduce soil temperatures, thus cooling water infiltrating to the subsurface. See the soil management portion of this table. The major additional element of crop management is the presence of roots and shoots of growing crops. These may alter the physical and hydrologic properties of soil. In addition, the primary production, exudation, decay, and imperfect uptake of fertilizer amendments which are characteristic of crop management practices may release additional organic and inorganic compounds to soil solution which, in turn, may leach into the subsurface habitat

Vanderzee and Boesten (1991)

Parkin and Meisinger ( 1989)

Isensee et nl. ( 1990)

Alfoldi (1983); Goodrich et al. (1991); Spalding and Exner ( I993 )

( continues)

Table Ill-Continued Key references documenting impact of agricultural practices on soil or groundwater

Type of agricultural practice

Practice

Impact on groundwater flux and types of solutes entrained

Pest management (exerts an indirect” influence on the hydrologic link between soil and the subsurface)

Physical weed control (tillage, burning, etc.), crop rotation, trap crops, other IPM procedures, insecticides, fungicides, acaricides and other pesticides, fumigation

See the water, soil, and crop management portions of this table. The major additional characteristics of pest management are the addition of pesticides and related carriers and residues to soil, soil solution, plants, and plant tissues. These may, under some circumstances, leach into subsurface environments.

Domagalski and Dubrovsky ( 1992): Jury and Gruber ( 1989, 1990); Lawrence et a/. (1993); Loague et crl. ( 1990); Ritter (1990); Shoemaker et d.( 1990); Varshney et crl. (1993): Villeneuve et ul. ( 1990)

Livestock management (exerts an indirect” influence on the hydrologic link between soil and the subsurface)

Water management, soil management (none, plowing, tilling, see above), crop management (none, forage, feed, see above), grazing, manure management, feeds, feed supplements (hormones, antibiotics, etc.), primary harvest ( i t . , biomitss of livestock animals), secondary harvest ( i t . , livestock products such as milk, fiber, eggs), pest management. livestock medicine

See comments for water, soil, crop, and pest management. Livestock management practices have additional effects such as soil compaction and erosion (imposed by various traffic and grazing regimes); nutrients released as manures and urine; and other logistical aspects of rearing, maintaining, transporting, and harvesting livestock populations

Headworth ( 1989); Sangodoyin and Ogedengbe (1991)

“References for agricultural practices include Loomis and Connor (1992); Tivy (1990); and Briggs and Courtney ( 1985) ’See Fig. 6 and text in Section 111.

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43

the surface followed by mixing into the topsoil by plowing); (3) sideband (fertilizer application in bands adjacent to the seed); (4) contact placement (fertilizer application in direct contact with the seed); (5) side-dressing (fertilizer placement in narrow rows at the surface after crop emergence); and (6) top-dressing (general application of fertilizer to the crop after emergence). During crop growth, a variety of crop and soil maintenance as well as weed removal practices may be undertaken. Again, the specific type of farm machinery actually used and overall management practices are site, farmer, and climate specific. But overall, weed control can be accomplished through several types of soil cultivation practices (see earlier). These include dense arrays of small spring tines, rotary hoes, and tractor-mounted arrays of spear- or sweep-pointed shanks designed to till in between crop rows (Loomis and Connor, 1992). Herbicides are also used widely for weed control (see the following discussion). Row 10 of Table 111 provides an overview of the input of crop management and the subsurface habitat. In essence, crop management practices influence the subsurface habitat by two independent mechanisms. First, the physical structure of soil (hence the infiltration rates of water) is altered by farm machinery traffic passing over the soil, by cultivation implements, and by the penetration of soil by roots and shoots of the growing crop plants. Second, the solutes in soil that may be conveyed to the subsurface by infiltrating water are determined by the organic and inorganic compounds present in the soil as a result of fertilizer amendments and crop growth and decay.

4. Pest Management The steps that are taken in agriculture to foster the growth of desirable organisms (crops) also foster the growth of undesirable organisms (pests). From an ecological viewpoint, the presence of pests in agriculture is unavoidable because the physical, chemical, nutritional, and hydrologic resources made available to crops represent a bounty of resources for opportunistic noncrop biota. These include weeds, animals (especially insects; but also slugs, nematodes, rodents, birds, and others), and microorganisms (especially plant pathogens that include fungi, bacteria, and viruses) (Briggs and Courtney, 1985; Tivy, 1990). According to Briggs and Courtney (1985), it has been estimated that weeds, animals, and pathogens may account for a reduction of global preharvest crop yields that approach 50%. The major mechanism of yield reduction is reducing leaf areas, hence photosynthesis. Weeds also compete with crop plants for sunlight, water, and nutrients. Clearly, pest management is an essential component of agriculture. Ecological methods of pest control are implicit in all agricultural practices and are explicit in integrated pest management (IPM) strategies. All pests are susceptible to their own arrays of environmental stresses, diseases, and predators. All pests generally do less damage to well-managed, rapidly growing crops than, for example, to

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those deficient in nutrients (Loomis and Connor, 1992). The wide variety of weeds, animals, and microorganisms which threaten agricultural crops demand a correspondingly wide range of crop protection methods. These include five main techniques, each of which may be used to suppress, deter, eradicate, or prevent infestation: ( I ) direct control (i.e., weeding); (2) crop cultivation methods; (3) chemical control; (4) biological control; and (5) habitat removal (Briggs and Courtney, 1985). Weed management strategies center on limiting the size of the seed bank through control of weed reproduction (Loomis and Connor, 1992). Sanitation is important because small untended weed populations in I year can cause crop losses over a number of subsequent years. Tillage is a major means for reducing weed plant populations that have germinated and grown within stands of agricultural crops (Loomis and Connor, 1992; tillage practices have been described earlier). Herbicides have reduced or replaced tillage weed control practices in many cropping systems. Herbicides enable weed control to be accomplished more quickly, more timely, less expensively, and with significantly less energy use than with tillage (Briggs and Courtney, 1985). Herbicides include synthetic organic chemicals that are highly selective to particular groups of plants (i.e., monocotyledonous vs dicotyledonous). Other herbicides are formulated to act as general toxins-by inhibiting physiological processes common to all green plants (i.e., photosynthesis and cell elongation and division). Depending on mode of action, target weeds, and cropping systems, herbicides may be applied with a variety of spraying implements (mounted on knapsacks, booms, tractors, and airplanes). The application may occur prior to weed growth (pre-emergence herbicides) or postemergence to weed leaves where they may act on contact or be translocated through the plant to roots and shoots. Animal and microbial pest management practices, like those of weed management, rely to a large degree on crop cultivation methods. Tillage, irrigation, crop rotation, burial of crop residues, and related practices alter the crop environment such that conditions are adverse to the growth and reproduction of insect and microbial pests. By understanding specific details of a pest’s life cycle (such as alternate hosts, overwintering stages, or susceptibility to desiccation), management practices can be directed to reduce the pest population, hence its detrimental effect on crops. Using much the same approach, biocides (directed toward insects, mites, nematodes, fungi, and bacteria, among others) have been developed which target essential physiological and biochemical functions specific to the pest of interest. For instance, organophosphorus and carbamate insecticides act on the nervous system of insect pests by inhibiting a key enzyme, acetylcholinesterase, at the nerve synapse. Both contact and systemic biocides are applied at relatively low concentrations (part per million, i.e., kilogram per hectare of land) by aerial application to plants or as solutions or granules applied to soil. To act, some pesticides must be ingested by the pest (i.e., by an insect consuming insecticide-

SUBSURFACE MICROBIAL ECOLOGY

45

coated plant leaves) while others must come into direct physical contact with the surface of the pest. Row 1 1 of Table I11 summarizes pest management practices and their impact on the subsurface habitat. Pest management practices influence the subsurface indirectly by affecting the chemical composition of waters that may percolate through soil to reach ground water.

5. Livestock Management By definition, the variety of agricultural animals (i.e., cattle, sheep, pigs, goats, buffalo, horses, donkeys, mules, and poultry) graze on products of agricultural crops. Livestock reside one trophic level above the crops, which carry out primary production. Thus, implicit in livestock management is a degree of complexity that surpasses that of the four types of management described earlier (Briggs and Courtney, 1985; Tivy, 1990). In order to support livestock, water, soil, crops, and pests need to be managed. This is true even if the livestock are supported by lowinput range land. Furthermore, animal-specific requirements must also be met. These pertain to animal growth, reproduction, physiology, behavior, maturation, and both primary (i.e., animal biomass) and secondary (i.e., milk and eggs) harvest. Livestock management techniques vary considerably with the type of animal, climate, soil type, moisture regime, and infrastructures for use, distribution, and marketing the animal products. As in crop management, the overall goal in livestock management is to maximize the yield of desired product and minimize input of time, energy, and materials, while maintaining the management system (i.e., soil, water, crops, and other related resources) in a sustainable state. Major considerations in livestock include growth of, harvest, storage, curing, and distribution of fodder (crops harvested then fed to livestock); growth of forage (crops grazed in situ by livestock); dietary considerations (especially to ensure mineral and lysine sufficiency for both ruminants and nonruminants); herd maintenance; disease development and transmission; animal reproductive cycles; growth and harvest of the animal and/or animal products; hygiene for the animals and animal products; hygiene for animal wastes (especially urine and manures); handling, distribution, and disposal of animal manures (especially for feedlot rearing of beef cattle); grazing regime for forage animals (this includes free range, set-stocking, rotational, strip grazing, and zero grazing); and minimizing damage to soil structure and vegetation caused by the trampling of soil by animals (Briggs and Courtney, 1985; Tivy, 1990). A full and detailed treatment of livestock management strategies and procedures is beyond the scope of this chapter. For additional information, readers are referred to Ensminger (1991) and Curtis (1983). The overview presented here and Row 12 of Table 3 succinctly indicate the complexity of livestock management

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practices and their primary mechanisms of altering surface environments; hence their potential impacts on groundwater and the subsurface habitat.

IV. IMPACT OF AGRICULTURAL PRACTICES ON SUBSURFACEMICROBIAL ECOLOGY Thus far, the fundamental and independent characteristics of subsurface microbial ecology (Section 11) and agricultural practices (Section 111) have been outlined. In discussing how these two entities overlap and interact, it is appropriate to remind the reader of information and concepts in Figs. 1 through 6 and the chapter overview presented in Section I.

A. A HISTORICAL PERSPECTIVE FOR INQUIRY INTO SUBSURFACE VERSUS SURFACE HABITATS Human curiosity, ingenuity, and material gain are among the primary motivations for scientific investigation of the variety of habitats in the biosphere. Clearly, grasslands, forests, mountains, oceans, lakes, ponds, rivers, and streams, their physical resources, and their flora and fauna have been critical for the development of civilizations (Ehrlich er al., 1977; Odum, 1971; Wetzel, 1983; Nybakken, 1988). Since early visualization of microorganisms by Robert Hooke in 1664, the ecological role of microorganisms in these key surface habitats has become evident (Atlas and Bartha, 1993; Brock er al., 1994; Odum, 1971). But why were both the subsurface ecosystem and its inhabitants left undiscovered (Ghiorse and Wilson, 1988) until the latter part of the 20th century? One answer to this question may be that, unlike much of the rest of the biosphere, subsurface ecology had little to do with human activities. After all, there is really nowhere for humans to go underground without miners’ tools. Furthermore, other than microorganisms, there is no wildlife to speak of in the subsurface. Also, unlike the rest of the biosphere the subsurface has until recently been considered to be an inert, lifeless zone (Ghiorse and Wilson, 1988; Madsen and Ghiorse, 1993; Chapelle, 1993). Aside from supplying bountiful, reliably pure sources of drinking water, the subsurface habitat has not traditionally been known to provide vital functions characteristic of other portions of the biosphere (e.g., photosynthesis, decomposition, and cycling of nutrients; see Section II.B.3 and Fig. 4). In the soil habitat, [it has virtually been impossible to overlook the microbially mediated annual dynamics of plant growth, biomass decay, cycling of carbon, nitrogen and phosphorous; uptake and release of other nutrients; humification; etc.]. In contrast, there has traditionally been no need to even suspect that subsur-

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face microorganisms carried out ecologically relevant processes (Section II.B.3). This is because the water in aquifers, though considered subject to geochemical evolution by geologists (Freeze and Cherry, 1979), has also been viewed as a virtually unchanging resource. Prior to the era of manufacture and improper disposal of toxic contaminant compounds, water that made the journey from the atmosphere to the soil and infiltrated through to the water table (Fig. 2) was almost unquestionably of drinking water quality. When subsequently drawn back to the surface through wells, this water was still of drinking quality. With no evidence (or need) for microbiologically mediated changes in the composition of water while it resided in aquifers, there was no reason to propose that microorganisms were present and metabolically active in the subsurface. Why then have the subsurface habitat and subsurface microbial ecology been recently explored? As mentioned in Section I, the major motivation for advancing the science of subsurface microbiology has been to alleviate groundwater pollution. The key hope has been, and continues to be, that the potential of subsurface microbial populations for eliminating toxic organic compounds will match that of microorganisms in more thoroughly studied habitats such as surface water, sediments, and soils.

B. How CANTHE IMPACTS OF AGRICULTURAL PRACTICES ON SUBSURFACE MICROBIAL ECOLOGY BE kkASURED? Perhaps the most efficient way to assess impacts of agricultural practices on subsurface microbial ecology is to look for changes in the normal composition and function of the native microflora. But “to look for change” requires two pieces of prior knowledge. First, one needs to have sufficient familiarity of the natural history of subsurface systems to know what “normal composition and function” are. Unfortunately, the complexity of microbial communities in virtually all natural habitats has defied complete understanding by microbial ecologists (Hobbie, 1993; Hobbie and Ford, 1993; Madsen, 1995). This general lack of understanding in microbial ecology is compounded in subsurface environments by this habitat’s physical inaccessibility and the relatively brief period of time since the inception of the modern era of subsurface microbial ecology in the 1970s (Ghiorse and Wilson, 1988; Chapelle, 1993). There simply is no robust data base on subsurface microbial communities that defines a “normal composition” of subsurface microflora. Moreover, the “normal function” of microorganisms in subsurface environments is uncertain because (as discussed earlier in Sections II.B.2, II.B.3, and 1V.A) the biogeochemical processes acting in groundwater between infiltration and discharge events have not traditionally been obvious. Thus, present knowledge of the natural history of subsurface habitats does not let us definitively state what “normal” structure and function are.

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The second piece of prior knowledge required for assessing the impact of agricultural practices on subsurface microbial ecology is possession of reliable tools to “look for changes.” In Sections 1I.B.I and 2 and Table I1 a hierarchy and potential responses of microorganisms (bacteria) to environmental changes were outlined. The rich and varied mechanisms by which bacteria adapt to external stimuli, especially at the gene, enzyme, and cellular levels of the hierarchy, are remarkable. But equally remarkable is the uncertain applicability of these molecular response mechanisms to ecologically relevant, mixed microbial communities such as those in groundwater. Most of the information presented in Table I1 and Section 1I.B.I was derived from pure cultures, unlikely to be present in subsurface microbial communities, that were grown under laboratory conditions that depart radically from those found in field sites. Thus, the usefulness of information from a few domesticated,pampered bacteria in interpreting the status of real-world ones is highly suspect. This discontinuity between “pure” and “applied” research is a common problem. For instance, Mayer et al. (1992), in discussing the use of biomarkers as indicators of anthropogenic stresses, have emphasized that the utility of biochemical, physiological, and histological measures in the field is unknown because the pertinent data base is derived from single organisms raised under laboratory conditions. Mayer et al. ( 1992) emphasized that testing and evaluation of biomarker methods in field settings are of critical importance. Although the gene, enzyme, and cellular level measures described in Table I1 and Section 1I.B. 1 are of uncertain applicability to subsurface microbial communities, the population and community level measures have a long history of usage in soil microbiology. Hicks et al. (1990) have reviewed both conceptual and practical aspects of attempts to evaluate the responses of microorganisms in soil to environmental change (specifically to exposure to xenobiotic chemicals). As mentioned earlier in this section, the complexity of naturally occurring microbial communities is overwhelming and soil microbial communities are perhaps the best example of this rule. The fundamental challenge in attempting to devise useful procedures for testing for ecological effects of environmental change is selecting meaningful parameters to measure. Naturally occurring microbial communities are so diverse, resilient, and dynamic that individual tests are often woefully inadequate because they characterize the responses of only a small proportion of the total microbial community present in any given environmental sample. The strategy advocated by Hicks et al. (1990) maximizes the relevance of a testing procedure by including a multiplicity of measures performed on soils in the laboratory and in the field. To achieve the desired breadth of tests, one should focus on processes of major ecological relevance including carbon cycling via respiration; nitrogen cycling via nitrogen fixation, denitrification, nitrification, and ammonification; l4CO2 production from radiolabeled amendments of pesticides, plant residues, or readily metabolizable carbon compounds such as glucose and acetate; extracellular enzyme assays such as phosphatase, dehydrogenase, and

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peroxidase; enumeration of common microbiological groups (i.e., bacteria, fungi, actomycetes, protozoa, algae); and measures of overall metabolic status (ATP concentration, adenylate energy charge, etc.). Hicks et al. (1990) closed their review article with several conclusions. Notable among these were: 1. “Agricultural chemicals do not appear to have any long-term harmful effects on soil microbial activity when applied at recommended field levels.” 2. “When exposed to xenobiotic compounds, various segments of the soil microbial community are affected to different extents. The degree to which a xenobiotic affects microbial activities is largely dependent upon the chemical, its dosage and method of application, and the particular physicochemical characteristics of the soil.” 3. “The most pressing issue is deciding which of the available (measurement) methods are the most valid for investigating xenobiotic effects and how can they be applied for best results.” 4. “A comprehensive system for evaluating the effects of xenobiotics on soil microbial activity should be established.”

These four conclusions reflect the general condition of “effects testing” in microbial ecology. Regarding the specific objectives of this chapter, one can confidently modify these four conclusions by substituting “agricultural practices” for both “agricultural chemicals” and “xenobiotics” and by substituting “subsurface microorganisms” for “soil microbial activity.” In other words, methods for measuring responses of subsurface microorganisms to environmental change are tenuous.

c. MEASURES OF THE IMPACT OF AGRICULTURAL PRACTICES ON SUBSURFACE MICROORGANISMS

1. Agricultural Practices That Cause Physical Changes in the Subsurface Habitats Table IV provides a framework for understanding the types of agriculturally induced changes in the subsurface habitat and their potential and documented impacts on subsurface microorganisms. There are essentially two different categories of changes that can be imposed on the subsurface by agricultural practices: physical and geochemical. Physical changes are direct effects of water infiltration (as discussed in Section I11 and Fig. 6, water infiltration is the primary means by which agriculture influences the subsurface habitat), independent of solutes that may be present. Geochemical changes result from solutes deposited in the subsurface after being entrained in water infiltrating through from the surface. A widespread agricultural practice, surface irrigation, can readily increase the flux of water infiltrating through to the subsurface. When Brockman et al. (1992)

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E. L. MADSEN Table IV

Types of Agriculturally Induced Changes in the Subsurface Habitat and Their Potential and Documented Impacts on Subsurface Microorganisms

Type of change

Physical changes Increased volume of water infiltrating to the subsurface

Decrease volume of water infiltrating to the subsurface

Alteration of soil structure to increase or diminish permeability

Temperature

Geochemical changes Increased ionic strength

PH

Potential or documented impact on subsurface microorganisms

Shift in microbial community away from desiccation-resistant populations Transport of microorganisms from soil into and within the subsurface habitat

Shift in microbial community toward desiccationresistant populations Diminished transport of microorganisms from soil into and within the subsurface habitat

Increased or diminished flux of water infiltrating from the soil may alter microbial populations (as above) Warm surface waters infiltrating to shallow aquifers may stimulate microbial activity strictly because of increased temperatures. Deposition of salts from irrigation waters may select for salt-tolerant populations Solutes present in infiltrating waters may affect acid/base equilibria which, in turn, may stimulate or inhibit various populations in the microbial community

Selected references

Brockman er a/. (1992)

Ijzerman et d.( 1993); Harvey and Garbedian (1991 ); Powelson er a/. (1990); Scholl and Harvey ( 1992); Tim and Mostaghimi (1991); Wan ri a/. ( 1994) Kieft et N / . ( 1993)

Ijzerman P I ol. ( I 993); Harvey and Garbedian (1991); Powelson er a/. ( 1990); Scholl and Harvey ( 1 992); Tim and Mostaghimi ( I 99 I); Wan et a/. (1994) Undocumented

Undocumented

Undocumented

Undocumented

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Table IV-Conrinurtl ~

Type of change Specific solutes Nitrate introduction

Pesticide introduction

Introduction o f other organic compounds

Introduction of other nutrients

~~

Potential or documented impact on subsurface microorganisms

Nitrate leaching into the subsurface may satisfy nutritional nitrogen requirements and/or act as final electron acceptor under anaerobic conditions Pesticides may serve as carbon and/or nitrogen sources, act as toxins, or have no impact on subsurface microorganisms Crop root exudates and compounds released from decay of crop residues may serve as carbon and energy sources Fertilizer components (especially phosphorus, perhaps potassium and urea) may relieve nutrient limitations, hence stimulate microbial growth and activity

Selected references

Smith er ul. (1991a,h); Starr and Gillham (1993); Wilson Pt a / . ( 1990)

Agertved (1992); Dippel et cil. (1991); Konopka (1993); Konopka and Turco ( 199 I ); McMahon et a/. ( 1992) Undocumented

Undocumented

(Table IV) compared the microflora of two cliff face-derived sediments that were geologically similar but had different moisture regimes, the viable microbial communities were also quite different. In these Columbia River Basin sediments, the “control” field sediment (which received no irrigation water) was rich in actinomycetes (Gram-positive spore-forming bacteria). However, the field sample of sediment that had received irrigation water was dominated by Gram-negative nonspore-forming bacteria. Thus, infiltration of water at this arid field site appears to have produced a substantive change in the composition of the subsurface microbial community. It is not certain precisely how the change in subsurface community was achieved. In all probability the community composition was altered both because Gram-negative bacteria were transported into the sediment with the water and because the altered moisture regime may have favored growth and survival of the introduced and indigenous Gram-negative bacteria.

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The potential for microorganisms (especially bacteria and viruses) to be transported from surface to subsurface environments in both saturated and unsaturated systems has been reasonably well documented (Table IV). The quantity of microorganisms entrained and transported is considered to be roughly proportional to the flux of water. As discussed in Section 111 and Table 111, the converse of irrigation is also a routinely implemented agricultural practice. Under certain conditions it is conceivable that direction of moisture away from agricultural soils could cause a shift in subsurface microbial populations toward those that tolerate desiccation. Although Kieft et al. (1993) (Table IV) did not work beneath agriculturally active soil (instead the study area was approximately 400 m beneath the Nevada test site), these researchers showed that the ambient low-moisture conditions found at the study area correlated well with the physiological capacity of sample-derived bacteria to tolerate desiccation. The final two entries of the Physical Changes section of Table IV are purely speculative. A variety of soil tillage and crop growth practices, especially passage of farm equipment over the soil surface and deep penetration of plant roots, routinely alter soil structure, hence permeability characteristics (Section 111). Such alterations in permeability can undoubtedly influence the volume of water reaching the subsurface and alter microbial populations either toward or away from desiccation resistance as already discussed. Depending on climatic conditions, heat capacities of subsurface sediments, and infiltration rates, it is also conceivable that the warmth of rapidly infiltrating irrigation waters could stimulate microbial activity in cooler subsurface zones. Such a temperature effect has never been documented, however.

2. Agricultural Practices That Cause Geochemical Changes in the Subsurface Habitat In the Geochemical Change section of Table IV, increased ionic strength is a type of environmental perturbation that is certain to have occurred throughout the world in arid field sites under irrigation. Soil salinization represents one of the major problems implicit in many modern agricultural practices (Frenkel and Meiri, 1985; Magaritz and Nadler, 1993; McTernan and Mize, 1992; Umali, 1993). Despite the near certainty of salt deposition in arid climate subsurface environments, the impact of increased salinity on subsurface microorganisms has not been investigated. However, at least one study designed to assess the impact of salt stress on soil microorganisms has been completed. Kilham (1985) tested a variety of stresses, only one of which (NaCI) was relevant to salinization processes. The investigator amended soil samples with "C-labeled glucose and measured both evolved W O z and extracted ['T]glucose after exposure to 0.3 M NaCI. An additional metabolic measure was dehydrogenase activity. Kilham

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53

( 1985) found that the NaCl amendment decreased both glucose respiration and

dehydrogenase activity. These same effects are likely to occur for subsurface microorganisms. Much as was the case for increased ionic-strength effects on subsurface microorganisms above, it is likely that other solutes entrained in infiltration waters may also alter the geochemical composition of groundwater. These compositional alterations may stem from mechanisms such as acidbase reactions or via contribution of both crop-derived and fertilizer-derived organic and inorganic nutrients. However, neither of these geochemical changes nor their effects on subsurface microbial communities are well documented (Table IV). Nitrate contamination of groundwater is one of the most widespread environmental problems associated with agricultural practices (Spalding and Exner, 1993). Agricultural sources of nitrate include fertilizer application and animal manures. The major physiological impact of enriched concentrations of nitrate for subsurface microorganisms is its utilization under anaerobic conditions as a final electron acceptor. When used as a final electron acceptor, nitrate may undergo microbial denitrification reactions to N2 or may be completely reduced to ammonia (Tiedje, 1988; 1994). Although the denitrification potential for subsurface microorganisms is well documented (see Table I), only relatively rarely has in situ denitrification activity by groundwater microorganisms been demonstrated (see Korom, 1992). A selection of representative subsurface denitrification studies is cited in Table IV. These studies are discussed next. At a field site contaminated with sewage treatment effluent, Smith et al. (1991 b) documented in situ denitrification activity by subsurface microorganisms by determining trace amounts of N,O (a key denitrification intermediary metabolite) in water chemistry profiles from the site. An innovative study by Starr and Gillham ( 1993) examined denitrification by subsurface microorganisms in two agricultural areas in Ontario. The acetylene block technique (which allows denitrification activity to be measured via accumulation of relatively high concentrations of N 2 0 ) was applied to cores of subsurface sediments incubated in the laboratory and to sealed volumes of well water incubated in the field. In unamended in situ assays at one of the study sites, groundwater microorganisms converted NO,- to N 2 0 , providing convincing evidence that the agriculturally derived NO, - underwent microbially mediated denitrification reactions in situ. Starr and Gillham ( 1993) also found that denitrification rates were sometimes limited by organic carbon (which serves as an electron donor for the reaction). In addition, the authors concluded that the active subsurface microbial community was responsible for preventing migration of nitrate by removing it as it entered the site. Additional evidence for in situ denitrification activity in groundwater beneath fertilized agricultural soils has been obtained by Wilson et al. (1990). These researchers used measurements of trace gases and stable nitrogen isotopes to compute N2 to Ar ratios, microbial fractionation of I5N versus I4N, and historical re-

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charge temperatures for groundwater samples along a landscape gradient. Wilson et al. (1990) found that N2 to Ar ratios increased markedly down gradient; this and related data allowed these investigators to infer that microorganisms had reduced as much as 33 mg/liter of nitrate in the anaerobic zone of a limestone aquifer. The microbiological fate of synthetic pesticides in groundwater systems has also been investigated to a limited extent (Table IV). Both Konopka and Turco ( 1 99 1) and McMahon et al. (1 992) conducted laboratory studies which incubated subsurface sediments in the presence of radiolabeled herbicides (atrazine and/or metolachlor). Despite the considerable duration of the experiments (up to 128 days) and the presence of [ 14C]glucosemineralization activity, neither Konopka and Turco ( I99 1 ) nor McMahon et al. (1992) were able to demonstrate microbially mediated conversion of the herbicides to COz. Using an innovative and challenging field approach, Agertved et al. (1992) examined the behavior of two herbicides, MCPP and atrazine, injected into groundwater beneath the Canadian Forces Base in Borden, Ontario. The fate of the herbicides was monitored using a conservative tracer (chloride), multilevel piezometers, and a partially enclosed chamber installed in the field. Furthermore, laboratory incubations of sediment-groundwater mixtures amended with the herbicides were also prepared. After 74 days of incubation in the laboratory microcosms, Agertved et al. (1992) failed to discern any herbicide biodegradation activity. Similarly, using a decline in the ratio of atrazine to chloride in field tests to indicate microbial metabolism, no subsurface biodegradation activity for atrazine was detected. However, Agertved et al. ( 1992) did report a decrease in the field concentration of MCPP relative to chloride. Despite lack of corroboration by other tests, the authors attributed the MCPP decline to in situ metabolism by subsurface microorganisms.

3. Testing Integrated Effects of Agricultural Practices on SubsurfaceMicroorganisms It is evident from these series of reports (Table IV) that nitrogen transformations, particularly denitrification reactions, have been thoroughly studied in nitrate-enriched groundwaters beneath agricultural lands. Furthermore, these reports indicate that pesticide biodegradation activity in groundwater has been sought but, in general, not found. There is an analogy here between subsurface denitrification/pesticide research and broader pollution abatement research already discussed in Sections II.A.2, II.B.2, and 1V.A. For denitrification, pesticide metabolism and microbial detoxification processes as a whole, the concern motivating scientific investigations has not been “preservation of known and beneficial subsurface microbial ecosystem processes” but rather “now that the contaminants have escaped, are the indigenous subsurface microorganisms capable of detoxifying their habitat?” Implicit in these types of research approaches is a very narrow focus for discerning the impact of agricultural processes on subsurface micro-

SUBSURFACE MICROBIAL. ECOLOGY

55

bial ecology. Often the only aspect of subsurface microbial ecology that is truly of interest is a single process (e.g., denitrification activity or biodegradation activity) that will undo the single environmental insult of interest (e.g., groundwater contamination by nitrate, pesticides, or other organic compounds). Section 1V.B and portions of Sections 1I.B.I and 2 reviewed the spectrum of measures that have (somewhat unsatisfyingly) been developed and applied to soil systems for evaluating impacts of environmental perturbations on microbial communities. As may be evident from the discussion in Sections 1V.C. 1 and 2, and the paucity of listings in Table IV, such broad-based biochemical impact studies have not been conducted on groundwater microbial communities. However, Van Beelen et al. ( 199 I ) have conducted limited tests on the effects of environmental pollutants on subsurface microflora. In this report, the field site from which the subsurface samples were derived was not thoroughly described, but uncontaminated soils from depths up to 4 m were examined. Van Beelen et al. (199 1) used aerobic and anaerobic respiration of I4C-labeled acetate and glucose to assess the response of subsurface microorganisms to four model pollutant materials: acid (hydrogen chloride), cadmium, chlorite, and a wood preservative (pentachlorophenol). Although inhibitory concentrations of these “environmental stressors” were measured, they had little, if any, relevance to agriculture practices. In light of the dearth of data directly addressing the response of subsurface microbial communities to agricultural practices, mention of related studies using soil is deemed appropriate. Soil microbial communities are generally more robust and diverse than subsurface communities (see Section II.A.2); thus, the results of studies described next should be viewed as insensitive approximations of the behavior of subsurface microbial communities. Wardle and Parkinson (1991) applied the herbicides 2,4-D and glyphosate to soils in field plots; removed soil samples 1, 5, 15, and 45 days later; and at each sampling time conducted laboratory-based respiration assays as well as enumerations of bacteria and fungi. After an extensive statistical analysis, Wardle and Parkinson ( 1 991) concluded that glyphosate had no effect on soil microorganisms and that the applied 2,4-D stimulated respiration for only the first 5 days. Beyond this time period, 2,4-D had no influence. In another study designed to evaluate the side effects of the herbicide Dinoseb on soil microorganisms, Malkomes and Wohler (1983) applied Dinoseb to two different soils (a loamy sand and a sandy loam) in the laboratory and in the field. After a 1-month exposure period these investigators measured respiratory activity, dehydrogenase activity, total ATP, nitrification, total mineral nitrogen, and decomposition of added straw. Malkomes and Wohler (1 983) found that the herbicide had no measurable effect on the sandy loam soil. However, in the loamy sand, all activities except straw decomposition were somewhat inhibited. Smith et af. ( I99 la) provided an impressive and extensive review of long-term (33-35 year) effects of herbicide application (2,4-D and MCPA) to agricultural soils. Microbiological properties (including total biomass; respiratory activity; ni-

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trogen transformation; urease and dehydrogenase activity; numbers of bacteria, fungi, and actinomycetes; and pesticide metabolism assays) were measured for soils that did and did not receive herbicide applications in the field. Smith et al. ( 1991a) concluded: ( I ) that minor short-term soil biochemical effects were seen (slight inhibition of soil enzyme activities, bacteria, actinomycetes, and slight stimulation of soil respiration by 2,4-D); but (2) that long-term effects of herbicide usage had not affected soil microbiological populations to a significant extent; and (3) that the exposed soil microorganisms had become metabolically adapted to the herbicides, thus showing enhanced biodegradation activity.

V. CONCLUDING REMARKS Throughout this chapter an attempt has been made to elucidate relationships between seemingly disparate disciplines that fall within a continuum from ecosystem ecology, geology, hydrology, agriculture, nutrient cycling, ecological effects testing, and the ecology, physiology, and molecular biology of microorganisms. An additional objective has been to use the relationships between these disciplines to weave a coherent fabric addressing the impacts of agricultural practices on subsurface microbial ecology. An itemized list describing the status of current knowledge pertinent to impacts of agricultural practices on subsurface microbial ecology is described next. These listed items are supported in sections of this chapter noted in parentheses. 1. The methods of environmental microbiology and microbial ecology provide only a “primitive ability” (Hobbie, 1993) for describing (a) the organisms present in their microhabitats in nature; (b) what these organisms are actually doing; and (c) what controls their activity and growth (Section 1V.B). 2. Given the relatively recent interest in exploring subsurface microbial ecology (aseptic sampling procedures were developed only in the late 1970s), the admittedly limited methodologies (item No. 1) have not been applied for a sufficient period to provide a robust data base for understanding the “normal” composition and function of subsurface microbial communities (Sections I, II.B.3, and IV.A), 3. The lack of understanding in subsurface microbial ecology that follows from item No. 2 poses obstacles for assessing the impact of any and all environmental perturbations of subsurface microbial ecology, including agricultural practices (Section 1V.B). 4. Despite limitations of the information describing the inhabitants of subsurface environments and both their potential and actual metabolic activity (item Nos. l-3), new data and new types of data are arriving at an accelerating rate. Thus our level of understanding is rapidly improving (Sections II.A.2, II.B.2 and 3, and 1V.C).

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5. Ongoing developments in disciplines such as molecular biology and biochemistry (describing cellular- and subcellular-level processes) and ecosystemeffects testing (describing population- and community-level processes) offer much promise for investigating the status of subsurface microbial communities in field sites. Application of the new molecular biology and biochemical methods (derived from laboratory-grown single organisms) to real-world microbial communities needs to be aggressively pursued. When perfected these methodologies will provide far-reaching insights (Sections 1I.B. 1 and 1V.B). 6. One simple reason for limited knowledge of the impacts of agricultural practices on subsurface microbial ecology is that very few scientific investigations that directly address these issues have been conducted. This interdisciplinary research area has not traditionally been a high priority in governmental research programs, although this policy is probably changing both in North America and Europe (Section 1V.C). 7. Regardless of the limitations of current knowledge describing how agricultural practices affect subsurface microbial ecology, decent progress in this area has already been made. We know that infiltrating water is the primary means by which materials are transported from soil into the subsurface. Furthermore, the water itself and many of the entrained materials (microbial cells, salts, organic or inorganic nutrients, pesticides, etc.) affect the status of subsurface microorganisms. In light of promising developments in microbial ecological methods and increasing interest in the interface between agriculture and groundwater resources, rapid advancements in this important interdisciplinary area are imminent (Sections LA, II.A.l, 111, and 1V.C).

ACKNOWLEDGMENTS The author expresses his appreciation for provocative and insightful discussions with W. C. Ghiorse. These discussions assisted in the development of many ideas presented in this chapter. Research support during preparation ofthis manuscript was provided by the Air Force Office of Scientific Research (Grants AFOSR-9 1-0436 and 93-NL-073) and the Cornell Biotechnology Program, which is sponsored by the New York State Science and Technology Foundation (Grant NYSCAT 92054). and consortium of industries and the National Science Foundation. 1 am grateful to Patti Lisk and Shirley Cramer for expert manuscript preparation.

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HERBICIDE-RESISTANT FIELD CROPS Jack Dekker ' and Stephen 0.Duke? Agronomy Department Iowa State University Ames, Iowa 5001 1 'United States Department of Agriculture Agricultural Research Service Southern Weed Science Laboratory Stoneville, Mississippi 38776 I

I. Introduction 11. Mechanisms of Herbicide Resistance A. Exclusionary Resistance Mechanisms B. Altered Molecular/C~ellularSite (Target) of Herbicide Action C. Site of Action Overproduction 111. Selection for Herbicide-Resistant Variants A. Sources of Resistance Genes and Traits B. Traditional Plant-Breeding Techniques C. Biotechnological Techniques W. Herbicide-Resistant Crops by the Herbicide Chemical Family A. Triazines B. Acetolactate Synthatase Inhibitors C. Acetyl-CoA Carboxylase Inhibitors D. Glyphosate E. Bromoxynil F. Phenoxycarboxylic Acids (e.g., 2,4-D) G. Glufosinate (Phosphinothricin) H. Other Herbicides V. Summary References

I. INTRODUCTION Traditionally, herbicidal chemicals have been selected for their weed-killing characteristics as well as for their effects on crops. One of the primary limitations in this search has been lack of tolerance to the chemical by one or more of the major world crops (e.g., rice, maize, soybean, wheat, rapeseed). Also, this process 6') Advonces in Agmnom.v Vor'ume 14 Copyright 0 1995 by Acadernlc Press. Inc. All rights of reproduction in any form reserved.

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has led to the development of herbicides that often control only part of the weed species present in that crop, necessitating the use of other herbicides or management strategies. Herbicides that control a broad spectrum of weed species often have limited utility because they also injure crops. Additionally, often the most desirable herbicides for weed control and crop safety have other less desirable characteristics (e.g., environmental, economic). For these and other (e.g., regulatory) reasons, herbicide development has been very expensive, time consuming, and has yielded a relatively small number of chemicals with desirable agronomic, environmental, and economic qualities from a potentially large group of candidates. With the availability of many new biotechnological tools to incorporate herbicide resistance into crops, this traditional approach has been reversed in some instances. The possibility now exists to select herbicides for desirable weed control, as well as environmental and economic qualities at the beginning of the discovery process, and incorporate resistance into the crop(s) after these herbicides have been identified. The development of herbicide-resistant crops (HRC) could provide many advantages in the efficient, safe, and economical production of crops, although there may be risks involved in the development of specific technologies (Bright, 1992; Dekker and Comstock, 1992; Duke et al., 1991; Dyer et al., 1993b; Goldburg et al., 1990; Goodman, 1987; Hindmarsh, 1991; Kline, 1991; Miller, 1991; Williamson, 1991). Assessment of the risks and benefits of individual HRCs is complex, and value judgements about the appropriate utilization of HRCs have been purposefully avoided in this chapter. Several environmental advantages may result from the development of HRCs. Herbicides and their associated HRCs could be developed with less persistence in the environment (e.g., herbicide “carryover” to subsequent crops in a rotation; accumulation in other sites in the landscape, biosphere), less damage to off-site targets (e.g., adjacent susceptible crops; homes and farmsteads; surface waters), decreased undesirable movement in the environment (leaching downward through the soil profile to subsoil, groundwater sites; volatility and movement to off-target sites), and low acute and chronic toxicity to humans and animals. Also, these technologies could result in several advantages to crop producers, including less expensive production costs. HRC systems could result in increased production options to growers by providing more weed control strategies, resulting in an increased likelihood of the use of multiple, integrated approaches to weed management. For these and other reasons, crop germ plasm improvement now also includes selection and incorporation of desirable herbicide-resistancequalities. Conversely, production and utilization of crops resistant to herbicides with undesirable characteristics could have adverse toxicological and environmental consequences. However, there is comparatively little effort being expended to generate and develop crops resistant to herbicides that might raise such concerns.

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The term herbicide resistance is used to describe the ability, trait, or quality of a population of plants within a species or larger taxon, or of plant cells in culture, to withstand a particular herbicide at a dosage that is substantially greater than the wild type of that plant is able to withstand, with a near normal life cycle. Herbicide resistance can be conferred in many ways and includes the pre-existing lack of susceptibility, selection of resistant variants from within a diverse species, or resistance achieved by genetic manipulation (e.g., crops). Because of the diversity of possible mechanisms, a complete description requires the use of appropriate qualifiers to the term “resistance.” This chapter reviews information about how crop plants resist herbicides and how resistance is selected for in plants and surveys specific herbicide-resistant crops by chemical family. The scope of this discussion includes HRCs derived from both traditional and biotechnological selection methodologies. The toxicological and environmental concerns that have been raised and discussed in other reviews (e.g., Dekker and Comstock, 1992; Duke et al., 1991; Dyer et al., 1993a,b) will not be discussed here. The following discussion reviews a very large literature, but emphasizes that information relevant to field crop improvement.

II. MECHANISMS OF HERBICIDE RESISTANCE Plants avoid the effects of herbicides they encounter by several different mechanisms (Holt et al., 1993; Vaughn and Duke, 1991). These mechanisms can be grouped into two categories: those that exclude the herbicide molecule from the site in the plant where they induce the toxic response (exclusionary resistance) and those that render the specific site of herbicide action resistant to the chemical (site of action resistance). Several of these mechanisms often act in concert to produce whole-plant resistance. Although it is difficult to generalize over the many plant species affected by the use of herbicides in crop production (weeds and crops), differential resistance is primarily due to herbicide metabolism, secondarily due to site of action mechanisms, and lastly due to differences in interception and absorption (Devine ef al., 1993a.b). In general, our understandings of plant physiology, morphogenesis, and biochemistry are significantly less than our understanding of herbicides. This situation limits the options available for crop improvement by enhancement of herbicide resistance. Herbicides with a single site of action in plants have been the focus of the first HRCs being developed because they rely on single gene mutants, or single gene transformants, for resistance. As our understanding of plant biology increases, more targets for crop improvement, especially quantitative traits, will become available.

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A. EXCLUSIONARY RESISTANCE

MECHANISMS

The specific site at which herbicides act is protected to some extent by the morphology and physiology of the individual plant species and subspecific variant. The structural characteristics of the leaf, root, and vascular system influence the movement of herbicides into and through the plant. The metabolic activities in living plant cells also are capable of detoxifying herbicides. At the present time, the only exclusionary strategy for HRC production is to transform plants with genes encoding enzymes that degrade herbicides. 1. Herbicide Uptake

Herbicides typically are encountered by the plant at the soil-root, or leaf-air, interfaces. Differential absorption, adsorption, and uptake of the herbicide into the plant at these interfaces can occur (Hess, 1985). Herbicides must pass the nonliving portions (apoplast) of the plant and enter the living parts (symplast) of the plant to have an effect. Herbicide resistance can be conferred in individual plant species by structures capable of excluding the entry of these chemicals into the living part of the plant. The first plant structures that encounter herbicides are nonliving and include those associated with the leaf, stem, and root surfaces. Movement of a herbicide across these nonliving structures is complex and involves the nature of the herbicide applied (including the formulation ingredients), the physical properties of the cuticle (epicuticular wax, cuticular wax, cutin, pectin fibers, cell walls, and the cuticular “peg” between cell walls), the species and age of the plant, and the environment (Devine et al., 1993a,b). Herbicide absorption by the plant from the soil solution can occur through root, shoot, or seed tissue. Root absorption occurs through passive diffusion of the soil solution through the epidermis (suberized in older root tissue) and cortex. These outer structures are separated from the root endodermis (containing the vascular tissues in the stele) by a suberized layer, the Casparian strip. Herbicide uptake can occur anywhere in the root system, but absorption primarily occurs at the apical end (Jacobson and Shimabukuro, 1982; Strang and Rogers, 197 1). It is in this area of the root system that most water and ion uptake takes place, the place where the Casparian strip is least developed (Tanton and Crowdy, 1972). Despite the important role herbicide uptake through shoots and roots plays in the total resistance of a plant to a herbicide, it is not generally regarded as a primary crop improvement target for enhancing herbicide resistance. It more likely plays a secondary role in conjunction with other plant factors, the sum of which act to produce the whole-plant level of herbicide resistance. Once the complex chemical nature of leaf waxes (i.e., epicuticular, cuticular) is better understood, and once the exact nature of herbicide

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uptake by roots in specific crop species is well characterized, it is conceivable that structures modified by single (or few) genes could be the focus of selection schemes.

2. Translocation After herbicides are taken into the plant they often are transported to the site of activity. This movement can be either in nonliving (apoplast; eg., cell walls, xylem) or in living tissues (symplast; e.g., cell plasmalemma, phloem). This translocation of herbicides can either be over relatively short distances (e.g., paraquat activity in living cells near the point of entry) or it can be accomplished by the vascular system over relatively longer distances (e.g., glyphosate translocation in the phloem). Whether a particular herbicide is moved over short or long distances, and what plant structures facilitate or retard its movement, is a function of the chemical nature of the herbicide, the plant species, the condition of the plant (e.g., age, stress, nutrition, etc.), and the environment in which both are found. Although much of the physiological and morphological effects of the plant on herbicides are understood (Devine et al., 1993a), it has been far easier to modify the chemical properties of the herbicide (Crisp and Look, 1978; Crisp and Larson, 1983; Lichtner, 1986) for resistance than it has been to alter the translocation factors in the plant. Additionally, differential translocation of herbicides within different plant species is intimately related to concurrent herbicide metabolism, a confounding factor when studying translocation resistance mechanisms. For these reasons, crop improvement by alteration of solute translocation factors in crop plants will probably remain a low priority for the enhancement of herbicide resistance. In few cases of herbicide resistance has translocation proven to be a significant factor.

3. Compartmentation Herbicides can be sequestered in several plant locations before they reach the site of action. Some lipophilic herbicides may become immobilized by partitioning into lipid-rich glands or oil bodies (Foy, 1964; Stegink and Vaughn, 1988). Sequestration of herbicides in vacuoles, followed by metabolism, is another possible mechanism of resistance by immobilization (Coupland, 1991). Differential metabolism of herbicides has been reported in cell vacuoles from soybean suspension cultures of both susceptible and resistant variants (Schmitt and Sandermann, 1982). Sequestration (Fuerst et al., 1985; Norman et al., 1993), metabolism (Amsellem eral., 1993; Shaaltiel eral., 1988), or both (Lehoczki eral., 1992) has been suggested as the possible basis of paraquat resistance in weedy Conyza spp. As with uptake and translocation, herbicide resistance enhancement by manipulation

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of these complex, poorly understood, physiological phenomena will probably remain an insignificant objective for crop improvement for some time.

4. Metabolic Detoxification One of the most useful mechanisms of resistance for crop improvement is the enhancement of herbicide metabolism to detoxify the chemical before it reaches the site of inhibition. Metabolic detoxification of herbicides and other chemicals by plants are the major mechanisms providing resistance in crops, and this topic been extensively reviewed (e.g., Baldwin, 1977; Casida and Lykken, 1969; Cole et al., 1987; Devine et al., 1993a; Fedtke, 1982; Hatzios and Penner, 1982; Kearney and Kaufman, 1975; Lamoureux and Frear, 1979; Menzer, 1973; Owen, 1987; Sanderman et al., 1977; Shimabukuro et al., 1982; Shimabukuro, 1985). Crop enhancement by herbicide detoxification can be accomplished either by selection for variants or mutants with increased levels of specific metabolic activities or by introduction and transformation of crop plants with genes from other organisms. Herbicide safeners, antidotes, antagonists, protectants, or synergists are herbicidally inactive chemicals that are applied to crops and weeds for improved weed control. Many types of these chemicals exist, but often they are used to enhance the action of a herbicide by either interfering with weed metabolism (hence improved weed control) or enhancing crop metabolism (hence crop protection) (Ezra et al., 1985; Lamoureux and Rusness, 1986). The reader can refer to some related reviews (Devine et al., 1993a; Pallos and Casida, 1978) for a discussion of this approach to crop improvement. The biochemical reactions that detoxify herbicides can be grouped into four major categories: oxidation, reduction, hydrolysis, and conjugation. Oxidation of herbicides is among the most important detoxification reactions providing resistance in plants. These reactions are catalyzed by monooxygenases known as mixed function oxidases and include alkyl oxidation, aromatic hydroxylation, epoxidation, N-dealkylation, 0-dealkylation, and sulfur oxidation. Much of the biochemistry of these reactions has not been characterized, but aryl hydroxylation may be the most common reaction leading to herbicide detoxification (Shimabukuro, 1985). Reduction of herbicides is of much lesser importance in plants compared to other metabolic reactions conferring resistance. Aryl nitroreduction is an important reaction in herbicide degradation, but probably plays a minor role in herbicide detoxification, and may compete with the more important glutathione conjugation reaction (Lamoureux and Rusness, 198I). Hydrolysis of herbicides is a common plant reaction and is important in detoxification of several herbicides, including bromoxynil (Buckland et al., 1973), cyanazine (Benyon et al., 1972), and propanil (Lamoureux and Frear, 1979), as

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well as other ester, amide, and nitrile-containing herbicides. Carboxylic acid ester herbicides such as 2,4-D (ester; Loos, 1975), as well as several graminicides such as diclofop-methyl (Fedtke and Schmitt, 1977; Shimabukuro et al., 1979), are hydrolyzed to the active, free acid once in the plant leaf (Loos, 1975). Conjugation of herbicides to glucose, amino acids, or glutathione is a major reaction in detoxification and is a potentially major objective for crop resistance improvement. Conjugation in plants is the reaction in which a herbicide metabolite formed in earlier reactions is joined with an endogenous substrate to form a new, larger compound. Typically this reaction converts a lipophilic herbicide molecule into a more water-soluble compound. This more polar compound is then subsequently metabolized, leading later to a bound herbicide residue. Glucose conjugation occurs to many herbicides (or their metabolites) with amino, carboxyl, or hydroxyl functional groups. Examples of glucose conjugation include 0-glucosides (e.g., chlorpropham; Still and Mansager, 1972), N-glucosides (e.g., propanil; Still, 1968), and glucose esters (e.g., diclofop-methyl; Shimabukuro et al., 1979). More complete treatment of this area can be found in Frear (1 976) and Hatzios and Penner ( 1982). Amino acid conjugation occurs primarily with acidic herbicides, through an aamide bond (Mumma and Hamilton, 1976). For example, 2,4-D forms major glutamic (2,4-D-Glu) and aspartic (2,4-D-Asp) acid conjugates, as well as minor conjugates with alanine, leucine, phenylanaline, tryptophan, and valine. Glutathione conjugation often involves the reaction of the active parent herbicide molecule and glutathione (GSH), and is one of the most important types of conjugation conferring resistance in plants and has been extensively reviewed (Baldwin, 1977; Hatzios and Penner, 1982; Lamoureux and Frear, 1979; Lamoureux and Rusness, 1981; Shimabukuro et al., 1978, 1982). This type of detoxification is important because of the wide range of potential substrates, or herbicides, that can be conjugated. This conjugation is accomplished primarily by glutathione-S-transferases with different specificities to different herbicide substrates. GSH conjugation involves a nucleophilic displacement reaction between GSH and the herbicide, resulting in direct detoxification of the active molecule. GSH conjugation reactions can also proceed nonenzymatically due to the high degree of reactivity of some herbicides (Lamoureux ef al., 1973; Leavitt and Penner, 1979). Many herbicide groups are conjugated by GSH and include a-chloroacetamides (e.g., metolachlor), diphenylethers, thiocarbamate sulfoxides, and 2-chloro-s-triazines. For example, atrazine is directly detoxified by nucleophilic displacement when a conjugation reaction occurs between it and GSH (Lamoureux et d., 1973). The appearance of herbicide-resistant weeds due to enhanced GSH conjugation has been reported (e.g., velvetleaf, Abutilon theophrasti; Gronwald ef a/., 1989), as well as evidence of inter- and intraspecific variation in GSH conjugation in Setaria spp. (Wang and Dekker, 1994).

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There is relatively little interest in generating HRCs by manipulation of genes encoding plant enzymes that detoxify herbicides. The reason for this is unclear. However, the movement of microbial genes that encode herbicide-detoxifying enzymes into crops by genetic engineering is a strategy being used to produce HRCs for use with glyphosate, giufosinate, bromoxynil, dalapon, and 2,4-D. Details of the production of these HRCs can be found in the next section.

B. ALTEREDMOLECULARKELLULAR SITE(TARGET) OF HERBICIDE ACTION Resistance in plants can be due to differential sensitivity of molecular target sites, usually sites of herbicide activity and inhibition. Resistance is conferred on a plant by alteration or mutation of the different target site protein structures. Much of what is known about the molecular, biochemical, and physiological nature of these important sites of action comes from understandings gained from herbicide-resistant weeds (e.g., Holt and LeBaron, 1990; LeBaron and Gressel, 1982; Smith et al., 1988). Variability in the functional qualities of these target site mutants exists and they may be equally (e.g., sulfonylurea resistance) or less competitive (s-triazine resistance) than their wild types with the wild type site of action protein (e.g., Beversdorf et al., 1988). Examples of some important molecular sites of action, and the herbicides that interact with them to cause inhibition, follow. In each instance, a more complete presentation can be found in subsequent sections dealing with specific herbicide groups. The best characterized is the site of s-triazine inhibition of photosynthesis in the chloroplast. s-Triazine herbicides (e.g., atrazine, cyanazine) inhibit photosynthetic electron transport at the reducing side of photosystem 11. These herbicides bind to the reaction center D- 1 protein, the native binding site for plastoquinone (Arntzen et a/., 1987; Barber, 1987; Mattoo et al., 1989; Trebst, 1986, 1991). A key enzyme in lipid biosynthesis, acetyl-coenzyme A carboxylase (ACCase; Hanvood, 1988a) is the molecular site of inhibition of the aryloxyphenoxypropionates (e.g., diclofop-methyl) and cyclohexenedione (e.g., sethoxydim) herbicides (Burton et al., 1987; Hanvood, 1988b; Harwood et al., 1987; Secor and Czeke, 1988). These are important groups of herbicides used to control graminaceous plants. Several groups of herbicides target amino acid biosynthesis (Devine et al., 1993a; Kishore and Shah, 1988). Branched-chain amino acid synthesis (isoleucine, leucine, and valine) is inhibited by three classes of herbicides: the imidazoh o n e s (Shaner and O’Connor, 1991), sulfonylureas (Beyer et al., 1988; Blair and Martin, 1988; LaRossa et al., 1987), and triazolopyrimidine sulfonanilides (Gerwick et al., 1990; Subramanian and Gerwick, 1989). All three groups have as their

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molecular site of action the enzyme acetolactate synthase (ALS; also known as acetohydroxy acid synthase, AHAS).

c. SITE OF ACTIONOVERPRODUCTION Resistance can be conferred by overproduction of the target site, diluting the herbicide that reaches the target site, thus allowing enough additional target protein to remain to complete normal functions and growth (Devine et al., 1993a; Goldsbrough et al., 1990; Rogers et al., 1983; Shah et al., 1986). Overproduction of the molecular site of action can confer resistance by either multiple copies of the gene encoding the target site protein (gene amplification; Steinrucken et al., 1986) or (and) by increased target site protein gene expression (Hollander-Czytko et al., 1988). For example, glyphosate resistance is conferred by the overproduction of 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS), a key enzyme in the pathway synthesizing many plant aromatic compounds. This strategy has been used to develop resistance, but has not been entirely successful in engineering glyphosate-resistant crops (Kishore and Shah, 1988).

111. SELECTION FOR HERBICIDERESISTANT VARIANTS The selection for herbicide resistance for HRCs can be accomplished by both traditional plant breeding and biotechnological techniques. Before the advent of these newer approaches, few HRCs were developed using traditional plant breeding methodologies. HRCs derived from biotechnological techniques provide more ways for crop and cultivar improvement; these techniques have been reviewed previously (Botterman and Leemans, 1988; Fincham and Ravetz, 1991;Goodman and Newell, 1985; Gressel, 1987, 1989, 1992, 1993; Mazur and Falco, 1989; Oxtoby and Hughes, 1989, 1990; Quinn, 1990; Schulz et al., 1990). Herbicideresistant mutants probably occur in populations of all plant species, but the frequency of their occurrence is unknown (Warwick, 1991). Single sites of action herbicides provide the most likely candidates, often allowing for single loci mutants, or single gene transformants. Multiple sites of action resistance are the hardest to select for and their development awaits more complete information of complex quantitative traits. Some multiple resistance plants have been observed in weedy populations (Powles and Howat, 1990). The following sections briefly review the several approaches used in the selection and incorporation of resistance in HRCs.

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A.

SOURCES OF RESISTANCE

GENESAND TRAITS

Microorganisms and higher plants and animals are potential sources of resistant genes. Each of these organisms presents its own set of advantages, problems, and technical considerations. The isolation of genes from microorganisms is often easier than from higher plants, but the right organism must be found first (Gressel, 1993).Crop plants are inherently resistant to many herbicides, and improvements can be made with proper selection efforts in many instances. One of the richest sources of resistance is in herbicide-resistant weeds. Many species of weeds have resistant populations, and prolonged selection enriched their frequency in many agricultural fields (Holt and LeBaron, 1990; LeBaron and Gressel, 1982; Powles and Howat, 1990). Studies of these resistant mutants have revealed many new insights about both the resistance mechanisms themselves, as well as new information about plant biological systems (e.g., atrazine: Dekker, 1993: Hirschberg et al., 1984; Trebst, 1986).

B.

TRADT IO INAL

PLANT-BREEDING TECHNIQUES

Historically, few HRCs have been developed with traditional plant-breeding approaches (Beversdorf, 1987; Beversdorf and Kott, 1987; Snape et al., 1987, 1990, 1991; Van Heile et al., 1970). This is probably due to the length of time needed to develop resistant cultivars relative to the patent life of a herbicide. Direct herbicide selection of variants within a species with enhanced resistance has been successful in many crops (Fedtke, 1991; Johnston and Faulkner, 1991). In many cases sufficient variability in herbicide response among plant populations exists from which to select improved lines (Boerboom e? aZ., 1991; Dekker and Burmester, 1988; Hartwig, 1987; Tranel and Dekker, 1992), but in other instances the amount has been insufficient (Kibite and Harker, 1991). Traditional plantbreeding methodologies (with resistance derived from weedy sources) have produced HRCs with resistance to s-triazines in rapeseed (Beversdorf and Hume, 1984) and lettuce (Mallory-Smith et al., 1993), with others coming in the future.

C. BIOTECHNOLOGICAL TECHNIQUES Selection for herbicide resistance traits, and their transfer into crops by biotechnological techniques, promises to speed development of HRCs considerably. Techniques that rely on rapid in vivo or in vitro selection and subsequent crop transformation may permit shorter times to achieve this type of crop improvement. Transformation by genetic engineering can also be rapid; however, development

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of some HRCs by these methods has been slower than originally anticipated. These methodologies include cell and tissue culture selection, hybridization, microspore and seed mutagenesis, and plant transformation techniques.

1. Cell and Tissue Culture Selection Somaclonal variation in cultured plant cells has been exploited to select herbicide resistance traits for crop improvement. Utilization of plant cell and tissue culture has provided one of the most important selection techniques for the development of HRCs (Chaleff, 1988; Chaleff and Ray, 1984; Hughes, 1983; Maliga et al., 1987). Selections for resistance using callus (maize; Anderson and Georgeson, 1989; Tubersosa and Lucchese, 1990), microspores and protoplasts (rapeseed; Swanson et al., 1988), and plant cell suspension cultures (maize; Parker et al., 1990a,b) have been used. HRCs resistant to herbicides inhibiting the ALS site of action have been developed using these techniques (Newhouse et al., 1991a,b), but in some instances resistance has been insufficient for complete crop safety (Bauman et al., 1992).

2. Hybridization The transfer of herbicide resistance from weedy relatives to crops has been used to develop HRCs. Protoplast fusion techniques have been used to incorporate striazine resistance between Solanum species (Austin and Helgeson, 1987), as well as to transfer it into cytoplasmic male sterile Brassica variants (Barsby et al., 1987). s-Triazine resistance was transferred from weedy bird’s-rape (Brassica campestris; Beversdorf et al., 1980) to several Brassica crop species, including rapeseed and rutabaga (Beversdorf and Hume, 1984). Similar approaches have been used to transfer s-triazine resistance from the weedy green foxtail (Setaria viridis, subspp. viridis Briquet) to the foxtail millet crop (S. viridis, subspp. italica Briquet) (Darmency and Pernes, 1989). Sulfonylurea-resistant prickly lettuce (Lactuca serriola) has served as the source of ALS inhibitor resistance in a new cultivar of lettuce, ID-BR1 (Mallory-Smith et al., 1993).

3. Microspore (Gametophytic) and Seed Mutagenesis One of the most powerful means of deriving novel herbicide-resistant variants for HRCs is mutagenesis. Several different approaches have been used and reviews of these techniques are available (e.g., Christianson, 1991). Mass selection of mutagenized soybean seed has been used to find herbicide-resistant crop variants (Sebastian et al., 1989). Similar approaches have been used in other crops (Dyer et al., 1993b). Microspore mutagenesis and selection have been used in rapeseed (Swanson et al., 1989).

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4. Plant Transformation The transfer of herbicide resistance genes from various sources into crop plants has been performed using several techniques. These transgenic products rely on both target site and metabolic detoxification resistance mechanisms. A s-triazineresistant gene construct composed of the mutant resistance coding sequence, expression level control sequences, and a transit-peptide encoding sequence resulted in a resistant transgenic tobacco plant line (Cheung et al., 1988). Glyphosateresistant crop development has relied on a mutant E. coli gene fused to a EPSPS enzyme chloroplast transit sequence to create transgenic plants (Della-Cioppa er al., 1987). Transgenic, glyphosate-resistant cotton, rapeseed, soybean, tobacco, and tomato crops are currently in various stages of development and commercialization (Dyer et al., 1993b). Crops resistant to ALS-inhibiting herbicides have been developed by transfer of resistant genes between different higher plant species (Falco et al., 1989; Haughn et al., 1988; Miki et al., 1990). Metabolic detoxification resistance has been transferred from microbial species to crop plants. The bromoxynil-specific nitrilase gene, encoded by the bxn gene, has been transfered into cotton, potato, tomato, and rapeseed (Dyer el a]., 1993b). The cyanamid hydratase-encoding gene from the soil fungus Myrothecium verrucaria has conferred resistance in the transgenic tobacco product (Maier-Greiner et al., 1991). Detoxification of glufosinate by acetylation is accomplished by acetyl transferase, encoded by the bar gene from Streptomyces. This gene was fused to high expression promoters and was used to produce high levels of glufosinate resistance in transformed alfalfa, poplar, rapeseed, potato, sugar beet, tobacco, and tomato crops (De Block et al., 1987). Genes for herbicide resistance have been used as selectable markers in transformation studies (Yoder and Goldsbrough, 1994). It is not likely that such genes would be left in a crop that has not been approved as herbicide resistant.

IV. HERBICIDE-RESISTANTCROPS BY THE HERBICIDE CHEMICAL FAMILY A. TIUAZINES 1. Introduction The triazine herbicide family is a large and important group of herbicides that was first discovered in 1952, and first introduced commercially in 1957. The most important member of this family is atrazine, and its introduction revolutionized weed control in maize. Other triazine herbicides include ametryne, cyanazine, prometryn, and simazine. Herbicides in this group are used to control many broad-

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leaf and grassy weeds, and are applied to the soil or foliage (often with oil-based adjuvants). Members of this group are used in many crops, including maize and sorghum. Atrazine is a relatively persistent herbicide in the environment, and environmental and health problems have been found with this chemical.

2. Mode of Action The biochemical and physiological effects of atrazine in plants are similar to those caused by many of the s-triazines. Atrazine is the most commonly used striazine herbicide in agriculture. It is rapidly absorbed by plant roots and, to a lesser extent, by plant shoots (Esser and Marco, 1975). Once in the plant it translocates readily (apoplastically) in the xylem and cell walls. When translocation ceases, it diffuses into the cell cytoplasm and chloroplast. In the presence of light it preferentially attaches to a high-affinity binding site on a rapidly turned over 32kDa protein known as the D-1 protein (Chua and Gillham, 1977). This protein is the product of the psbA gene and is a component of the photosystem I1 (PS 11) reaction center located in the thylakoid membranes (Callahan et al., 1989; Mattoo et al., 1989). Atrazine competes with quinone for separate, but overlapping, domains of this binding site (Pfister and Arntzen, 1979; Tischer and Strotmann, 1977; Velthys, 1981; Vermaas er al., 1983). In susceptible tissue, atrazine binding blocks electron transport on the reducing side of PS I1 from Q A to QBwhich causes an increase in variable chlorophyll fluorescence (Trebst, 1980). The redirection of electrons away from the blocked site results in the generation of toxic oxy-radicals and other highly reactive radical species (Bolhar-Nordenkampf, 1979; Dodge, 1982). These radicals are primarily quenched by membrane lipids (autocatalytic peroxidation), and the death of localized tissue results. If enough tissue is destroyed, homeostasis cannot be maintained and the death of the plant follows. In many resistant species atrazine is metabolized by one of three initial reactions before it reaches the chloroplast: nonenzymatic hydroxylation, N-dealkylation, and conjugation with glutathione (Ashton and Crafts, 1981; Lamoureux et al., 1973).

3. Plant Resistance Resistance to the s-triazine herbicides is a function of both alterations to the site of action as well as of metabolic exclusion before reaching that target site. Many of the insights and technologies that have been utilized for s-triazine resistance of both kinds come from understandings gained for studies of resistant weeds. a. Site of Action Resistance The most important resistance mechanism in plants to the s-triazine herbicides is that conferred by alterations to the site of action, the D-1 protein in the chloro-

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plast. Much of what we know of this type of s-triazine resistance comes from information gained as a result of the discovery of resistant weeds. s-Triazine resistance in weeds and higher plants was first discovered in 1969 in Senecio vulgnris L. (Ryan, 1970). Since that time resistance has been found in 107 weed species, infesting over 3 million hectares worldwide (Dekker et al., 1991; Holt and LeBaron, 1990; Le Baron, 1991; Le Baron and Gressel, 1982). Resistance usually appeared in agricultural and industrial situations wherein s-triazine herbicides were used continuously for at least 5- 10 years and were the only weed control method used. After the discovery of s-triazine resistance, research revealed that resistance was not a function of differential herbicide uptake, translocation, accumulation, metabolism, or by differential membrane permeability (Radosevich, 1977; Radosevich and Appleby, 1973; Radosevich and DeVilliers, 1976). Genetic analyses indicated that the resistance mechanism was maternally inherited at the whole plant level (Sousa Machado et al., 1978b).This cytoplasmic inheritance was subsequently found to be at the level of the chloroplast membrane components (Darr et al., 1981). Photoaffinity labeling studies (Gardner, 1981; Pfister et al., 1981) showed that atrazine had reduced binding to the 32-kDa chloroplast protein (D-1) in the resistant biotype (Arntzen et nl., 1982; Bowes et al., 1980; Hirschberg and McIntosh, 1983; Pfister and Arntzen, 1979; Pfister et al., 1979, 1981; Steinback er al., 1981). s-Triazines do not have a high affinity for this altered site and hence electron transport in PS I1 is not blocked (Sousa Machado et ul., 1978a). Resistance can result from point mutations to the psbA gene at several sites leading to amino acid substitutions in the D-1 protein product (Trebst, 1991). In addition to the direct effects, several structural and functional pleiotropic effects are associated with s-triazine resistance. Many of the structural changes in the chloroplast of these s-triazine-resistant mutants are similar to those in shadeadapted leaves (Boardman, 1977): increased thylakoid grana stacking, decreased starch content, lower chlorophyll a/b ratios, greater amounts of the chlorophyll a/b light-harvesting complex, and relatively lower amounts of the P700 chlorophyll a protein and chloroplast coupling factor (Burke et al., 1982; Vaughn, 1986; Vaughn and Duke, 1984). Chloroplast lipids differed between the resistant mutant and wild types (Blein, 1980; Burke etal., 1982; Pillai and St. John, 1981). These changes in lipid composition in the chloroplast membranes in the resistant mutants were correlated to enhanced resistance to lower temperature stress and greater fluidity at low temperatures (Pillai and St. John, 1981). The mutation to the psbA gene results in functional changes in resistant variants. These functional changes probably result as a consequence of the conformational changes in the quinone-binding niche and as a consequence of the secondary structural changes noted earlier. The quinone-binding pocket alterations in the mutant not only decrease atrazine-binding properties, but they also result in changes in the electron transfer properties in photosystem 11. The rate of electron

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transfer in PS I1 from the acceptor Q A to QHis reduced in the resistant mutant (Arntzen et al., 1979; Bowes et a/., 1980; Burke et al., 1982; Pfister and Arntzen, 1979). Several studies have demonstrated lower carbon assimilation efficiency, or lower productivity, in the resistant mutant compared to the wild type (Ahrens and Stoller, 1983; Beversdorf et a/., 1988; Conard and Radosevich, 1979; Holt, 1990; Holt et al., 1981; McClosky and Holt, 1989, 1991; Ort et al., 1983; Warwick, 199 1). Other studies have shown that the photosynthetic activity in these two may be similar (Ahrens and Stoller, 1983; Van Oorschot and van Leeuwen, 1984) or have found it greater in the resistant mutant relative than in the wild type under some environmental conditions (Dekker, 1993; Dekker and Burmester, 1992; Dekker and Sharkey, 1992). b. Metabolic Resistance In many resistant species atrazine is metabolized by one of three initial reactions before it reaches the chloroplast: nonenzymatic hydroxylation, N-dealkylation, and conjugation with glutathione (Ashton and Crafts, 1981; Lamoureux et al., 1973). Several weed species are resistant to s-triazines because of enhanced metabolism (Gronwald et al., 1989; Le Baron, 1991). The reduced rates of photosynthesis associated with site of action mutants is not present in these metabolicresistant populations.

4. s-Triazine Herbicide-Resistant Crops a. Traditional Plant Breeding s-Triazine resistance ( pshA gene mutant) has been incorporated into several crops. Hybridization methods have been used to transfer this type of resistance from weedy bird’s-rape ( B . campestris) (Beversdorf et al., 1980); to rutabaga and rapeseed (Beversdorf and Hume, 1984), as well as from weedy green foxtail (S. viridis, subspp. viridis Briquet) to foxtail millet (S. viridis, subspp. italica Briquet) (Darmency and Pernes, 1989). Agronomic performance has in many cases been less in the resistant crop compared to that in the susceptible crop (Beversdorf e t a / . , 1988; Dekker, 1983). b. Biotechnological Techniques s-Triazine-resistant crops have been developed utilizing tissue culture selection and protoplast fusion techniques. Tissue culture selection has led to resistant tobacco (mutant D-1; Pay et al., 1988; Rey et nl., 1990) and potato (mutant D-I; Smeda et al., 1989) HRCs. Protoplast fusion transfer of the psbA gene to potato was accomplished with a weedy relative as the source of resistance (Gressel et al., 1990). Protoplast fusion methodologies were also used to incorporate s-triazine resistance in rapeseed and in several other Brassica species, including broccoli (B. oleracene, var. italica; Christey et al., 1991).

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Three triazine-resistant canola cultivars have been released in Canada (cv. ‘OAC Triton,’ “OAC Tribute,” and “OAC Triumph”) with little commercial success. Farmers have generally only used these cultivars when they were willing to trade a significant yield and quality reduction for control of weeds that could be managed with triazine herbicides better than with other methods (Duke et al., 1991; Hall et al., 1995). Efforts to produce cultivars with triazine resistance and normal photosynthetic productivity have been unsuccessful (Hall et al., 1995).

B. ACETOLACTATE SYNTHATASEINHIBITORS 1. Introduction Three groups of herbicides whose site of action is acetolactate synthatase (ALS) are either available or soon will be. They are the sulfonylureas, imidazolinones, and the triazolopyrimidine sulfonanilides (Devine et al., 1993a; Hawkes et al., 1989). Reviews are available on the sulfonylureas (Beyer et al., 1988) and triazolopyrimidines (Subramanian and Gerwick, 1989), and an entire book is available on the imidazolinones (Shaner and O’Connor, 1991). Compounds from other chemical families that act at this site are under development. ALS inhibitors are generally highly active and selective and are used for both soil-applied and postemergence weed management. Particular compounds have been designed with particular crops in mind. Thus, certain ALS inhibitors are favored for each crop, resulting in the marketing of a relatively large and growing number of ALS inhibitor herbicides. Examples of sulfonylureas include bensulfuron methyl, chlorsulfuron, nicosulfuron, sulfometuron, and primsulfuron. The imidazolinones include imazaquin, imazapyr, and imazethapyr, and the newer triazolopyrimidine sulfonanilides are represented by flumetsulam.

2. Modes of Action and Resistance ALS (also known as acetohydroxy acid synthase or AHAS) is the first enzyme in the branched chain amino acid pathway that produces valine, leucine, and isoleucine (Devine er al., 1993a). This enzyme and others in the pathway are found only in the plastid. It is nuclear encoded and, thus, requires a transit sequence to be properly imported and processed. A large number of compounds have been found to be effective inhibitors of ALS, apparently binding a vestigal ubiquinonebinding site (Schloss et ul., 1988). In many ways the herbicide-binding site of ALS is similar to the herbicide-binding site of D-1 of PS I1 (see the section on triazine). The ALS inhibitor herbicides stop growth and then kill the plant relatively

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slowly compared to some of the older contact herbicides. Their mechanism of action was discovered by studies in which the effects of the herbicide could be prevented by feeding cell cultures leuine, valine, and isoleucine. ALS appears to be a particularly good molecular target site for herbicides, considering how little of the best ALS inhibitors is needed to kill plants. Only a few grams per hectare of certain sulfonylureas are needed to kill target weeds. Crops that are naturally unaffected by these compounds are resistant due to rapid metabolic degradation of the herbicide (Beyer et al., 1988; Shaner and Mallipudi, 199l). However, weeds that have evolved resistance to ALS inhibitors almost always have evolved an ALS that is resistant to the herbicide (Devine et al., 1993, Chapter 13; Schmitzer et al., 1993). Within the same species, every sort of cross-resistance pattern imaginable seems possible with ALS herbicide resistance, whether selected in tissue culture or in the field. As with the D-1 protein of PS 11, there appear to be various herbicide-binding domains on ALS, which can overlap to provide cross-resistance (Mourad and King, 1992). Cross-resistance can be due to a single mutation or to combined mutations, each conferring resistance to only one ALS inhibitor class (Hattori et al., 1992). There are at least 10 mutation sites in the ALS-encoding gene that confer herbicide resistance without compromising enzyme activity (Mazur and Falco, 1989). Resistance evolves comparatively rapidly to ALS inhibitor herbicides compared to other herbicides. Resistance has appeared in weed populations in only 3 to 5 years of selection with sulfonylureas (Thill et al., 1991) and imidazolinones (Schmitzer et al., 1993). Some sulfonylurea herbicide-resistant weed biotypes may have some altered physiological characteristics (Dyer et al., 1993a; AlcoerRuthling et al., 1992); however, it is not clear as to whether this affects fitness. The ALS enzyme efficiency is not significantly affected by most of the mutations that result in resistance. This may be due largely to the fact that the herbicidebinding site is different from the active site of the enzyme. From the large number of good inhibitors and the different types of mutations conferring different patterns of cross-resistance to different ALS inhibitors, one can infer that the herbicide-binding site of ALS is a very plastic molecular domain. X-ray crystallography studies have not yet been conducted with ALS, so an accurate description of the binding site remains to be elucidated.

3. ALS Inhibitor-Resistant Crops Both sulfonylurea- and imidazolinone-resistant crops have been produced. It has been relatively easy to tailor these herbicides to specific crops that metabolically degrade them. It has also been quite easy to generate crops resistant to ALS inhibitors that lack natural resistance. Because of the plasticity of the enzyme, selection out at the seed or whole plant (e.g., Sebastian et al., 1989), organ (e.g.,

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Harms et a/., 1991), and tissue or cellular level (e.g., Hart et al., 1993) has been a very simple and successful strategy to produce such crops. At the whole plant level, mutagens have proven helpful in providing sufficient genetic diversity. a. Sulfonylureas The topic of sulfonylurea-resistantcrops has been specifically reviewed previously (Saari and Mauvais, 1994). In only one case has sulfonylurea herbicide resistance been transferred from a evolved resistant weed (prickly lettuce; MallorySmith et al., 1991) to a crop (lettuce) by conventional breeding methods (Mallory-Smith et al., 1993). Mutant selection has created sulfonylurea herbicide-resistant lines of barley (Baillie et al., 1993), tobacco (Chaleff and Ray, 1984), canola (Tonnemaker et al., 1992), sugarbeet (Hart et al., 1993; Saunders et al., 1992), soybean (Sebastian et al., 1989), rice (Terakawa and Wakasa, 1992), and flax (Jordan and McHughen, 1987), as well as some horticultural crops. The trait is always a single gene mutation and is usually inherited as a semidominant or dominant trait. Almost all mutants selected are resistant because of resistant ALS, although resistant mutants without resistant ALS have been selected once (Sebastian and Chaleff, 1987) and a partially resistant ALS that was amplified was found in another case (Harms et al., 1992). Genetic engineering has produced sulfonylurea-resistant crops (Falco et al., 1989). Arabidopsis thaliana chlorsulfuron-resistant ALS has been transferred to chicory (Vermeulen et al., 1992), tobacco (Gabard et al., 1989), poplar (Brasileiro et al., 1992), canola (Brassica napus; Miki et al., 1990), flax (McSheffrey et al., 1992), and rice (Li et al., 1992). A mutant tobacco ALS has been used to transform cotton (Saari and Mauvais, 1994) and sugarbeet (D’Halluin et al., 1992), and a resistant maize ALS has been used to transform maize (Fromm et al., 1990). Several field tests of sulfonylurea-resistantcrops created by biotechnology have been reported. Transgenic flax expressing a resistant ALS was as productive as untransformed varieties (McHughen and Holm, 1991; McSheffrey et al., 1992) whereas, in the absence of the herbicide, there appeared to be a yield penalty in lines of tobacco transformed with a resistant ALS (Brandle and Miki, 1993). A rapeseed line derived by selection in cell culture was less productive, and produced harvestable seed later, than the wild type (Magha et al., 1993). Because of the large number of available sulfonylureas for most crops, there is not as much interest in creating crops resistant to them as there is in creating crops resistant to other herbicides. b. Imidazolinones The topic of imidazolinone-resistant crops has been reviewed previously by Newhouse er al. (199 1b) and Shaner et al. (1994). The first imidazolinones registered for use in the United States, imazaquin and imazethepyr, were used in

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soybeans. These herbicides are very effective on many of the problematic weeds in maize, but maize is susceptible. Furthermore, maize is often planted in rotation with soybeans so that residual imidazolinone herbicides can cause crop damage. Selection for imidazolinone-resistant maize began in 1982 (Shaner et al., 1994). Imidazolinone resistance was sucessfully selected for in tissue culture (Newhouse era/., 1991b) and by pollen mutagenesis (Shaner e t a / . , 1994) to produce imidazolinone-resistant maize. Maize seed with this trait is the only commercially available herbicide-resistant crop in the United States at this writing. Work is in progress to produce imidazolinone-resistant wheat (Newhouse et a/ . , 1992) and canola (Swanson et al., 1988, 1989). Initial field data confirm that both imidazolinone-resistant canola and wheat have the same yield as susceptible varieties in the absence of the herbicide and that the selected varieties are resistant to rates of imidazolinones recommended for weed control (Shaner et al., 1994). Commercial varieties of imidazolinone-resistant wheat and canola are expected to be available in the late 1990s.

C. ACETYL-COA CARBOXYLASE INHIBITORS 1. Introduction This group of herbicides includes the aryloxyphenoxypropionates and the cyclohexanediones (Devine et al., 1993a; Duke and Kenyon, 1988). Some commonly used aryloxyphenoxypropionates are diclofop, haloxyfop, and fluazifop and some commercial cyclohexanediones are sethoxydim and alloxydim. Hence, some people refer to the two herbicide classes as the “fops” and “dims.” Both herbicide classes are postemergence grass killers that are used extensively in agronomic crops. Although chemically different, they both target the same molecular site and produce similar effects on grass weeds. They are generally active at lower rates than many older herbicides (e.g., atrazine).

2. Modes of Action and Resistance The ACCase form inhibited by herbicides is a plastid-localized enzyme that catalyzes ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA in the lipid synthesis pathway of plants. ACCase is a pivotal enzyme in the plant lipid biosynthesis pathway, exerting strong flux control, especially in light-stimulated biosynthesis (Page el al., 1994). Another cytosolic form of ACCase is not inhibited by herbicides and apparently plays no role in their mode of action (Egli et al., 1993). Both aryloxyphenoxypropionates and cyclohexanediones strongly inhibit ACCase, and this enzyme appears to the the primary site of action for these herbicides (Devine et a[., 1993a; Harwood, 1991).

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A direct effect on membrane function has been proposed as a site of action of these herbicides (Shimabukuro and Hoffer, 1992); however, the action at this site does not appear to kill plants at herbicidal doses (DiTomaso et al., 1991 ; Dotray et al., 1993). The membrane effect appears to be antagonism of auxin-stimulated proton effux at the receptor level (Barnwell and Cobb, 1993). Auxin antagonism of these compounds has been known since they were first discovered (Duke and Kenyon, 1988). Wild oat with resistance to diclofop and fenoxaprop, two ACCase inhibitor herbicides, had neither enhanced herbicide degradation nor resistant ACCase, but was able to reverse herbicide effects on transmembrane proton fluxes (Devine et al., 1993b). Crop resistance to these herbicides appears to normally be due to an insensitive ACCase (Burton et al., 1987; Devine et al., 1993a). Some grasses such as wheat are resistant to some of these herbicides because they can metabolically degrade them rapidly (Shimabukuro, 1990). Red fescue is naturally resistant by virtue of a herbicide-resistant ACCase (Stoltenberg et al., 1989). Weeds appear to have evolved at least three different mechanisms of escaping the phytotoxicity of members of this herbicide group. Some biotypes have evolved a herbicide-resistant ACCase (e.g., Marles et al., 1993; Tardiff et al., 1993). Others appear to more rapidly degrade certain ACCase inhibitors (Kemp et al., 1990), although this has not yet been rigorously proven. In at least one case, the resistance appears to be due to an altered membrane response (Devine et al., 1993b).

3. ACCase-Resistant Crops Maize resistant to both aryloxyphenoxypropionates and cyclohexanediones has been produced by selection for mutations conferring resistance in tissue culture and then regenerating the plant (Marshall et al., 1992; Parker et al., 1990a,b; Somers, 1994). This approach has also been used to select for herbicide-resistant wheat and Kentucky bluegrass (Somers, 1994), although these efforts have not yet been well documented or have been unsuccessful. Several types of mutations have been generated, as characterized by varying degrees of cross-resistance to ACCase inhibitors not used for selection of the mutant. For example, the maize mutants Acc 1-S 1, Acc 1-S2, and Acc-S3 are resistant to both sethoxydim and haloxyfop, whereas the Acc I -H 1 mutant is resistant to only haloxyfop, and the Accl-H2 mutant is very resistant to haloxyfop, but only partially resistant to sethoxydim (Marshall er al., 1992). In the absence of herbicides, ACCase levels in the S lines are similar to the wild type and those of the H lines are only slightly lower. Whole plant resistance of several mutant lines closely parallels the ACCase resistance (Somers, 1994). Field trials with sethoxydim-resistant maize have demonstrated that no injury occurs to the crop at 0.88 kgha of sethoxydim, a rate in excess of that required to control grassy weeds (Dotray et al., 1992). Herbicide treatment had no adverse

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effects on grain yield or quality. The germ plasm for resistance to ACCaseinhibiting herbicides was transferred to commercial maize breedingcompanies in 1990, and backcrossing and inbred development are in progress (Somers, 1994).

D. GLYPHOSATE I. Introduction Glyphosate [(N-phosphonomethyl)glycine] is the only herbicide in its class. The physical and biological characteristics of glyphosate have been reviewed (Duke, 1988) and an entire book has been devoted to this one compound (Grossbard and Atkinson, 1985). Glyphosate is a nonselective, postemergence herbicide that is used extensively prior to crop emergence, as a harvest aid, and as a directed spray. It is used extensively in forests and orchards where understory vegetation can be sprayed without contacting the foliage of the crop. It is also used in landscaping and lawns for edging and borders. Very few weeds are resistant to glyphosate and there are no reported cases of evolved resistance. It is toxicologically and environmentally benign (Duke, 1988). Upon contact with the soil, it is immobilized by binding to soil components, where is it is rapidly degraded by soil microbes.

2. Modes of Action Glyphosate is normally a slow-acting herbicide that can take several days to weeks to kill a plant. It is translocated readily from sites of uptake (normally foliage) to metabolic sinks, such as meristems, developing leaves, and storage organs (Duke, 1988). Most plants do not metabolically degrade glyphosate. The shikimate pathway and, more specifically, the EPSPS is the primary site of action of the herbicide. EPSPS is a nuclear-coded, plastid enzyme. The shikimate pathway is the biosynthetic source of the three aromatic amino acids: phenylalanine, tryptophan, and tyrosine. These amino acids are necessary for protein synthesis as well as for biosynthesis of auxin, most plant phenolic compounds, and other secondary compounds. Furthermore, blockage of the shikimate pathway at EPSPS leads to deregulation of the pathway, resulting in accumulation of huge, possibly phytotoxic, concentrations of shikimate and benzoic acid derivatives of shikimate (Lydon and Duke, 1988). This deregulation and enhanced carbon flow into the shikimate pathway drains other biosynthetic pathways of necessary building blocks (Killmer et al., 1981; Jenson, 1985). Thus, the blockage of the shikimate pathway can lead to a large number of potentially damaging physiological effects. There is no good evidence of any other primary site of action of glyphosate (Duke, 1988), although very rapid effects of glyphosate on photosynthesis in

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some species are difficult to explain by an effect on EPSPS (Madsen er al., 1995; Sheih et af., 1991). Designing HRCs around a single target site by site modification requires that no other sites of action can play a significant role in phytotoxicity under any field conditions. No plants are considered to be naturally resistant to glyphosate, although there is considerable variation in sensitivity. The physiological bases for these variations in susceptibility are poorly understood.

3. Glyphosate-ResistantCrops The production of glyphosate-resistant crops has been the focus of much research for over a decade. A major problem in the production of glyphosateresistant plants is that its glyphosate is readily translocated to rneristems and other metabolic sinks where it is concentrated to levels many times that found in leaves. Furthermore, it is not metabolically degraded to a significant extent. So, although the plant may be resistant at the foliar level, the concentrations that accumulate in meristems, flower buds, and other metabolic sinks may overwhelm the resistance mechanism. Even if this is not the case, unacceptable residues of the herbicide might accumulate in the harvested portions of the plant. Overcoming these problems has slowed the development and introduction of glyphosate-resistant crops. Research to produce glyphosate-resistant crops has gone through three phases. The first approach was to select for glyphosate resistance in tissue or cell culture and then to regenerate glyphosate-resistant plants from resistant cells or tissues by selection on glyphosate-containing media. The resulting selections were generally found to have more EPSPS than the wild type due to gene amplification (e.g., Goldsbrough et af., 1990; Shah er al., 1986); however, the EPSPS was equally susceptible to glyphosate as the wild type. The amplification is generally stable in the absence of the herbicide, and plants regenerated from cell cultures with amplified EPSPS maintain amplified EPSPS genes (Shyr et af., 1992; Wang el al., 1991). Amplification of EPSPS can occur at the gene, mRNA (enhanced transcription), or enzyme (reduced turnover) levels (Hollander-Czytko et af., 1992).Glyphosate is simply diluted by a larger number of enzyme molecules. The level of resistance obtained by this approach was not useful for commercial application. In at least one case, selection with glyphosate in cell cultures resulted in resistance due to a glyphosate-resistant EPSPS. Forlani et al. (1992) selected maize cell cultures with glyphosate and produced a cell line with a glyphosate-resistant EPSPS. However, the cell line still had a tolerant form of EPSPS and the resistant form had reduced enzymatic efficiency, thus making it of no commercial value. The second approach to the generation of glyphosate-resistant crops has been to transform them with genes encoding glyphosate-resistant EPSPS. Several

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EPSPSs have been produced with reduced sensitivity to glyphosate (Padgette et al., 1991; Sost and Amrhein, 1990; Stalker et al., 1985); however, these forms of EPSPS are generally less efficient than the wild type, resulting in unacceptable pleiotropic effects unless the level of the inefficient but resistant EPSPS is increased. Initial attempts to transform plants with resistant EPSPS were also hampered by the fact that no transit peptide to target the gene product to the plastid was included (Comai et al., 1985; Fillati et al., 1987). These plants were only slightly resistant to glyphosate. The introduction of a chloroplast transit signal improved the level of resistance (Della-Cioppa et al., 1987). An EPSPS from strain CP4 of Agrobacterium sp. was found with a high level of glyphosate insensitivity and good enzymatic efficiency (Barry et a f . , 1992). Plants transformed with the gene encoding this enzyme, along with a chloroplast transit peptide to target the enzyme for the plastid, resulted in highly resistant canola and soybean. The leading transgenic glyphosate-resistant soybean line expresses the CP4 EPSPS (Padgette et al., 1994). There appears to be no yield penalty from this gene and it confers a high level of glyphosate resistance. The third and most recent approach to glyphosate-resistant crops has been to introduce genes from microbes that degrade glyphosate. Plants do not generally degrade glyphosate and no plant enzyme has been found to have such activity. However, many soil microbes readily degrade glyphosate. Certain species of Pseudomonas and other soil microbes convert glyphosate to sarcosine and PPi with a C-P lyase activity (e.g., Kishore and Jacob, 1987). The enzyme itself has not been isolated and despite good progress in understanding the molecular genetics of C-P lyase, this enzyme was abandoned because of the complexity and number of gene products (Barry et al., 1992). The predominant degradation pathway in soil appears to be an initial conversion of glyphosate to aminomethylphosphonic acid (AMPA) and glyoxylate (Tortensson, 1985). Although this enzyme has not been isolated, the gene from an Achromobacter sp. strain taken from a glyphosate waste stream treatment facility has been cloned and used to transform crops (Barry et al., 1992). A region from the DNA of this microbe encodes glyphosate oxidoreductase (COX) which cleaves the C-N bond of glyphosate, producing AMPA and glyoxylate. Expression of COX in plants imparts glyphosate resistance, and targeting GOX for the plastid with a chloroplast transit peptide improves the level of resistance (Barry et al., 1992; Padgette et al., 1994). At this writing, there are no published accounts of weed management experiments with glyphosate-resistant crops. However, Madsen and Jensen ( 1 995) found that with glyphosate-resistant sugarbeets, glyphosate alone in three applications (720 g a.i./ha total) was just as effective as a conventional application of 4 kg a.i.1 ha of other herbicides (phenmedipham, ethofumesate, and metamitron) applied in three sprayings.

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E. BROMOXYNIL 1. Introduction Bromoxynil is a postemergence benzonitrile herbicide used on barley, oats, wheat, flax, rice, and in grass seed production. Dicotyledonous crops are especially sensitive to bromoxynil and, thus, the generation of a dicotyledonous bromoxynil-resistant crop would extend its use, especially in crops such as cotton for which there are presently no good postemergence herbicide options. A related nitrile herbicide, ioxynil, is also commercially available. Bromoxynil is a relatively short-lived herbicide, with a soil half-life as short as 1 week, due to microbial degradation (Stalker et al., 1994).

2. Modes of Action and Resistance Bromoxynil is a photosystem I1 inhibitor with the same mode of action as triazines (see earlier discussion) (Devine et al., 1993a). The crops with which bromoxynil is used are apparently naturally resistant through rapid metabolic degradation of the herbicide (Ashton and Crafts, 1981; Schaller et al., 1991, 1992), although this is not a well-studied topic. Bromoxynil-resistant weeds have been reported (LeBaron, 1991), although the mechanism of resistance has not. In some cases, resistance to triazine herbicides can impart greater susceptibility to bromoxynil; a case of negative cross-resistance (Durner et al., 1986). Thus, bromoxynil could be useful in managing triazine-resistant weeds.

3. Bromoxynil-Resistant Crops Bromoxynil-resistant tobacco and cotton have been generated by transformation with a plasmid gene from the bacterium Klebsiella ozaenae that encodes a bromoxynil nitrilase enzyme (Stalker et al., 1988a,b, 1994). Resulting plants are resistant to up to 10-fold the recommended field rates of commercial formulations of bromoxynil. In cotton, the gene was introduced via Agrobacterium and was expressed as a dominant Mendelian gene. At rates up to 1 1.2 kg a.i./ha (0.56 kg a.i./ha is a normal field rate), no phytotoxicity to the transgenic cotton varieties is seen. Metabolic studies have shown that all of the bromoxynil is converted to nonphytotoxic degradation products in bromoxynil-resistant cotton, whereas more than 99% of the bromoxynil extracted from susceptible, nontransformed cotton remained as bromoxynil (Stalker et al., 1994). Bromoxynil-resistantcotton may be the first transgenic herbicide-resistant crop introduced to the market. It has been successfully tested throughout the cotton-

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growing areas of the United States, except California (e.g., Arkansas, Beaty and Guy, 1994; South Carolina, Murdock, 1994; Georgia, Richburg el al., 1993). No undesirable pleiotropic effects of the nitrilase gene have been noted. Bromoxynilresistant cotton was approved by the U.S. Department of Agriculture in 1994; however, at this writing it still awaits clearance by the U.S. Food and Drug Administration and the U.S. Environmental Protection Agency.

F. PHENOXYCARBOXYLIC ACIDS(E.G., 2,4-D) 1. Introduction The phenoxycarboxylic acids (PCAs) such as 2,4-D and 2,4,5-T are among the oldest synthetic herbiciges. These compounds are used primarily for postemergence management of dicot weeds in grass crops, pastures, forests, and lawns. The butryric acid derivative of 2,4-D, 2,4-DB is a proherbicide (a herbicide that is inactive in the form applied) that is metabolized to 2,4-D in sensitive species. Some legumes that are sensitive to 2,4-D are tolerant to 2,4-DB because they lack the P-oxidation activity required to activate it. Phenoxycarboxylic acids have been off patent for many years and are very effective and inexpensive. Sensitive crops and ornamentals are often injured by 2,4-D spray drift from applications to tolerant crops or turf. Introduction of 2P-D-resistant crops could eliminate damage to these crops from such a source-the purported or implied objective of some research to generate 2,4-D-resistant cotton (Bayley et af., 1992; Lyon et al., 1993). This herbicide would be extremely useful and cost effective as a postemergence herbicide in cotton, a crop for which there is presently no adequate foliar-applied herbicide.

2. Modes of Action and Resistance Despite many years of study the actual molecular site of action of this family of herbicides is unknown. The PCAs are sometimes called hormone-type herbicides because they mimic in many ways lethal doses of the plant hormone indoleacetic acid (IAA) (Devine et af., 1993a). IAA and the PCAs are both thought to act by influencing plasma membrane properties by acting at a molecular site in the plasma membrane. Resistance of grasses to these herbicides appears to be related to rapid metabolic conversion to irreversible products, whereas in dicotyledonous species the herbicide is often found in the form of reversible conjugates (Devine et af., 1993a,b).Several weed species have evolved resistance to 2,4-D (LeBaron, 1991). Where studied, the resistance appears to be due to metabolic detoxification.

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3. 2,4-D-Resistant Crops The half-life of 2,4-D in soil is relatively short, due to several microbes that readily degrade it (Llewellyn and Last, 1994). Certain Alcaligenes eutrophus (a bacterium) strains contain a large plasmid (75 kb) containing genes for enzymes that fully degrade 2,4-D. The first step in the enzymatic degradation of 2,4-D is its oxidation to dichlorophenol (DCP) by a dioxygenase encoded by the tfdA gene. DCP is 50- to 100-fold less phytotoxic than 2,4-D (Llewellyn et al., 1990), so the gene encoding this dioxygenase is apparently the only gene needed to confer resistance to crops. There has been some concern about the safety of DCP as a food residue in 2,4-D-resistant crops. The latter genes of the Alcaligenes plasmid 2,4-D degradation pathway have been cloned and sequenced (Perkins et al., 1990) and could be used to provide a more complete degradation of 2,4-D in plants. Cotton and tobacco have been made resistant to 2,4-D by genetic transformation with the tfdA gene (Bayley et al., 1992; Llewellyn and Last, 1994; Lyon et al., 1989, 1993; Streber and Willmitzer, 1989). All three groups who have made these transformants have used the 35s promoter of the cauliflower mosaic virus, in some cases with additional promoters. The resultant transformants are resistant to more than three times the highest recommended doses of 2,4-D for wheat, corn, sorghum, or pasture. In the case of cotton, this represents a 50- to 100-fold increase in tolerance to 2,4-D compared to untransformed controls. Transgenic cotton seed with 2,4-D resistance has been distributed to private and public plant breeders in the United States for possible incorporation into commercial germ plasm (Llewellyn and Last, 1994). In Australia the 2,4-D tolerance gene is currently being transferred to the best commercail cotton varieties with expectations of commercial release as 2,4-D drift-tolerant cultivars by the year 1998 (Lyon et al., 1993).

G. GLLJFOSINATE (PHOSPHINOTHRICIN) 1. Introduction Glufosinate, the chemically synthesized version of the microbial product (phosphinothricin), is used in Europe and other parts of the world as a nonselective herbicide (Devine et al., 1993a). It is a toxicologically and environmentally benign herbicide that does not persist in the environment. Streptomyces spp. produce phosphinothricin and a precursor of phosphinothricin, bialaphos. Bialaphos can be converted by plants to phosphinothricin, thus generating a phytotoxin. Bialaphos produced in fermentation culture is sold in Japan as a herbicide. These products are foliar-applied, contact herbicides that act faster than gly-

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phosate, but slower than paraquat. Although some plant species are more tolerant of glufosinate than others, it is generally used as a nonselective herbicide in much the same way as glyphosate. Resistant crops would greatly increase the utility of this herbicide.

2. Modes of Action and Resistance Glufosinate is the most potent known inhibitor of glutamine synthetase (GS) (Devine et al., 1993a). GS is critical to the assimilation of nitrogen by plants, and its inhibition leads to several immediate metabolic dysfunctions. Initially, it was thought that most of the phytotoxicity caused by GS inhibitors was due to accumulation of toxic levels of ammonia, a GS substrate. However, it now appears that the rapid cessation of photosynthesis brought about by glyoxylate accumulation is the more important phytotoxic effect. Glyoxylate accumulates in GS-inhibited plants because the levels of amino acids required in photorespiratory glyoxylate transamination are reduced. No weeds have yet evolved resistance to glufosinate. However, this is a relatively new herbicide and it is a contact herbicide with a very short selection pressure duration. There is considerable natural variation between species in sensitivity to glufosinate, and this variation does not appear to be based on differential sensitivity to GS (Ridley and McNally, 1985). Lines of oat that are insensitive to the GS-inhibiting glufosinate analog, tabtoxin, have a tabtoxin-resistant GS isozyme (Knight et al., 1988); however, no plants with glufosinate-resistant GS have been reported.

3. Glufosinate-Resistant Crops Glufosinate-resistant crops have been the focus of at least two reviews (Mullner et al., 1993; Vasil, 1994). Two genes that encode enzymes that metabolically inactive glufosinate have been used to produce resistant plants by transgenic methods. The bar gene from Streptomyces hygroscopicus andlor the pat (phosphinothricin-aceytl transferase) gene from S. viridochromogenes has been used to transform about 20 crops, including carrot (Droge et al., 1992), oats (Somers et al., 1992), beet (Botterman and Leemans, 1988), oilseed rape (DeBlock et al., 1989), tall fescue (Wang et al., 1992), barley (Wan and Lemaux, 1994), tomato (DeBlock et al., 1989), alfalfa (D’Halluin et al., 1990), rice, (Cao et al., 1992; Datta et al., 1992; Rathmore et al., 1993), potato (DeBlock et al., 1987), sorghum (Casas et al., 1993), wheat (Vasil et al., 1993), tobacco (Droge et al., 1992), sugarbeet (D’Halluin et al., 1992), and maize (Fromm et al., 1990; Laursen et al., 1994). Both of these similar genes encode a phosphinothricinacetyl transferase. Sugarcane has been transformed with the pat gene in callus

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culture, but no plants have yet been regenerated (Chowdhury and Vasil, 1992). The Emu promoter, which drives high levels of gene expression in cereals, works well with the pat gene (Chamberlain et al., 1994). There is apparently no crop yield or quality sacrifice for either of these genes. Comparing glufosinate use with glufosinate-resistant crops with standard weed management methods in agronomic crops, the total volume of herbicides used can be reduced substantially (Mullner et al., 1993). An additional benefit of glufosinate-resistantcrops is that bialaphos and glufosinate are toxic to some pathogens, so the herbicide might also act as a fungicide to protect the crop, as has been found against sheath blight in rice (Uchimiya et al., 1993). Whether the bar or pat genes will also protect against pathogens that produce analogs of glufosinate (e.g., tabtoxin) as virulence factors has not been reported. The risk of outcrossing of transgenic glufosinate-resistant rapeseed has been assessed with the conclusion that the risk for gene dispersal is limited (Kerlan et al., 1992). The bar and pat are used extensively as selectable markers. The advantage of these genes is that the selection agent is inexpensive and highly efficient. Thus, the production of a glufosinate-resistantcrop per se has not always been the major objective of the many published examples (Yoder and Goldsbrough, 1994).

H. OTHERHERBICIDES 1. Cyanamide

Cyanamide is a herbicide used in Europe. Its mode of action and mechanism of selectivity are unknown. Tobacco has been made resistant to cyanamide by transformation with a gene from the fungus Myrothecium verrucaria that encodes cyanamide hydratase (Maier-Greiner et al., 1991), an enzyme that converts the herbicide to urea. Whether this capability will be exploited to expand the use of cyanamide is unknown. 2. Dalapon

Dalapon is a chlorinated aliphatic acid. Its mode of action is unclear, perhaps because it has several molecular sites of action. One of its physiological effects is to inhibit the synthesis of very long chain fatty acids (Devine et al., 1993a,b). Plants do not readily metabolically degrade dalapon (Foy, 1975), and the mechanism of natural resistance is unknown. This herbicide is or has been used extensively as a grass killer in asparagus, citrus, cotton, flax, peas, potatoes, and sugarbeets (Klingman and Ashton, 1982). However, there are several major

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crops with which this relatively inexpensive herbicide cannot be used because of phytotoxicity. Dalapon-resistant tobacco (Nicotinna plurnbaginifolia) has been produced by transgenic methods (Buchanan-Wollaston et a/., 1992). A gene from Pseudornonas putida encoding a dehalogenase capable of degrading dalapon was used in the transformation. Resulting transformants were resistant to five times the recommended field rate of dalapon. This study was not done to generate a commercial herbicide-resistant tobacco, and it is not known if a similar approach will be used to produce dalapon-resistant crops for commercial use.

3. Phytoene Desaturase Inhibitors Carotenoid synthesis is dependent on phytoene desaturase. Inhibition of this enzyme leads to albino plants because chlorophyll will not accumulate in the absence of carotenoids. Only two phytoene desaturase inhibitors, fluridone and norflurazon, are commercially available, and, of these, only norflurazon is used on the agronomic crop cotton. Diflufenican, another phytoene desaturase inhibitor, is being developed for use in wheat and barley. Norflurazon is generally used as a pre-emergence, soil-applied herbicide. Several important crops are not resistant to it and there is sometimes phytotoxicity to cotton. Furthermore, because of its persistence in soil, phytotoxicity can be experienced the next year in rotated crops. A major problem with phytoene desaturase inhibitors is their lack of selectivity. Thus, the advent of crops with resistance to these herbicides at the phytoene desaturase level would be especially useful. No cases of evolved resistance to these herbicides have been reported. Natural resistance of crops and weeds to these herbicides is not well studied. Metabolic degradation of norflurazon in cotton is not faster than in some susceptible weeds, and there is evidence that sequestration of norflurazon in the lysigenous glands that cover the hypocotyl and cotyledons of cotton imparts tolerance to cotton (Duke, 1992). Glandless cotton varieties are more susceptible to norflurazon than those with glands (Stegink and Vaughn, 1988). Crops that are resistant to phytoene desaturase inhibitors are under development (Sandmann et al.. 1995). A gene from the norflurazon-resistantbacterium Envinia uredovora that encodes a norflurazon-resistant phytoene desaturase has been inserted into tobacco to produce a herbicide-resistant crop (Misawa et al., 1993). The crtl gene of the bacterium was fused with the sequence encoding the plastid transit peptide for pea Rubisco (small subunit) and put under the control of the cauliflower mosaic virus 35s promoter. This construct was inserted via Agrobacteriurn. Resistance was dominantly inherited. Cross-resistance to several other phytoene desaturase-inhibiting herbicides was expressed. Using a naturally resistant gene product instead of one that has been selected

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for may increase the odds of the gene product being highly functional without pleiotrophic effects due to impaired catalytic function. Whether the success with tobacco will spur development of herbicide-resistant crops for use with phytoene desaturase inhibitor herbicides is an open question.

4. Protoporphyrinogen-Oxidase(Protox) Inhibitors One of the most patented groups of herbicides in recent years are the inhibitors of Protox (Anderson et al., 1994). Commercialized herbicides with this molecular site of action include acifluorfen, lactofen, oxadiazon, and fomesafen. These herbicides are contact herbicides that are applied as foliar sprays which cause very rapid cellular collapse and desiccation. The most effective of these compounds can be used at rates of only a few grams per hectare. One of the major problems with this group of herbicides is that only a few crops (primarily soybean and rice) Protox is the last enzyme in the porphyrin pathway that is common to both heme and chlorophyll synthesis pathways. These compounds cause massive levels of the enzyme product (not the substrate) protoporphyrin IX (Proto IX) to accumulate through a complex mechanism involving both a herbicide-susceptible chloroplast Protox and a herbicide-resistant extraplastidic Proto IX-oxidizing enzyme (Jacobs ef al., 1991; Jacobs and Jacobs, 1993; Lee et al., 1993; Nandihalli and Duke, 1993; Duke et al., 1994). Proto IX is a photosensitizing agent, generating highly reactive singlet oxygen in the presence of sunlight. Thus, Proto IX, a metabolic intermediate, is the acutely toxic agent causing phytotoxicity. There is a wide range in the natural resistance of crops and weeds to these compounds (Sherman et al., 1991; Matsumoto et al., 1994); however, there is no evidence that any weeds have become resistant to these herbicides as the result of selection pressure. There are apparently several mechanisms of resistance, including reduced sensitivity of Protox, metabolic rapid degradation of the herbicide, and resistance to singlet oxygen. Efforts are being made to produce a plant with a herbicide-resistant chloroplast Protox by either selection with Protox inhibitors (Che et al., 1993) or introduction of a resistant Protox from another organism (Sato et al., 1994). The ultimate practical result of this research would be the production of new crops resistant to Protox inhibitors.

5. Bipyridiliums This group of nonselective herbicides includes paraquat and diquat. Bipyridiliums are contact herbicides that cause very rapid desiccation of foliage. They are the fastest acting group of herbicides. They are acutely toxic to animals. These herbicides kill green plants by accepting an excited electron from photosystem I of photosynthesis to form the paraquat radical, which in turn gen-

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erates a highly destructive superoxide radical (Devine et al., 1993a). The paraquat radical is formed more slowly in animals through other mechanisms. Some weeds have become resistant to paraquat; however, the mechanism of resistance is in dispute. One group has evidence that paraquat resistance is due to greater expression of existing genes encoding enzymes that protect against superoxide and its peroxidizing products (e.g., Amsellem et al., 1993). The other view is that the resistant biotypes have evolved a mechanism to exclude paraquat from the molecular site of action (e.g., Norman et al., 1993). Paraquat-resistant crops have been produced by increasing protective enzyme levels by transgenic means. An E. coli gene encoding glutathione reductase when inserted into tobacco increased its resistance to paraquat (Aono et al., 1993). Genes for tomato superoxide dismutases transferred potato by use of Agrobacterium conferred enhanced tolerance to paraquat (Per1 et al., 1993). The gene from pea for Cu/Zn chloroplast superoxide dismutase imparted paraquat resistance to genetically engineered tobacco (Sen Gupta et al., 1993). These studies were done for purely academic reasons and it is unlikely that paraquat-resistant crops will be introduced. It is possible, however, that such modifications in crops will be used to prevent other types of oxidative stress, such as that caused by ozone or sulfur dioxide (Herouart et al., 1993; Tanaka, 1994). 6. Dihydropteroate Synthase Inhibitors Asulam is a broad spectrum herbicide that is chemically related to sulfonamide antibiotics. Asulam inhibits folate synthesis by inhibition of dihydropteroate synthase (DHPS) and thereby acts as a herbicide (Devine eta!., 1993a). Some bacteria have DHPS that is resistant to sulfonamide antibiotics (Wise and Abou-Donia, 1985). The gene for this enzyme has been fused with a plastid transit peptide sequence and has been used to transform tobacco leaf explants to produce asulamresistant plants (Guerineau et al.. 1990). Whether there will be any commercial development of this capability is not known. Also, whether this strategy will confer sufficient resistance for commercial use is not known, as asulam is also reported to be a plant mitotic inhibitor (Devine et al., 1993a).

7. Mitotic Inhibitors Several families of herbicides appear to have a primary effect on molecules involved in cell division (see Devine et al., 1993a). The dinitroanilines apparently bind to the protein subunit of microtubules, tubulin, to disrupt proper assembly of the microtubules required for cell division. Trifluralin, the most widely used dinitroaniline, is used as a preplant incorporated herbicide to control primarily grasses in broadleaf crops such as soybeans and cotton. It has been used for several years and some weed species such as

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goosegrass have evolved resistance (LeBaron, 199 1). Resistance in goosegrass is apparently due to a modified tubulin (Vaughn and Vaughan, 1990). Some species with natural tolerance to trifluralin, such as carrot, apparently owe their immunity to a modified tubulin, much like that found in resistant goosegrass (Vaughan and Vaughn, 1988). A patent for the production of trifluralin-resistant crops by genetically engineering with a gene encoding the modified tubulin of trifluralin-resistant goosegrass has been filed (Cronin er al., 1993). Whether such crops will be produced and developed is not known.

v. SUMMARY As a summary of current efforts in the development of HRCs, a list of herbicides and their resistant crops are provided in Table I. Resistant crops are listed

Table I

Herbicides and Their Resistant Crops Herbicide group

Herbicide

Resistant crops

AlSase inhibitors

Sulfonylureas

Imidazolinones ACCase inhibitors Bipyridiliums DHPS inhibitors Phenoxycarboxylic acids Phytoene desaturase inhibitors Triazines

Bromoxynil Cyanamide Dalapon Glufosinate

GIyphosate

Barley; chicory; cotton; flax; lettuce; maize; poplar; rapeseed; rice; soybean; sugar beet; tobacco Maize; rapeseed; wheat Kentucky bluegrass; maize; wheat Potato; tobacco Tobacco Cotton; tobacco Tobacco Foxtail millet (Setaria ifnlicn); potato; rapeseed; rutabaga; tobacco Cotton; tobacco Tobacco Tobacco Alfalfa; barley; beet; carrot; maize; oats; potato; rapeseed; rice; sorghum; sugar beet; tall fescue; tobacco; tomato; wheat Maize; rapeseed; soybean

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regardless of whether they have been commercialized or were developed for experimental purposes only, and are provided regardless of their “success” as resistant plants. They are listed whether their development is complete or research is still in progress. In most cases, literature citations for these HRCs have been provided in the text.

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ACIDSon, TOLERANCE IN W~IEAT Brett F. Carver' and James D. Ownby2 Departments of 'Agronomy and 'Botany Oklahoma State University Stillwater, Oklahoma 74078

I. The Problem: Causes, Symptomatology,and Severity A. Causal Elements of Soil Acidity B. Phytotoxicity of Acid Soils C. Severity and Extent of Soil Acidity 11. Physiology of Aluminum and Manganese Tolerance in Wheat A. Uptake and Distribution of Aluminum in Roots B. Aluminum Tolerance Mechanisms C. Uptake and Distribution of Manganese in Plants D. Mechanisms of Manganese Tolerance III. Genetic Mechanisms of Tolerance to Acid Soils A. Basis of Aluminum Tolerance B. Basis of Manganese Tolerance C. Relationship of Aluminum and Manganese Tolerance W. Breeding for Acid Soil Tolerance A. When Is Genetic Improvement Justified? B. Pools of Genetic Variation C. Screening Strategies D. Breeding Approaches E. Cultivar Development Efforts V. SustainableProduction in Acid Soils VI. Conclusions References

I. THE PROBLEM: CAUSES, SYMPTOMATOLOGY,AND SEVERITY Increasing soil acidification has attracted the attention of both researchers and producers of common wheat (Triticum aestivum L. em Thell.) to unprecedented levels. Relative to highly sensitive cereal crops like barley (Hordeum vulgare L.) and durum wheat (Triticum durum L.), common wheat is moderately sensitive to 117 Aduamer in .4pnomy, Volume $4 Copyright Q 1995 bykademic Press, Inc. All rights of reproductionin any form reserved.

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soil acidity. Reductions in forage and grain yields occur when soil pH ( 1 : 1 w/w ratio of soil and water) falls below 5.0 to 5.2 (Westerman, 1987). To ensure a margin of safety across soil types and cultivars, the minimum pH recommended for wheat production is 5.5. In practice, however, producers often fall short of that target pH, either in the subsurface or surface layers or both. This shortfall is experienced in wheat fields worldwide. Remediation of soil acidification can have the appearance of prevention, correction, or tolerance. The latter constitutes the theme of this chapter, not as an endorsement over other measures, but to recognize a gradual crescendo over the last two decades in wheat literature emphasizing acid soil tolerance. The first objective of this chapter is to provide a fundamental appreciation for the implications of soil acidity to wheat production on both sides of the equator, including the nutritional disorders associated with phytotoxicity. This chapter's intention is to bring to the view of plant scientists-including physiologists, breeders, and geneticists-a problem founded on principles of soil science. Second, why some genotypes of wheat are more physiologically equipped than others to tolerate soil acidity and how physiological adaptation is controlled genetically will be explained. A central thesis to this discussion is that toxic factors in acid soils initiate a cascade of stress responses in the wheat plant that demands multiplicity of tolerance mechanisms. Unfortunately, technology has not been applied to the same level of sophistication for examining genetic mechanisms of tolerance as for physiological mechanisms. When and how breeding solutions to acid-soil stress can be achieved will also be discussed, with considerable detail given to the arsenal of screening techniques available to the wheat breeder. The final objective will be to promote the concept that sustainability in a soil-acidifying wheat production system is balanced by affordable management practices on one hand and manipulation of genotype on the other.

A.

CAUSAL ELEMENTS OF SOIL ACIDITY

Soil acidity is determined by the amount of H' activity in soil solution and is influenced by edaphic, climatic, and biological factors of natural occurrence (Johnson, 1988). For example, soils which develop from granite parent materials acidify at a faster rate than soils derived from calcareous parent materials. Sandy soils with relatively few clay particles acidify more rapidly due to their smaller reservoir of alkaline cations (buffering capacity) and higher leaching potential. Excessive rainfall influences the rate of soil acidification depending on the rate of percolation of water through the soil profile. Organic matter will decay to form carbonic acid and other weak organic acids. The cumulative effect of these factors is practically immeasurable over the course of a few years and, therefore, may contribute relatively little to total soil acidity.

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Table I Vertical Distribution of pH in an Oklahoma Wheat Field (3.0 Hectare) Continuously Cropped with Winter Wheat and Sampled in a Grid Pattern to a 1.2-mDepth ~_________

~

~-

~~

~

pH" Depth (cm) 0- 15 15-30 30-45 45-60 60-90 90- I20

Minimum Maximum 4.1 4.4 5.6 6.2 6.5 6.2

6.0 6.4 7.0 7.4 7.6 7.7

Mean

Standard deviation

5.0 5.5 6.5 6.9 7.2 7.2

0.4 0.4 0.2 0.2 0.2 0.3

" A 1 : I soil: water solution, as reported by Guertal(l993).

Soil acidity shows not only chronological variation but also spatial (horizontal and vertical) variation. Highly weathered soils, such as Brazilian Oxisols, are acidic throughout the profile. In contrast, Mollisols cropped with continuous winter wheat in Oklahoma develop phytotoxic levels of acidity confined to the surface layer (Table I). The soil in this example is classified as a Pond Creek silt loam (Fine-silty, mixed, mesic Pachic Argiustoll). It received annual applications of anhydrous ammonia since 1982 and was limed 6 years prior to sampling. Typical of acid soils of the Oklahoma wheat belt, surface (0-30 cm) pH values were critically acidic, but subsurface ( 2 3 0 cm) mean pH values exceeded 6.5. Soil acidity was also found in the top 30 cm for wheatland soils in southern New South Wales (Fisher and Scott, 1987). Soil acidity is often accelerated in the surface layer by certain cropping practices, e.g., repeated applications of nitrogen in excess of crop uptake. Net production of H occurs by natural processes, including nitrification of ammoniacal N: +

+ 2H+ -+2NH,' 2NH4+ + 302 + 2NO2- + 4H' + 2H2O 2NH,

2N02-

+ O2 + 2N0,-

Some of this acidity is neutralized by NO,- uptake and the subsequent release of OH -. Other compromising factors are the denitrification of NO3-, NH, volatilization, or NH,' uptake by the plant. Management practices which optimize N-use efficiency and ultimately reduce the amount of NO,- lost by leaching could slow the rate of acidification (Robson, 1989). Soil acidification in the surface layer is also accelerated by the removal of basic cations (Ca, Mg, K, and Na) in the harvested product. Different wheat cropping

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practices increase or decrease the potential acidity caused by nitrification of applied N, depending on the relative amounts of forage, grain, and/or straw (and therefore basic cations) removed. Removal of straw alone depletes basic cations to the greatest extent and actually enhances acidification by nitrification (Westerman, 1987). Base depletion is less severe in forage removal (by grazing or hay) and least severe in grain removal. Most multipurpose cropping systems (e.g., forage-plus-grain removal) reduce potential acidity caused by ammoniacal N application, albeit to different degrees depending on the excess basem ratio in different plant parts. Therefore, soil acidity will develop, and soil pH will decline, at different rates depending on the cropping system. The severity of soil acidity increases as yields of vegetative or grain dry matter increase. This may explain why soil acidity has reached unprecedented levels in some intensively managed soils of Oklahoma and Kansas.

€3. PHYTOTOXICI'I-Y OF ACIDSOILS Acid soils are phytotoxic to wheat, not because of the deficiency or excess of any one chemical element, but as the result of a complex of nutritional disorders: deficiency of essential nutrients like Ca, Mg, and Mo; decreased availability of P; and toxicity of Al, Mn, and H +.The relative importance of each of these factors is difficult to generalize across soils with inherently different soil solution chemistry. The chemical and biological components of soil acidity, and their effects on plant growth, are examined in a broader framework by Adams (1984) and Robson (1989). That part of the literature which pertains exclusively to wheat deserves some reiteration for the benefit of the reader with less familiarity. The more experienced reader might continue with Section I.C. It is widely accepted that as pH decreases, both Al and Mn increase in solubility and, consequently, in their relative toxicities (White, 1970; Marion er al., 1976). The classic review by Foy et al. (1978) pushed Al toxicity to the center of attention among soil scientists, physiologists, and geneticists. However, the precise form(s) of Al which restricts wheat root growth and subsequently hinders shoot growth by interfering with nutrient uptake and transport remains a subject of debate. The various forms of potentially available Al, such as the relatively nonphytotoxic amorphous precipitates [AI,SiO,, AI(OH),, and AIPO,] and organically complexed forms of Al, or the potentially toxic inorganic monomers [Al t3, AI(0H) + 2 , and AI(OH),+] have been summarized by Kinraide (199 1 ) and Blamey and Asher ( 1993). Most Al in soils is present as solid, nontoxic forms. Typically at pH > 5.5, exchangeable Al decreases dramatically with exception of exchange sites associated with organic matter; consequently, Al toxicity also declines. Baligar et al. (1992) found that the amount of Al extracted by 0.01 M CaCl, best predicts wheat seedling root growth in a range of acid soils of the Appalachian

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region. This result is expected if CaCl, primarily extracts only phytotoxic A1 species available in soil solution plus some exchangeable Al. Similar results can be found in other acidic soils and plant species (de A. Machado and Gerzabek, 1993). Immediate effects of A1 are observed in meristematic root tissue as elaborated in Section 11, with root elongation ultimately retarded and root ion transport disrupted. The degree of retardation is subject to ameliorative effects of Mg, Ca, and K (Kinraide and Parker, 1987; Tanaka er al., 1987; Baligar et al., 1992). Consequently, higher base saturation in surface horizons may lead to less inhibition of root elongation compared to subsurface horizons. Aluminum-stressed wheat roots typically appear shortened and thickened, while the effects of A1 toxicity on wheat foliage are not as clearly defined and are often confused with poor N, P, Ca, or Mg nutrition, and/or drought stress. Signs of A1 toxicity are unusually prostrate growth, leaf chlorosis resembling N deficiency, and leaf or stem purpling similar to P deficiency (Unruh and Whitney, 1986). Aluminum stress decreases total chlorophyll concentration and photosynthetic rate in wheat, but the decline in transpiration rate is the most severe (Ohki, 1986). The aluminum concentration in the leaf may reach 0.3 mmol kg - when A1 is supplied in excessive amounts for an extended period (0.30 mM for 28 days after transplanting seedlings; Ohki, 1986), but tissue concentrations in the foliage may not accurately reflect A1 stress (Keisling et al., 1984). Because leaf concentrations constitute
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Foy, 1988). Vegetative growth of the Al-tolerant cultivar was much less reduced than that of the sensitive cultivar. Likewise in soybean, Glycine max (L.) Merr., the combined effect of water-deficit stress and A1 stress on leaf water status exceeded predictions based on additive effects (Goldman er al., 1989a). Intuitively, root systems impaired by A1 stress have limited ability to withstand drought stress. Goldman er al. (1989b) recommended that soybean breeders select for A1 tolerance when developing drought tolerant cultivars because A1 tolerance may impart some degree of drought tolerance in areas limited by low rainfall and subsoil acidity. Testing of similar hypotheses are apparently lacking in wheat. Aluminum activity in soil solution may not totally explain phytotoxicity of soil acidity. The availability of Mn may exceed basal plant requirements for enzyme activation in the tricarboxylic acid cycle and for other physiological functions. Toxicity typically develops in acid soils with pH 6 . 5 or at higher pH values when soils are poorly drained or waterlogged, conditions which promote reduction of Mn4+and Mn3+to the more available Mn2+(Westerman, 1987). Thus, it is possible to find symptoms of Mn toxicity at a pH too high to cause A1 toxicity (Foy, 1983). Unlike Al, which is an integral part of the clay structure in acid soils, Mn is not part of the clay structure and originates in Mn-oxide parent materials or as an impurity. Thus, in addition to pH, total Mn content inherent to the soil largely determines potential Mn toxicity. Visual symptoms of Mn toxicity recorded for wheat under greenhouse conditions include stiffness and stunting of leaf tissue, with general chlorosis and some leaf necrosis, purpling, white flecking, and tip burn (Keisling et al., 1984; Ohki, 1984). In contrast to A1 stress, Mn toxicity is primarily expressed in the foliage and may not appear immediately following exposure to toxic amounts of Mn. The Mn concentration for wheat shoot tissue associated with a 10% reduction in growth or activity (critical toxicity level) is approximately 7 to 8 mmol kg - I (Fales and Ohki, 1982; Ohki, 1985; Keisling er al., 1984); this level increases almost threefold for leaf photosynthesis, chlorophyll concentration, and transpiration (Ohki, 1985). Why Mn-induced stress reduces dry weight production before reducing photosynthesis and transpiration is not documented, but may be explained by a concomitant increase in the maintenance component of respiration in response to Mn toxicity. Tissue Mn concentrations which exceed the critical toxicity level also cause reductions in Ca and Mg concentrations, with potential consequences to forage digestibility (Fales and Ohki, 1982). In turn, Mg availability influences the severity of Mn toxicity.

c. SEVERITY AND EXTENTOF SOIL ACIDITY Once considered a problem confined to tropical agricultural regions or high rainfall areas with highly weathered soils, soil acidity now attracts global attention

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in nearly every major wheat production region. Wheat producers must contend with acid soils in the United States, Australia, Canada, the Southern Cone region of South America, the Carpathian basin region of Europe, Central Africa, and, more recently, South Africa. The traditional view in the United States is that soil acidity is the most detrimental to crop production in the southeastern states. For wheat production in particular, it is the more densely cultivated region of the southern Great Plains where soil acidity takes its toll on vast acreages. Although virgin soils were slightly acidic (pH 5.5-6.5), surface soil pH has gradually declined to 4.0 or below over the last 40 years under continuous wheat production. The severity of acidity is greater in wheatland soils of the Southeast, but the number of cultivated wheat hectares impacted by acid soils is greater in the southern Great Plains. A statewide soil test survey revealed critically acid conditions (pH <5.0) in the surface layer of 15% of the total wheat area of Oklahoma (Johnson, 1986). This would equate to about 0.5 million hectares of hard red winter wheat in Oklahoma alone (authors’ estimate) which suffer potential production losses due to soil acidity. Soil acidity in the southern Great Plains extends across Oklahoma, from northcentral Texas to southcentral Kansas, and reduces production primarily due to toxic amounts of Al and, to a lesser extent, Mn. Soil test surveys conducted in Kansas indicated four intensively cropped counties in southcentral Kansas with alarming frequencies of low pH soils (Unruh and Whitney, 1986). The actual pH level is highly variable in this tri-state region, largely depending on liming practices adopted by different producers or landowners. Like the southern Great Plains, mildly acidic soils of the eastern edge of the wheat belt in southern Australia have become increasingly acidic over the past 40 years (Fisher and Scott, 1987; Chartres et al., 1990; Scott et al., 1992). Some 1 million hectares devoted to wheat production in the Albury, Wagga, and Cootamundra districts of New South Wales are threatened by acid soils and A1 toxicity. More than one-third of the total agricultural land area in New South Wales has severely acidic surface soil with pH <4.5 (Scott et al., 1992). Also affected by acid conditions are the eastern wheat areas of Western Australia (Davidson, 1987). Acidity is not necessarily confined to the surface layer, depending on soil classification. Cultivated soils have developed surface acidity throughout Alberta and northeastern British Columbia (Penney et al., 1977) in northwest Canada. Extensive subsoil acidity also occurs in the Peace River region of Alberta and British Columbia (Zale and Briggs, 1988). To what extent soil acidity in this region influences wheat production specifically could not be determined from these reports, but a review by Briggs and Taylor (1994) indicates that 5.0 million hectares of Canadian arable soil are acidic, with over 2 million in western Canada where wheat is widely produced. In Europe, A1 tolerance is desired in the intensively cropped wheat areas of

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Hungary (Bona and Carver, 1992) and in Poland, where 60% of all agricultural land is critically acidic (Aniol, 1984b). Rengel and Jurkic (1993) recognized the importance of A1 toxicity to wheat production in Croatia and Yugoslavia, where about 50% of all agricultural land is affected. Another major wheat production region limited by increasing soil acidification is composed of the three principal wheat producing areas of South Africa. Based on a personal communication (1994) with 0. J. Bosch, Grain Crops Institute, Bethlehem, approximately 0.4 million hectares of wheat produced in the summer rainfall region of South Africa are considered critically acidic with pH(KC1) <4.5. Most of those hectares are in the higher rainfall areas. In the winter rainfall region of South Africa, another 0.07 million wheat hectares have critical soil acidity. Aluminum toxicity is the primary limiting factor to wheat production in acidic soils of South Africa; Mn is not normally available in toxic quantities. The severity and extent of soil acidity in worldwide wheat production warrants continued investigation of the plant’s defense mechanisms against low-pH-induced stresses and the genes which regulate those mechanisms.

11. PHYSIOLOGY OF ALUMINUM AND MANGANESE TOLERANCE IN WHEAT As discussed earlier, reduction in wheat growth and yield in acidic soils is generally a consequence of A1 and/or Mn toxicity. The chemistry of these two metals is quite different, as is their role in plant development. Manganese is an essential element whereas Al is not. Mechanisms by which plants tolerate toxic levels of A1 and Mn are likewise different. Wheat cultivars tolerant of one metal are not necessarily tolerant of the other. The next section describes physiological mechanisms by which plants may tolerate A1 and Mn, with particular emphasis on insight gained from recent work with wheat.

A.

UPTAKE AND

DISTRIBUTION OF ALUMINUM IN ROOTS

The primary response to Al stress in wheat occurs in roots, where reduced elongation at the tip, followed by swelling and distortion of differentiated cells, as well as root discoloration, has been described for a wide variety of taxa (Foy et al., 1978; Bergmann, 1992, pp. 282-289). Within meristematic and root cap cells, A1 toxicity is associated with increased vacuolation and turnover of starch grains (de Lima and Copeland, 1994), as well as disruption of dictyosomes and their secretory function (Bennet et al., 1985; Puthota et al., 1991). In Zea mays L., Bennet et al. (1985) observed that cortical cells of Al-damaged roots were swollen, with

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grossly distorted walls; rupture of the cells of the epidermis and outer cortex was noted at high Al concentrations. The ability of Al-damaged roots to synthesize and translocate growth-promoting hormones such as cytokinins does not appear to have been investigated. An understanding of the dynamics of root growth inhibition by Al is important in designing experiments to elucidate Al tolerance mechanisms. Recent studies have shown that reduction in elongation of Al-challenged wheat roots commences after a lag period that may be as short as 2 hr (Ownby and Popham, 1989; Ryan ef al., 1992). Parker (1 994) has concluded from analysis of wheat root growth that there are two responses to Al: an initial “acute” inhibition of growth that is followed by a later “chronic” A1 effect on root growth. In his study, some, but not all, wheat cultivars became acclimated to low levels of Al and resumed growth after the initial shock. Surprisingly, the acclimation phenomenon appeared in Alsensitive cultivar ‘Scout 66’ as well as in Al-tolerant ‘Atlas 66,’ and no correlation between acclimation and Al tolerance was observed. The concept of acute vs chronic growth inhibition by Al is useful in evaluating various approaches to the study of Al toxicity. As Parker ( 1994) noted, short-term experiments by physiologists may reveal mainly acute effects of Al on growth, while long-term field work and breeding studies are more likely to observe chronic phytotoxicity of Al, which could have a different physiological basis. Likewise, short-term tests for Al tolerance such as root growth (Kerridge et al., 1971) and hematoxylin staining (Polle et af.,1978) may not invariably predict long-term performance of crops in acid soils. Indeed, examples of this have been observed (Scott and Fisher, 1989). This concept may also be useful in resolving what appears to be contradictions in the plant response to Al. Among the effects of Al that often appear after its initial effect on growth are inhibition of DNA synthesis (Wallace and Anderson, 1984), alteration of cell membrane potential (Kinraide, I988), and reduction of root apex H + efflux (Ryan ef al., 1992). These effects eventually contribute to cessation of root growth (i.e., the chronic effect) but they appear to succeed an initial response with which they may not be primarily involved. Two major unresolved questions about Al in plants concern its chemical form and cellular distribution. The general assumption that Al 3 + , the major form of Al,,,,,,,,at pH <4.75, is phytotoxic has been confirmed in wheat (Parker et al., 1988). However, as Taylor (1991) pointed out, the level of A13+that may exist in cell cytoplasm can be no greater than 10 l o M , and is probably much lower due to inorganic phosphate and other ligands. The results of studies in virro that assume reaction with Al 3 + thus may not apply to cytoplasmic conditions. Mononuclear hydroxy-Al does not appear to be very toxic in wheat but was suggested to be more toxic than A13+ in dicots (Kinraide and Parker, 1989; but see Kinraide, 199 I). The polynuclear hydroxy-Al (A1 species may be a major toxic Al species in the root apoplast. Parker et al. (1989) reported that Al,, was as toxic to AItolerant wheat cultivar ‘Seneca’ as to Al-sensitive cultivar ‘Tyler’; however, Kin~

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raide (1991) later suggested that Al,, was less likely to form in wheat than in dicots. In a study using purified cell walls, Bertsch et al. (1994) concluded that Al,, is more mobile and is bound with less affinity to carboxyl groups of the cell walls of Al-sensitive wheat cultivar ‘Caldwell’ than Al-tolerant cultivar ‘Yecora Rojo.’ Clearly, the task of characterizing and developing Al-tolerant plant lines is complicated by the diverse chemical species of Al that can form in plants and the possibility that not all may act at the same cellular sites. Zhang and Taylor (1989) examined Al uptake kinetics in wheat and observed an initial 30-min period of binding of Al to cell walls, followed by an extended period of linear uptake, the magnitude of which was about the same in both Alsensitive and Al-tolerant cultivars. However, the authors later concluded that this linear phase consisted of two separate processes: fixation of Al in the apoplast that was dependent on metabolism and permeation of the cell membrane (Zhang and Taylor, 1990). Although Taylor and co-workers did not observe marked differences in A1 accumulation between Al-tolerant and Al-sensitive wheat cultivars, other studies indicate that there is less total uptake of Al into roots of Al-tolerant wheat cultivars compared to sensitive cultivars, particularly in the zones of cell division and elongation. Rinc6n and Gonzales ( 1992) found that A1 accumulation in the terminal 2 mm of Al-sensitive wheat cultivar TAM 105 roots was about seven- to eightfold greater than in tolerant Atlas 66 during the critical first 6 hr of exposure. Likewise, Delhaize et al. (1993a) observed, within 4 hr of exposure, a markedly increased uptake of Al in an Al-sensitive wheat genotype compared to a sibling tolerant genotype. Attempts to localize Al in root tissue, however, have not produced consistent results, apparently varying according to the methods used. Early work with X-ray microanalysis suggested that Al was detectable in the cell, especially in the nucleus (Naidoo et al., 1978). In more recent studies using better fixation procedures, X-ray microanalysis has revealed Al only in cell walls. Aluminum was below the limit of detection by X-ray microanalysis in root cells of wheat (Ownby, 1993) and oat (Avena sativa L.) (Marienfeld and Stelzer, 1993) that were treated for 24 hr with growth-inhibiting levels of Al. In contrast to X-ray microanalysis, other methods indicate that considerable Al crosses the plasmalemma of Al-intoxicated root cells. Aluminum was readily detected in the nuclei of Al-treated wheat root cells by the putative Al-specific dye hematoxylin (Rinc6n and Gonzales, 1992) and the fluorochrome morin (Tice et al., 1992). The latter study also used procedures to elute Al from the apoplast (extracellular space) and concluded that, when Al-tolerant cultivar Yecora Rojo and Al-sensitive cultivar Tyler were treated with sufficient Al to inhibit growth by 50%, about 55 to 70% of total tissue Al was in the symplast (inside the plasmalemma) after 48 hr. Partitioning of Al between the apoplast and symplast was the same for both cultivars. Considering that initial root growth inhibition occurs

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within the first 2 to 6 hr of treatment (the “acute” response), it is possible that Tice ef a/. ( 1992) were observing the accumulation of A1 in the cytoplasm of Aldamaged cells. It cannot be concluded from this work that entry of A1 into the cytoplasm was the sole cause of growth inhibition. The ability to exclude A1 from shoots as well as roots also appears to be a trait of Al-tolerant wheat cultivars. Foy and Peterson (1994) noted that when 10 wheat lines differing in A1 tolerance were grown in Al-toxic Tatum soil (pH 4 . 3 3 , there was a strong positive correlation between accumulation of A1 in shoots and growth inhibition. Al-tolerant cultivars also contained two- to fourfold more shoot potassium than sensitive lines; however, there was no correlation between tolerance and the level of shoot Ca, Mg, or P. In summary, evidence now suggests that Al-tolerant wheat cultivars exclude A1 from root and shoot tissue better than Al-sensitive cultivars. This presumably results from the ability of tolerant cultivars to better exclude A1 from the root symplast; however, mechanisms of A1 exclusion remain unclear. Several physiological processes by which plants could exclude A1 from the tissue as a whole or the symplast in particular are described next.

B. ALUMINUMTOLERANCE MECHANISMS During the past two decades there has been no shortage of hypotheses to explain differential tolerance to A1 among plants. The reader is referred to reviews by Roy et al. (1988), Haug and Shi (1991), and Taylor (1991) for a more complete description of these hypotheses as well as earlier work on which they are based. Rao et al. (1 993) have reviewed physiological and genetic aspects of breeding for A1 tolerance in crops of the American tropics. In general, strategies that various plants use to tolerate A1 fall into two categories: (1) external tolerance mechanisms, by which A1 is excluded from plant tissue, especially the symplastic portion of root meristems; and (2) internal tolerance mechanisms, where A1 that has permeated the plasmalemma is sequestered or converted into an innocuous form. Work published during the last 3 to 5 years indicates that exclusion of A1 from plant tissue and cells is probably more important than internal mechanisms for A1 tolerance in wheat and most other crop plants. Specific responses in Al-tolerant plants that have generated enthusiasm among workers in this area include the following. 1. Accumulation and/or Secretion of Organic Acids

The ability of various di- and tricarboxylic acids to form strong complexes with A1 has led to various studies attempting to show that plants use this as a defense mechanism against A1 toxicity. Galvez e t a / . (1991) observed that Al-tolerant sor-

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ghum [Sorghum bicolor (L.) Moench] cultivar ‘SC283’ increased root organic acid content more than Al-sensitive ‘ICA-Nataima’ in response to Al. In the latter study, however, the total tissue level of potential Al-complexing organic acids such as citrate and malate was ca. 400 and 1600 pM, respectively (authors’ estimate), even in the Al-sensitive cultivar. This is probably much higher than the concentration of tissue Al; thus, even Al-sensitive plants would seem to have sufficient organic acids to complex Al. Perhaps more convincing are studies on the ability of Al-tolerant plants to release organic acids into the root environment when challenged by Al. Miyasaka et al. (1991) observed that Al-tolerant snapbean (Phaseolus vulgaris L.) cultivar ‘Dade,’ when grown under sterile conditions for relatively long periods (8 days), exuded citric acid to a level that reached 26% of initial A1 (mol/mol). This response was not seen in Al-sensitive cultivar ‘Romano.’ The authors concluded that exudation of citric acid into the medium provided Al tolerance in snapbean, either by chelating external AI and thus preventing its entry into the root (see Bartlett and Riego, 1972) or by mobilizing phosphate that had been precipitated with Al in the root apoplast. The idea that exudation of organic acids may function as an Al tolerance mechanism in wheat is supported by Delhaize and co-workers. When two nearisogenic lines of wheat were challenged with Al, the Al-tolerant but not the AIsensitive line released malate into the medium (Delhaize et al., 1993b). Exudation of malate was not associated with Al-induced phosphate deficiency; the Alsensitive line did not release significant amounts of malate when grown in the absence of phosphorus. The time course of release of malate, shown in Fig. 1, demonstrates that this phenomenon is rapid enough to account for resistance to an initial acute phase of growth inhibition by Al. The amount of secreted malate corresponded to about 35% of the initial All,,,,,, (mol/mol) in the medium. Although malate release was quite dramatic in response to high levels of Al, the difference in malate exudation between Al-sensitive and Al-tolerant lines was much less pronounced at lower Al levels that inhibited root growth only in the sensitive line (Delhaize et al., 1993a). Recent work has reinforced the concept that Al tolerance in wheat may be based on exudation of malate and its chelation of Al. In a survey of 36 wheat cultivars, there was a strong correlation between long-term (7 day) tolerance to Al and shortterm (80 min) efflux of malate (Ryan et al., 1994b). In the Al-sensitive genotype ES3, root growth inhibition by 3 pM Al was partially reversed by 10 pM malate and completely reversed by 20 pM malate. The activity of two enzymes presumably involved in malic acid synthesis, PEP carboxylase and NAD-malate dehydrogenase, was the same in Al-tolerant wheat genotype ET3 and Al-sensitive ES3, and was not altered when roots were grown in Al (Ryan et al., 1994a). However, they observed that a variety of anion channel blockers inhibited malate efflux, suggesting that Al 3 + -induced malate efflux involved activation of these channels

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T

0.41

d I

d

0. 0

5

Al-sensitive

5

Time (h)

Figure 1. Time course of malic acid secretion by two near-isogenic lines of wheat, one Al tolerant and the other AI sensitive. Five 6-day-old seedlings were incubated in flasks containing 20 ml of sterile nutrient solution, pH 4.1, to which 50 pA4 A1 as AIK(S0,)2 was added at zero time. This treatment reduced root growth of the Al-tolerant line to 70% of control and the Al-sensitive line to less than 10% of the control during a 5-day exposure to Al. The two lines of wheat were derived from a cross between Al-tolerant cultivar Carazinho and Al-sensitive cultivar Egret. Adapted from Delhaize et al. (l993b) by permission.

in Al-tolerant wheat lines. K appears to serve as a counter-ion to Al-stimulated malate efflux. Malate most likely functions in the apoplast and the unstirred boundary layer surrounding roots by chelating A1 to form Al-malate and thus shielding potential sites of injury such as the plasmalemma (M. Delhaize, personal communication). At present, exudation of organic acids is the most promising mechanism of Al tolerance yet studied. Preliminary evidence showing that organic acid release correlates with Al tolerance in crosses between tolerant and sensitive cultivars further supports this model (Delhaize et al., 1993b). The demonstration that mutant lines deficient in organic acid exudation also become more A1 sensitive would further bolster this mechanism of Al tolerance. +

2. Binding or Fixation of Aluminum in the Cell Wall Region The interaction of A1 with cell wall constituents remains a relatively unexplored aspect of Al phytotoxicity. It is generally thought that binding of A1 to charge sites on the cell surface is a prerequisite for uptake and toxicity. Plants with a high root cation exchange capacity (CEC) are generally more sensitive to A1 than similar lines with low CEC (Blarney er al., 1990). In terms of a specific interaction with Al, Blarney and co-workers have provided evidence that Al displaces Ca from cell

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wall pectic acids, which reduces the movement of water and mineral nutrients through the cell wall interstices (Blarney er al., 1993). This rapid response to Al, at least in model systems, is consistent with the time course of “acute” effects of A1 on root elongation. Hunter and Bertsch (1994), using cell wall fractions isolated from wheat cultivars differing in A1 tolerance, showed that Al may disrupt the hydrogen bonds between cellulose molecules; the extent of this response correlated well with the degree of sensitivity to Al among wheat cultivars. In contrast to these reports, Kinraide et al. (1992) have concluded that cell surface negative charges, derived from cell wall pectins as well as charge sites on membrane lipids and proteins, do not play a significant role in differential Al tolerance in wheat. They based this conclusion on the observation that cultivars Atlas 66 and Scout 66, the latter much more sensitive to Al than the former, exhibited about the same level of root growth inhibition when treated with La3+.The two cultivars were expected to show the same relative response to La3+as to A13+ if surface charge was the basis of differential sensitivity. Many early studies found an association between A1 toxicity and accumulation of A1 phosphate precipitates in the apoplasm (Clarkson, 1967). It is still not clear if Al-tolerant plants actively release phosphate to immobilize A1 in the apoplast. Evidence for active efflux of cell phosphate in Al-tolerant sugarbeet (Beta vulgaris L.) cultivars was provided by Lindberg (1990). However, it should also be noted that cellular phosphate often leaks into the cell wall region as part of the Al stress effect, seen for example in the Al-sensitive wheat cultivar ‘Victory’ (Ownby, 1993). In fact, the higher levels of total Al observed in roots of Alsensitive wheat cultivars (see earlier discussion) could be due in part to the accumulation of relatively innocuous Al phosphate complexes in the apoplasm of Aldamaged roots. Such precipitation would reflect cell damage by Al, not an Al tolerance mechanism per se. Various studies have suggested, moreover, that tolerance of low phosphate, and high efficiency in uptake and distribution of phosphate, may be characteristics of Al-tolerant wheat cultivars (Foy el ul., 1978). De Miranda and Rowell ( 1990), for example, have provided evidence that Al-tolerant wheat cultivars are better able to absorb and translocate phosphate to shoots in the presence of Al.

3. Production of Root Mucilage Horst et al. (1982) demonstrated that in cowpea (Vigna unguiculuta),removal of root cap mucilage caused an increase in Al uptake and phytotoxicity. Among 10 cultivars of winter wheat, Henderson and Ownby (1991) noted a strong correlation ( r = 0.82) between root mucilage volume and A1 tolerance as determined by root growth assays. The mechanism of protection by mucilage is not clear. Although it is generally assumed that mucilages contain Al-binding pectic acids,

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mucilage droplets of Al-treated wheat and cowpea did not stain with hematoxylin even under conditions where the root surface was readily stained (Henderson and Ownby, 199 I ). Because Al-organic acids complexes do not react with hematoxylin (Ownby, 1993), it was suggested that A1 in the mucilage was not associated with pectic acids, but rather with organic acids released by the root. The mucilage droplet would thus create a “boundary layer” in which diffusion of Al to the root surface is slowed and where the organic acid/Al ratio would likely be much more favorable than in the rhizosphere as a whole (Henderson and Ownby, 1991).

4. Exclusion of Aluminum at the Plasmalemma Considering its essential role in cell metabolism and growth, it is not surprising that the plasmalemma has been postulated to be the site of selective Al toxicity. The effects of Al on membrane integrity and function include binding of A1 to membrane lipids (see Haug and Shi, 1991), as well as inhibition of ATPase activity (Matsumoto and Yamaya, 1986), NADH-linked electron transfer (Loper et al., 1993). and ion channel functions (Rengel and Elliott, 1992). In sugarbeet, Al toxicity was associated with an increase in the ratio of phosphatidylcholine to phosphatidylethanolaminewhich could increase membrane permeability (Lindberg and Griffiths, 1993). Caldwell (1989), using the luminescent cation terbium [Tb(lll)], observed that wheat root membranes isolated from Al-sensitive cultivar ‘Anza’ appeared to bind more Al than did tolerant cultivar ‘BH 1136.’ He also inferred that Al could displace Ca from membrane protein-binding sites. However, it has been suggested that the plasmalemma continues many of its functions well after initial Al toxicity effects are noted. These functions include maintenance of membrane potential and H + efflux (Kinraide, 1988) and K + absorption (Petterson and Strid, 1989; but see Nichol et al., 1991). Huang et al. (1993) observed that A1 could inhibit the uptake of Ca in wheat seedlings, although it was demonstrated in a later part of this study that levels of A1 sufficient to inhibit growth did not affect Ca uptake (Ryan et al., 1994~). Although Pifieros and Tester (1993) found that 70 puM A1 completely blocked the Ca channels of plasmalemma-enriched fractions from wheat roots, the Ca channels of membranes isolated from Al-tolerant wheat cultivar Atlas 66 and Al-sensitive cultivar Scout 66 were equally sensitive to Al, indicating that differences in channel proteins did not account for differential Ca absorption (Huang et d , 1994). The idea that the plasmalemma is the primary target for Al toxicity remains attractive; however, the evidence is certainly not unequivocal. It remains to be shown if fundamental differences in membrane organization contribute to differential A1 tolerance in plants. Long-term effects of A1 toxicity, however, certainly do involve disruption of plasmalemma integrity and function as part of the overall disruption of cell metabolism (Meharg, 1993). Many of the cellular stress re-

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sponses observed in Al-intoxicated roots, for example, are elicited not only by Al, but also by other factors such as heavy metal toxicity and pathogen invasion in which damage to the plasmalemma occurs.

5. Synthesis of Aluminum Tolerance Proteins Aniol ( 1984a) suggested that plants could develop A1 tolerance through the synthesis of proteins that bind or sequester A1 and render it innocuous within the symplast. Since then there has been an intense effort by a number of laboratories to identify proteins synthesized in tolerant but not sensitive cultivars during AI challenge. As A1 tolerance is determined by potentially many genes, the task of identifying specific proteins that might confer Al tolerance is difficult. What is clear from recent work, however, is that Al-challenged roots synthesize a considerable number of proteins as part of the cellular stress response itself. Table I1 lists proteins whose synthesis or activity is either induced or upregulated in roots experiencing A1 stress. In addition to those proteins listed in the table, Snowden and Gardner (1993) also identified, by screening a wheat cDNA library from Al-treated roots, three other gene products whose deduced amino acid sequence was not homologous to any known protein. The pattern of expression of these genes, however, was not consistent with a role in Al tolerance. Table I1 also suggests some possible functions of these “A1 stress” proteins in Al-intoxicated roots, although in none of the examples given have their roles been unequivocally established. Slaski (1990) observed that Al-tolerant cereal species had more total NAD kinase activity than Al-sensitive cereal species. Among ditelosomic lines of

Table I1 Proteins Whose Synthesis or Activity Is Increased in Response to A1 Challenge in Plant Roots Protein ( I ,3)-/3-glucan synthase” NAD kinase

TAIL18 Phenylalanine ammonia lyase-like protein Metallothionein-like protein

Possible function Synthesis of callose as part of wounding response Upregulation of pathway of synthesis of secondary compounds (?) Pathogenesis-related protein; defense against fungal pathogens (?) Synthesis of Al-binding flavonoids (?)

Unknown

“Evidence from cowpea, not wheat.

Reference Horst er rrl. ( 1991) Slaski ( 1990) Cruz-Ortega and Ownby (1993) Snowden and Gardner (1993) Snowden and Gardner (1993)

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‘Chinese Spring’ wheat, however, the elevation of NAD kinase activity in response to A1 treatment was about the same in both sensitive and tolerant lines. The PAL homologue was suggested to function in synthesis of flavonoids that could bind Al. None of the other proteins appear to have any direct role in conferring Al tolerance to plant roots. Cruz-Ortega and Ownby (1993) have suggested that roots experiencing Al stress are more susceptible to other toxic elements and to pathogens. As a defense response, the cells may thus synthesize PR proteins such as TAI- 18 (Cruz-Ortega and Ownby, 1993) as well as callose (Horst et al., 1991) and possibly the MLPs that could function in binding heavy metals (Snowden and Gardner, 1993). To date there has been no unequivocal demonstration of a protein that is the product of a gene conferring Al tolerance. Picton et al. (1991) used twodimensional PAGE to identify five proteins that were more abundant in Al-tolerant wheat cultivar ‘Waalt’ than in Al-sensitive ‘Warigal’ in the absence of Al. These proteins subsequently appeared in Warigal during Al stress, but none have yet been characterized. Likewise, Delhaize et al. ( 1991) observed polypeptides specific to Al-tolerant wheat cultivar ‘Carazinho,’yet none cosegregated with the Altolerant phenotype when Carazinho was crossed with Al-sensitive cultivar ‘Egret’. Basu et al. (1994) described two forms of a 51-kDa protein, called RMP51, in the microsomal fraction extracted from the terminal 5 mm of wheat root tips. RMP51 was rapidly induced in Al-tolerant cultivar ‘PT741’ but not in sensitive cultivar ‘Neepawa’ during challenge with Al. Although also present in Cdstressed plants, RMP5 1 was not induced by heat shock or toxic levels of Mn and Cu, and turned over when Al was removed from the growth medium. From the preliminary data, RMP51 seems to fit the criteria of an “A1 tolerance” protein. Much more characterization of this protein is needed. To summarize, various proteins that are elicited during Al toxicity have been identified, but none have been identified whose presence can account for differential A1 tolerance among plants. It is assumed but not proven that proteins conferring Al tolerance are inducible. The possibility that products of genes providing Al tolerance are constitutive or that they encode protein isoforms that are indistinguishable from those in Al-sensitive plants on one- or two-dimensional gels cannot be ruled out. Some of the properties of a novel, inducible “A1 tolerance” protein would include: (1) consistently high concentrations in various tolerant lines and reduced levels or absence in all sensitive lines; (2) cosegregation with tolerant phenotypes when tolerant and sensitive cultivars are crossed; (3) relatively specific to Al toxicity; and (4) a physiological role that is consistent with proposed mechanisms for metal tolerance (e.g., a protein involved in production or secretion of chelating ligands or one that is part of an Al efflux pump or some other exclusion process). As this discussion illustrates, we are still a long way from completely understanding the physiological basis of Al tolerance. Current work suggests, however,

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B. F. CARVER AND J. D. OWNBY

that if one could design an Al-tolerant wheat cultivar, it would secrete organic acids for complexing Al, produce copious mucilage, and probably have a low CEC. Future work should identify other traits that play a major role in the physiological tolerance to Al in plants.

c. UPTAKE AND DISTRIBUTION OF MANGANESE IN PLANTS Like Al, Mn is a potential source of phytotoxicity in acid soils. However, many soils, including highly weathered acidic soils of the tropics, are generally low in Mn (Marschner, 1986). Manganese toxicity has not been considered the threat to crop growth and yield that Al toxicity is, especially in wheat-producing areas. Only recently has Mn toxicity in wheat begun to be examined in physiological terms. Two very thorough reviews that discuss general aspects of Mn toxicity in crop plants are Foy er al. (1988) and Mukhopadhyay and Sharma (199 I). Manganese toxicity usually occurs when low soil pH, low soil aeration, or both prevent the oxidation of Mn2+ to MnO, by soil microorganisms (Mengel and Kirkby, 1982). Manganese toxicity is usually aggravated by Fe deficiency. Recent studies suggest that this may be less important in grasses than in dicots, presumably because of the different modes of Fe uptake by the two groups. Iron-chelating phytosiderophores released by Fe-deficient cereals appear to have little affinity for Mn (Zhang, 1993). However, in iron-deficient peas, the inducible iron-reductase system reduced Mn at a rate 1 1-fold greater than in Fe-sufficient plants, which correlated with a 2.7-fold increase in leaf Mn in Fe-deficient plants (Norvell ef al., 1993). Manganese as Mn2+in the soil solution is readily absorbed into root tissue and is translocated to the shoot, where most toxicity symptoms are observed. Thus, while exclusion mechanisms seem to play a major role in A1 tolerance, there is general agreement that Mn tolerance is based almost entirely on internal mechanisms. Why do plants treat these two metals that are characteristic of acid soils so differently? For one, Mn is an essential element. It participates in redox reactions as manganoprotein on the water-splitting side of photosystem I1 (Hoganson and Babcock, 1988) and also functions in mitochondria1 Mn-containing superoxide dismutase (Sevilla er al., 1980). Second, the biological and chemical similarities between manganese and magnesium may enable Mn to enter the plant by the same absorption pathway used by Mg, so that any Mn exclusion process would run the risk of likewise reducing uptake and transport of Mg. For most plants, the best strategy under conditions of high soil Mn seems to be to allow Mn to accumulate to supraoptimal levels in the shoot and then to use internal mechanisms to isolate it from sites of cell metabolism. [For discussion of plants that do seem to tolerate Mn through exclusion, see Mukhopadhyay and Sharma (1991)l.

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Once Mn is absorbed into the root, its movement in the xylem stream seems to vary among species studied. Mn was reported to move as free Mn2+ in tomato (Lycopersicon esculentum Mill.) (Tiffin, 1967) and ryegrass (Lolium perenne) (Bremmer and Knight, 1970). White et al. (198 1) calculated that 63 and 28% of xylem sap Mn moved as complexes with citric and malic acid in soybean and tomato, respectively. Manganese is only slightly mobile in phloem and typically does not accumulate in regions of growth. Visible toxicity symptoms generally develop in mature leaves, including chlorosis in tobacco (Petolino and Collins, 1985) and spring wheat (Macfie and Taylor, 1992). In the latter study, the rate of photosynthesis decreased more than could be accounted for by the decrease in chlorophyll, especially in Mn-sensitive cultivar ‘Columbus.’ Cheniae and co-workers concluded that the reduction in photosynthesis observed in Mn-stressed tobacco was most likely due to interference by Mn in the Mg activation of rubisco (Houtz et a[.. 1988). Although a three-fold increase in leaf polyphenol oxidase was observed in the same study (Nable et al., 1988), nonspecific inactivation of chloroplast proteins by polyphenols does not seem to play a major role in the reduction of photosynthesis. The most diagnostic Mn toxicity symptoms are dark necrotic spots, usually <3 mm in diameter, that develop near margins and midveins (Bergmann, 1992). These necrotic lesions are reported to be rich in MnO, (Marschner, 1986), but Wissemeier and Horst (1992) observed that brown spots in cowpea were also comprised of oxidized phenolic compounds, consistent with the idea that the spots represent sites of immobilization of Mn 2+ through oxidation and precipitation as Mn-phenolic complexes. Early work by Morgan et al. (1976) showed that Mn-stressed cotton had elevated levels of IAA oxidases, which could lower the amount of the growth hormone IAA in the shoot. Reduction in shoot IAA probably affects growth more than leaf necrotic lesions, which are sometimes seen in plants exhibiting little reduction in growth or yield. The authors are not aware of any recent work corroborating this interesting hypothesis. With regard to other hormones, Wilkinson and Ohki ( I 988) demonstrated that supraoptimal levels of Mn in wheat reduced leaf gibberellic acid levels and also inhibited the in v i m synthesis of the GA precursor ent- kaurene. These responses, however, were observed at nutrient Mn levels considerably higher than those correlating with the onset of growth inhibition.

D.

MECHANISMS OF MANGANESE TOLERANCE

Progress in identifying Mn-tolerant wheat cultivars has been limited by lack of rapid, reliable assays for predicting Mn toxicity. Goss er al. (1991) observed a

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strong correlation between the soil and wheat shoot Mg :Mn ratio as well as reduced growth when the shoot Mg :Mn was below 20. They suggested that soil Mg:Mn ratios lower than 214, or shoot ratios lower than 20, could be used as indicators of potential growth-limiting Mn toxicity. In a study of seven wheat cultivars differing in Mn sensitivity, Burke et al. (1990) observed a 14-fold increase in aconitic acid, and lesser increases in malic and citric acid, in sensitive but not tolerant cultivars. These organic acids could serve as biomarkers of Mn toxicity. Other assays that might be useful in assessing Mn toxicity in wheat are the leaf disk assay used in cowpea (Wissemeier and Horst, 1991) and peroxidase activity reported in Mn-stressed cotton (Kennedy and Jones, 1991). An understanding of the physiological mechanism of Mn tolerance remains elusive. Although chelation of Mn by citrate, malate, and other organic acids seems to be a logical mechanism, the studies of Burke et al. (1990) and Macfie el al. ( 1994) showing that organic acids accumulate more in Mn-sensitive than in Mn-tolerant wheat cultivars are not consistent with this idea. Two factors that do seem to be associated with Mn tolerance are silicon uptake and the presence of leaf cell oxalate. Memon and Yatezawa (1984) observed that in the Mn accumulator plant Acanrhopanax, about half of the leaf Mn was associated with cytoplasm and vacuole. They offered the intriguing suggestion that Mn could be transported through the cytoplasm using malate as a “transport vehicle” and then deposited in the vacuole with oxalate as the “terminal acceptor.” In addition, it is commonly observed that Si alleviates Mn toxicity (Vlamis and Williams, 1967). In the presence of Si, Mn is more evenly distributed in the leaf and does not accumulate in necrotic brown spots (Horst and Marschner, 1978). Since Si (silicic acid) can stabilize plant cell walls and form complexes with phenols (Marschner, 1986), it is possible that Si prevents the complexion of Mn and phenolic compounds in necrotic brown spots, and thus allows Mn to be taken up and complexed within the cell in a more innocuous form. According to this conjecture, wheat lines that exhibit high rates of Si uptake and and high rates of oxalate accumulation in their mature leaves should be relatively Mn tolerant.

III. GENETIC MECHANISMS OF TOLERANCE TO ACID SOILS Increased demand by wheat farmers for cultivars with improved tolerance to acid soil stress has prompted numerous studies of its inheritance in the last decade. Wider recognition of A1 toxicity as the predominant growth-limiting factor in wheatland soils has swung the pendulum of attention to A1 tolerance and its genetic control, even though A1 and Mn phytotoxicities may be easily confounded

ACID SOIL TOLERANCE IN WHEAT

137

in a given soil or crop season. Given their physiological independence in wheat, the genetic mechanisms of A1 and Mn tolerance will be addressed separately in this section.

A. BASISOF ALUMINUMTOLERANCE The genetic control of Al tolerance in wheat, until recently, was poorly understood and clouded with conflicting evidence of simple vs. complex inheritance. One early report in wheat indicated that the moderately Al-tolerant cultivar ‘Druchamp’ and the sensitive cultivar ‘Brevor’ differed by a single gene controlling seedling root growth in a 0.06 mM Al solution (Kerridge and Kronstad, 1968). Additional genes were postulated for genotypes with tolerance to higher levels of Al toxicity such as Atlas 66. It is inconceivable that the wide genetic range in A1 tolerance, with varying intermediate degrees (Foy et al., 1965; Lafever et al., 1977), can be explained on the basis of single gene theory. Yet, single cross populations may segregate for a single gene pair at a prescribed level of stress, as in the Waalt (moderately A1 tolerant) X Warigal (A1 sensitive) cross examined by Larkin (1 987) and later verified by Wheeler et al. (1992). The bioassay for measuring tolerance, however, may not have the necessary precision to detect minor genes when a single gene pair with large effects segregates. Berzonsky (1992) recognized the potential ambiguity in classifying plants as sensitive and tolerant based on empirically derived criteria. The multitude of possible physiological mechanisms of A1 tolerance described in Section I1 should inspire geneticists to consider multiple genetic mechanisms. Different genetic systems may operate in seedlings vs. adult plants or at different levels of Al stress. Different systems may be detected by different bioassays (visual vs. quantitative). Not until genome and chromosome location studies were performed did it become certain that several genes can influence phenotypic expression of Al tolerance and that these genes may impart effects of varying magnitude with possible interactions.

1. Gene Number and Location Slootmaker (1974) first attempted to roughly locate genes for acid soil tolerance in wheat by comparing the response of diploid, tetraploid, and hexaploid species relative to their genome constitution. The various hexaploid genotypes (AABBDD) had the highest degree of tolerance. The A genome species exceeded the B genome species but not the tetraploids (AABB) at a lower acidity level. The importance of the D genome for acid soil tolerance was demonstrated by increased sensitivity of a tetraploid derivative lacking the D genome from ‘Canthatch,’ a

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B. F. CARVER ANID J. D. OWNBY

hexaploid cultivar, and restoration of tolerance in the reconstituted hexaploid by addition of the D genome from several sources. Even greater tolerance is provided by the R genome from rye (Secale cereale L.), either by itself or in combination with durum or hexaploid wheat genomes as hexaploid or octoploid triticales. Summarizing, the genomes can be ranked in decreasing order of tolerance: R > D > A > B. It could not be confirmed if the measured responses in acid soil were induced by Al toxicity alone. A similar experiment was conducted by Berzonsky and Kimber (1986) using several diploid, tetraploid, and hexaploid Triticum species, except plants were exposed to 0.44 mM Al in nutrient solutions. Surprisingly, no root regrowth (a measure of tolerance) was observed among diploid A or D genome species classified as tolerant by Slootmaker ( 1 974). This does not necessarily imply the lack of A1 tolerance genes on A and D genomes of hexaploid wheat; rather, the accessions sampled from the progenitor species merely lack tolerance. A unique source of tolerance was found in the tetraploid 7: ventricosum Ces. (Dun genomes) and in other species sharing the Un genome. Further evidence is needed to verify their hypothesis that a genetic mechanism different from 7: aestivum exists in T ventricosum. The prospect is encouraging for transferring tolerance from 7: ventricosum to 7: aestivum via a bridge cross to a tetraploid species. Tetraploid hybrids have been obtained by crossing 7: turgidum with 7: ventricosum (Maan, 1987). The genomic location of Al tolerance genes in the tolerant hexaploid cultivar Atlas 66 indicates that chromosomes other than those of the D genome confer tolerance (Berzonsky, 1992).Camargo (198 1) had previously concluded that two dominant genes controlled tolerance in Atlas 66 in relatively low Al concentrations (c0.22 mM Al). A tetraploid derivative of Canthatch was crossed with Atlas 66, and recombinant inbred lines comprised of D genome chromosomes exclusively from Atlas 66 were isolated. All lines should show tolerance to 0.44 m M Al (to which Atlas 66 is tolerant) if genes for tolerance are located strictly on the D genome chromosomes inherited from Atlas 66. However, some F, lines segregated for sensitive plants and backcrossing to the susceptible hexaploid parent produced an increased proportion of susceptible plants. Therefore, segregation likely occurred on chromosomes of the A and/or B genomes. Aluminum tolerance genes were more precisely located on individual chromosomes using aneuploid lines lacking a specific chromosome or chromosome arm. This method lacks some appeal for locating Al tolerance genes because cultivars for which aneuploid stocks are available do not possess desirable levels of tolerance for genetic improvement. Surprisingly, several genes exist in Chinese Spring for a cultivar generally regarded considerably less tolerant than Atlas 66 (Table 111). The B genome is the least important source of tolerance genes, at least for Chinese Spring, while the most frequent source of tolerance genes is the D genome. Tolerance genes have consistently been identified on chromosomes 2DL

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139

Table 111 Chromosomal Location of Genes ControllingA1 Tolerance in the Moderately Tolerant Cultivar, Chinese Spring Genome A

B

D

Reference

2L

2L, 4L 2L, 3L, 4L, 7 2L. 4L

Takagi et al. (1983) Aniol and Gustafson (1984) Aniol ( 1990)

4L,” 6L, 7s

5s”

6s‘

~

~~

~~

“Originally reported as 4BL. “Original citation indicated larger effect on tolerance (location of “major” gene). ‘ Suppresses Al tolerance of Chinese Spring.

and 4DL. All loci identified confer tolerance except one, 6BS, which in one report suppressed tolerance in Chinese Spring.

2. Gene Expression and Heritability Gene expression at one locus may not be independent of the gene(s) present at other loci, particularly when combined from different species or genera. For example, gene expression of A1 tolerance in wheat is altered in the presence of genes from rye and vice versa. Genes located on chromosomes 3R, 4R, and 6R confer Al tolerance, but the level of tolerance is greatly reduced when added to a wheat background (Aniol and Gustafson, 1984). Apparently, certain wheat genes suppress the expression of Al tolerance genes from rye, yet others allow expression of rye Al tolerance (Table IV). Most chromosome arms of wheat which control Al tolerance in wheat also activate or suppress Al tolerance of rye. On the other hand, some wheat chromosomes may not confer Al tolerance in wheat, at least in Chinese Spring, but influence rye gene expression. Gene interactions are not restricted to wheat-rye combinations. Epistatic interactions are implied when segregation in susceptible X tolerant parent crosses does not fit a simple additive-dominance model. Evidence of epistasis, however, does not surface with discrete classification of segregating progeny. Discrete classification of A1 responses may be an oversimplistic description of a complex segregation pattern. The traditional bioassay of root staining with hematoxylin is commonly used for the discrete classification of A1 tolerance (Polle et al., 1978), but genotypic differences in stainability may be more aptly described using a quantitative scale. Further, the inheritance of A1 tolerance is usually determined from F2 populations instead of by more sophisticated mating designs needed to

B. F. CARVER AND J. D. OWNBY

140

Table N Chromosomal Location of Wheat Genes Controlling

A1 Tolerance in Chinese Spring Wheat and/or Blanco Rye Activate Chromosome tolerance Genome arm in wheat 0 A

B

D

2L 4L 5L 5s 6L 7s 6s 7L 7s 1L IS 2L 3L 3s 4L 5L 5s 6L 7‘

Rye tolerance’ Activate

Suppress

X

X X

X X X X

X X X X X

X X

X X X X

X

X

X X X X X

aAs reported by Anion and Gustafson (1984) and Aniol (1990) for Chinese Spring. bAs reported by Gustafson and Ross (1990) for Blanco rye. PChromosomearm not reported.

detect gene interactions. Such was the case for the cross, ‘Cardinal’ (A1 tolerant) x ‘GK Zombor’ (A1 susceptible), in which epistatic effects at two loci were hypothesized based on root length measurements in nutrient solutions (Bona et al., 1994). Inheritance of root length in acidic soil was also not monogenic. Although Al tolerance in wheat is generally regarded as a dominant trait, the importance of additive gene action has also been emphasized (Aniol, 1984b; Campbell and Lafever, 1981; Ruiz-Torres and Carver, 1992; Bona et al., 1994). The relative magnitude of dominance effects is further complicated by changes in magnitude and direction of dominance for different allelic combinations. Two crosses examined by Bona et al. (1994) represented a wide range in degree of dominance for root length in acidic soil. Dominance was complete in one cross (Cardinal X Zombor) but absent in another (‘Becker’ X ‘GK Kincso’). Using a

ACID SOIL TOLERANCE IN w

w r

141

Table V Relative Root Length" of Four Tolerant and Three Susceptible Wheat Parents and Their F , Progeny Averaged across Two Al Concentrations",' Tolerant" parent (and RRL) Atlas 66 (83%) Dodge (80%) Wrangler (74%) Sandy (68%)

Susceptible" parent (and RRL) (%) Chisholm (61%)

Century (65%)

Siouxland (62%)

83 ( + ) '' 72

66(-) 77 ( + I

79 ( + ) 71

70 62(-)

I 3 (+) 68 ( + )

71 (+)

74 ( + )

"RRL, c/r of root length at 0 mM Al. "0.36 and 0.72 mM Al. I Data taken from Ruiz-Torres and Carver ( 1992). "Classification based on hematoxylin staining of seedling roots (Carver el d , 1988). ''Indicates positive (+) or negative ( - ) heterosis of F, relative to midparent; no sign indicates F , was not distinguished from midparent.

factorial series of tolerant X susceptible crosses, Ruiz-Torres and Carver (1 992) examined the consistency in gene expression for Al tolerance in a series of susceptible backgrounds. The genotype of the susceptible parent influenced the degree of tolerance expressed in the F,. For example, the phenotype of the F, resembled either the tolerant parent or the susceptible parent when Atlas 66 was crossed with either 'Chisholm' or 'Century,' two susceptible parents (Table V). In only three crosses, the hybrid phenotype was intermediate to the two parents. In most crosses where heterosis occurred, the hybrid resembled the tolerant parent in relative root length. This variation in heterotic pattern is not indicative of a single recessive gene governing sensitivity (Kerridge and Kronstad, 1968; Lafever and Campbell, I978), but may reflect allelic variation among susceptible parents (or variation in closely linked loci) since Chisholm has slightly more field tolerance in acid soils than Century (Carver et ul., 1993). Allelic variation at a single locus controlling A1 tolerance has been observed in Hordeum vulgare L. (Minella and Sorrells, 1992) and Zea mays L. (Rhue et af., 1978). Variation in heterotic pattern may also serve as evidence of additive X additive interactions, with or without dominance. Greater consistency was found for the F, phenotype relative to the midparent in a factorial series of soft wheat crosses (Campbell and Lafever, 1981). but their results are not directly comparable to those in Table V because lower Al concentrations were used in reporting root length per se (unadjusted for root growth in the absence of Al stress).

B. F. CARVER AND J. D. OWNBY

142

0 0

I

I

0.09 0.18

0.36

0.72

0.90

Al concentration(mM) Figure 2. Frequency of tolerant seedling plants detected by hematoxylin staining in two F2wheat populations at five Al concentrations ranging from 0.09 to 0.90 mM in solution culture. The hypothesized segregation ratio of tolerant: susceptible plants (or tolerant:intermediate:susceptible plants) is given for each Al concentration. Gene expression is influenced by the concentration of A1 in the nutrient solution and the unique combination of parents. Data taken from Bona er al. (1994).

The genetic expression of A1 tolerance is also influenced by the severity of A1 stress. Camargo (1981) noticed different inheritance patterns at different A1 concentrations,and Campbell and Lafever ( 1981) recognized the importance of characterizing A1 tolerance at more than one A1 concentration. More specifically, Aniol(1984b) observed a significant decrease (more than one-half) in the proportion of tolerant F3 plants from susceptible X tolerant crosses, as A1 concentration increased from 0.30 to 0.59 mM Al. Similarly, Bona ef al. (1994) observed a major break in the proportion of tolerant plants between 0.36 and 0.72 mM Al, using hematoxylin staining to classify segregating F2 plants as tolerant, intermediate, and susceptible (Fig. 2). A single genetic model did not consistentlyexplain the inheritance of A1 tolerance across A1 concentrations in one population. In another population, inheritance was monogenic, but as A1 stress increased, the direction of dominance changed from positive to negative, with no dominance at an intermediate A1 concentration. Minella and Sorrells (1992) also observed a change in the direction of dominance with increasing A1 stress in barley; this pattern has been reported for other types of stress resistance in wheat (Sutka and Veisz, 1988). Either expressivity of tolerance genes decreases at higher A1 stress or different levels of A1 stress induce expression of different gene systems. Obviously some lethal stress level exists at which expressivity ultimately dissipates;

ACID SOIL TOLERANCE IN WHEAT

143

the expression of different gene systems at different sublethal concentrations, however, would be of practical value in selection. Direct evidence of this phenomenon is provided by localization of tolerance genes to specific chromosomes at various Al concentrations (Aniol, 1990). The gene(s) located on chromosome 5AS of Chinese Spring was expressed in the range of 0.037 to 0.074 mM Al, whereas genes located on 2DL and 4DL were expressed only at 0.074 mM. Aniol ( 1 990) speculated that genes located on 5AS, 2DL, and 4DL control Al uptake, but 5AS may also regulate detoxification of A1 inside the root tissue. This study also provided evidence that different genes may impart different effects (major vs. minor) because aneuploid lines which lacked 5AS were classified susceptible even though tolerance genes on 2DL and 4DL were present. Genetic mechanisms of Al tolerance in wheat must be defined with reference to the level of A1 stress applied and the parental combinations used to develop experimental populations. The extent to which the Al tolerance phenotype is determined by genotype is well documented in wheat (Table VI). Estimates of heritability vary widely depending on the method of estimation and pedigree of the population. This further illustrates the genetic complexity of A1 tolerance in wheat and the futility in adopting a simple genetic model to uniformly describe it. Nonadditive gene action, particularly dominance, plays a major role in gene expression, but its value may be limited to F, hybrid breeding systems unless additive epistatic interactions can be fixed in a homozygous genotype. The potential role of epistasis warrants further investigation.

B. BASISOF MANGANESE TOLERANCE The genetic literature is much less developed for Mn tolerance of wheat than for A1 tolerance. There are several reasons for this information gap. The perception that Mn toxicity is less severe than Al toxicity and may be limited to poorly aerated soils has resulted in less concern to characterize genetic variation in Mn tolerance. Plants may develop symptoms of Mn toxicity (chlorotic, erect, or brittle leaves) with no obvious reduction in vegetative growth (Foy et al., 1988). The toxic effect of Al, on the other hand, can be observed much earlier in plant development due to immediate root damage and subsequent retardation of vegetative growth. Fisher and Scott (1993a) questioned whether Mn tolerance is a worthy wheat-breeding objective for southern New South Wales, given the neutral effect on grain yield and the difficulty in screening for Mn tolerance. Experiments designed to assess Mn toxicity often require a treatment period of several weeks before assessing plant damage (usually shoot and/or root yields). The lack of a rapid bioassay, which uses a readily identifiable characteristic(s) of Mn tolerance, has probably contributed to the information gap as well. Large germ plasm

Table V1 Estimation of Heritability for Al Tolerancein Nutrient Solutions (Refs. 1-4) or Acidic Soils (Ref. 5) Al concentration (mM"

Estimate(s)

Reference

0.41,0.79 0.86

1 2

0.57,0.91

2

Relative root length Relative root length

0.70 0.57.0.65

3 4

Relative root length Root length

0.26,0.66 0.57,0.60

4 5

TYPe

Method of estimation

Broad sense Narrow sense' Narrow sense

0.30 0.15, 0.44 0.15.0.44

Root length Root length Root length

0.36.0.72 0.02

Narrow sense

Correlation of F, and F2 Correlation of midparent and F, Variance components: General combining ability Correlation of midparent and F Variance components in F2, BC populations Variance components in F,, BC

Broad sense

Variance components in F,

(PH,,,

Narrow sense Broad sense

,

0.02 =

4.2)

Trait

'

"Lower concentration applied for longer duration ( 1 -8 days). ' ( I ) Lafever and Campbell (1978). (2) Campbell and Lafever (1981 ), (3)Ruiz-Torres and Carver (1992). (4) information reported in Proc.Australian Plant Breeding Conf., loth, Gold Coast, Australia, Vol. 2, pp. 78-79, and (5) Bona et al. (1944). ' Considered quasi-estimate over several crosses.

ACID SOIL TOLERANCE IN WHEAT

145

collections have not been screened to identify Mn-tolerant parents for selection programs or for constructing experimental populations. These populations, in turn, are needed to characterize inheritance of Mn tolerance. The largest collection screened thus far (Macfie er al., 1989) was composed of 30 genotypes primarily of Canadian origin previously assembled for A1 tolerance screening (Briggs et al., 1989). Genotypes showed a wide and continuous tolerance range to a 14-day treatment of 0.50 mM Mn (above basal level), based on relative root and shoot weights, The lack of distinct genotypic differences indicated that several genes control Mn tolerance. Whether these differences reflect the cumulative action of several genes, or possibly a few genes with large environmental effects, is still uncertain. The Mn treatment reduced root weight more than shoot weight relative to the corresponding control weights (39% vs. 68%, averaged across 30 genotypes), but differences in relative root weight were highly consistent with differences in shoot weight ( r = 0.88). Consistency in root and shoot weights after excess Mn treatment has been reported for a smaller set of genotypes of Brazilian and Australian origin (Burke et al., 1990), with greater biomass reduction occurring also in roots. These results suggest that a common gene system controls, at least in part, Mn tolerance in both root and shoot tissue. Foy et al. (1988) also recognized the continuous and wide variability for Mn tolerance in wheat, with the qualification that a few genes may explain the majority of the variation. This was demonstrated by backcrossing Mn tolerance from Carazinho to Egret; yet, transfer of tolerance was incomplete. Cultivars shown to have exceptional levels of Mn tolerance are the Brazilian cultivar Carazinho, the Australian cultivar Warigal, and the Canadian cultivar ‘Norquay’ (Macfie er al., 1989; Burke et al., 1990). All are classified as spring types. Manganese tolerance has not been widely surveyed in winter wheat.

C. RELATIONSHIP OF ALUMINUM AND MANGANESE TOLERANCE Depending on soil classification and pH, a wheat crop may experience both A1 and Mn toxicity over the course of a season. This possibility raises an important question relevant to wheat improvement: To what extent is tolerance to either factor genetically related? Notwithstanding the possibility of physical linkage of genes controlling tolerance, do genes which control A1 tolerance also influence Mn tolerance by pleiotropy? It is plausible that plant defense mechanisms may operate against both elements when present in toxic concentrations (e.g., exclusion, compartmentation, or detoxification). One line of indirect evidence suggests that genetic control is dichotomous. Accumulation of unusually high leaf tissue concentrations of Mn in some genotypes, without significant loss in dry matter yield, is a unique mechanism of Mn toleraiice which appears independently inherited.

I46

B. F. CARVER AND J. D. OWNBY

Cultivar comparisons also offer indirect evidence that different gene systems influence A1 and Mn tolerance (Neenan, 1960; Foy et al., 1973; Burke et al. 1990), but the converse is not necessarily proven, i.e., that no gene(s) exists which coregulates tolerance. Several examples can be cited to support differential control. Atlas 66 and BHI 146 are tolerant to A1 but sensitive to Mn toxicity, each to various degrees; likewise, Monon and Warigal are A1 sensitive but Mn tolerant. However, examples of concurrent tolerance can also be cited. Carazinho and Norquay are tolerant to both A1 and Mn. Obviously, all combinations exist, which may lead to different conclusions depending on which genotypes are sampled. Macfie et al. ( I 989) quantified the phenotypic relationship across 29 cultivars using relative root weight as an indicator of each tolerance. As might be expected, the correlation was positive but intermediate ( r = 0.57). Any genetic interpretation of a correlation estimated in this manner is not advised because it may reflect simultaneous selection pressures (inadvertent or intentional) during cultivar development, particularly if field testing occurred where A1 and Mn toxicities coexisted. A direct approach to ascertain a genetic relationship would be to apply selection pressure for only one element (A1 or Mn) and examine the correlated response in tolerance to the other element. Fisher and Scott (1993a) generated pairs of closely related lines differing for Mn tolerance but uniform for A1 tolerance, implying that tolerance to A1 and Mn was inherited independently from the original parent, Carazinho. Limited data suggest that selection should focus on both A1 and Mn tolerance if tolerance to acid soils is to be fully realized. Aluminum-tolerant genotypes might be identified first to reduce the number of genotypes to a manageable level before using more tedious procedures to screen for Mn tolerance.

IV. BREEDING FOR ACID SOIL TOLERANCE Genetic improvement of acid soil tolerance in wheat must be economically and biologically reasonable, as mandated for any plant-breeding objective (Simmonds, 1979, p. 29). While economic justification is not widely documented, the monetary benefit of acid soil tolerance measured in southern New South Wales (Fisher and Scott, 1993a) may be extended to other wheatland soils undergoing gradual acidification, like the southern Great Plains of the United States and the summer rainfall region of South Africa. Economic rationale for breeding acid soil tolerance can be predicated on other terms than monetary, as discussed in this section. The extent to which a useful level of heritable variation exists for components of acid soil tolerance and the availability of practical techniques to identify tolerance or susceptibility largely determine whether breeding for acid soil tolerance in wheat is biologically reasonable. Still, others may argue that short-term

ACID SOIL TOLERANCE IN WHEAT

147

genetic approaches to long-term environmental problems are unjustified. While the “Adapt-a-Plant” philosophy embraced by C. D. Foy in Kaplan (1989) is difficult to refute, sustainability of wheat production is eventually threatened as acidification increases with time, to the point that nutritional disorders in addition to Al and Mn toxicity could conceivably render genetic tolerance ineffective.

A. WHENIs GENETIC IMPROVEMENT

JUSTIFIED?

Genetic improvement is most often justified where corrective lime applications are ineffective or impractical in acidic subsoil layers (Foy et al., 1965; Aniol and Kaczkowski, 1979). Lime distribution below the surface layer is not impossible but is generally cost prohibitive. Liming the surface layer may offer a partial yield benefit, but genetic potential is still not reached if the root system does not penetrate the acidic subsoil. A wheat cultivar with improved tolerance may tap critical water and nutrient supplies below the surface layer until subsoil pH declines to levels which exceed the tolerance range of the genotype. The level at which pH stabilizes in the subsurface layers primarily depends on mineralogy of a particular soil, but pH (H,O basis) generally can be as low as 3 (Baas Becking et al., 1960). This lower pH limit corresponds to the hydrolysis of ferrous oxide minerals which are the ultimate product of mineral weathering. Genetic improvement of acid soil tolerance may also be justified even when soil acidity is ameliorated by surface applications of lime, as for some soils in southern and western Australia and the southern Great Plains of the United States (Westerman, 1981 ; Dolling er al., 199 1). Fisher and Scott (1 993a) estimate that the current benefit of Al tolerance to grain yield in the southern NSW wheat belt is l .4% and could increase to 3.2% if their soils acidify at the current rate over the next 10 years. No benefit was expected above pHca 4.4 ( 1 : 2 soil:O.Ol M CaCl,), and no apparent yield benefit was found with Mn tolerance in their soils. The economic benefit to grain yield derived from breeding for disease resistance still exceeded that of breeding for Al tolerance. The relative importance of acid soil tolerance is magnified if the economic benefit of increased forage production is also considered. Unfortunately, the effects on forage production have not been widely researched. A soil pH below 5.0 (H,O basis) in Oklahoma reduces forage production more severely than it reduces grain yield (E. G. Krenzer, Jr., 1994, personal communication). Preliminary data collected for hard winter wheat cultivars indicate that acid soil tolerance can dramatically improve potential grazing capacity (Fig. 3), particularly during early vegetative growth (prior to winter dormancy) when Al toxicity stress on shoot growth is most noticeable. As soil pH drops from 5.0 to c4.5, early season forage growth may be reduced by as much as 85%, hardly enough to support a wheat pasture program. Because even the tolerant genotype suffers some forage yield

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Figure 3. Yields of wheat forage produced prior to winter dormancy for three hard red winter wheat cultivars under varying soil pH (H,O) near Haskell, Oklahoma, during the 1992-1993 crop season. All cultivars are currently produced in the southern Great Plains and occupy almost 60% of the approximately 3.0 million wheat hectares in Oklahoma (Oklahoma Agricultural Statistics Service, 1994. Responses in forage yield coincide with hematoxylin stain ratings (authors’ data) for Al tolerance: Karl (very susceptible), Chisholm (moderately susceptible), and 2 180 (tolerant). 2 180 is one of only a few hard red winter cultivars currently recommended for production on critically acidic soils in Oklahoma. Unpublished data provided by E. G. Krenzer, Jr.

reduction below pH 5.0, lime application would still be yield beneficial. Since grain yield is primarily determined in late winter and spring, the differences between tolerant and susceptible genotypes can be obscured by the recovery of susceptible genotypes over time, as their root systems eventually penetrate subsurface soil layers with higher pH. Hence, genetic improvement of acid soil tolerance may be justified solely to support a wheat pasture program, notwithstanding a favorable response in grain production. Demonstrating a yield benefit of acid soil tolerance, whether for grain or forage, does not necessarily justify replacement of corrective measures, such as liming, with genetic tolerance. Genetic tolerance does not correct the problem of soil acidity but only postpones the need to take corrective action. On the other hand, lime application is often economically unfeasible, if not physically impossible. Besides the physical limitation of correcting subsoil acidity, several scenarios call for the inclusion of a breeding component to manage wheat production in acid soils. Scott and Fisher (1989) draw attention to several: ( 1 ) Nonuniformity of lime application or incorporation may leave pockets of acid soil stress in a wheat field; ( 2 ) naturally occurring spatial heterogeneity of soil pH may also leave pockets of acid soil stress even with uniform application and incorporation; (3) acid soil stress may be discovered too late before planting to allow complete amelioration by liming, forcing the wheat producer to rely on genetic tolerance for an interim period; and (4) the producer may wish to apply lime at a lower rate, and therefore

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at a reduced cost, than recommended for complete amelioration. This complementary approach of planting tolerant cultivars with the lowest practical rate of lime application makes the use of lime more economical. Lime recommendations for continuous wheat production in Oklahoma are based on grain yield performance of older and traditionally susceptible cultivars; the recommended pH is 2 5 . 5 , whereas rotation with a legume requires a higher target pH of 6.5 (Johnson et al., 1988). With increased efforts devoted to breeding acid soil tolerance in hard winter wheat, the target pH for continuous wheat could be lowered, thereby reducing lime requirements to more economical levels. Conditions unique to wheat production in the southern Great Plains justify the genetic improvement of acid soil tolerance. First, while lime is an inexpensive material relative to other soil amendments, the cost of transporting lime from distribution points to areas of greatest need is economically impractical (often $25 to 30 per ton of effective calcium carbonate equivalent). Second, the producer often leases land on short term (year to year) from landowners unwilling to at least share the long-term investment of liming.

B.

POOLS OF GENETIC VARIATION

The search for genetic variation in components (A1 or Mn) of acid-soil tolerance has garnered much attention in several gene pools demarcated by geographic boundaries or market class. This activity has affirmatively answered one question critical to the potential success for genetic improvement: Is genetic variation sufficient to allow improvement? The consensus has not changed since the earliest searches revealed valuable genetic sources of acid soil tolerance, specifically Al tolerance, among clusters of European wheat genotypes (Mesdag and Slootmaker, 1969; Aniol and Kaczkowski, 1979) and among soft winter wheat cultivars of the eastern United States (Foy et al., 1965; Lafever et al., 1977). The highest level of tolerance is found among cultivars of Brazilian origin or with Brazilian parentage. These genotypes remain today the standard of performance for A1 tolerance, apparently as a result of intense selection pressure for tolerance to highly Al-toxic soils during their development. Nontargeted selection (in the absence of acid soil stress) has, nevertheless, yielded germ plasms with useful levels of tolerance in practically every gene pool surveyed (Table VII). This level of genetic variation in locally adapted germ plasms should permit rapid gains in tolerance, without the usual barriers of distant hybridization so often encountered for improving stress tolerance. Assessment of genetic variation has disproportionately favored Al tolerance. Several germ plasms cited i n Table VII approach the tolerance level of Atlas 66, generally regarded as the winter wheat standard for Al tolerance. Some even surpassed the tolerance of Atlas 66 (Briggs et al., 1989). Where lineages were traced, the Al tolerance in contemporary cultivars is attributed to rye but, more often, to wheat

Table VII Evidence of Genetic Variabilityfor Al or Mn Tolerance in Several Adapted Gene Pools of Wheat Targeted gene pool

Screening medium"

Hawk, Bounty 203

0.18-0.72 mM Al, NS

Stain

0.36 mM Al, NS 0.075 mM Al, NS 0.50 mM Mn, NS

RRL RRW, RRL RRW

0.18-0.72 mM Al. NS

Stain

Soil, pH,,?, = 4.1 Soil, pH,,,, = 4.1 0.18-0.72 mM Al, NS 0.18- I .44 mM Al, SS 0.148 mM AI. NS

RRL RRL Stain Yield RRL

Wrangler, Hawk, Dodge, Frontiersman, Mustang HY 320, Vernon PT74 I, PT726, Norquay Norquay, PT742, PT726, PT329 GK Szoke, Jubilejnaja 50, Martonvlsiri 9 GR 855 D523, GK Pannondur Timgalen, Bencubbin, Songlen, Tincurrin, Halbred Sivka; germ plasms from Zagreb-PCH and Novi Sad programs

Soil, pH,?,

United States, hard red winter wheat Canada, spring wheat Canada, spring wheat Canada, spring wheat Hungary, bread wheat Eastern Europe, bread wheat Hungary, durum wheat Australia. spring wheat Croatia/Yugoslavia

Exceptional germ plasms'

Yield

United States, hard red winter wheat

=

4.7

Assessment of tolerance'

"NS, nutrient solution culture; SS, soil solution culture; exposure time generally longer in experiments with lower Al concentrations. 'Stain, hematoxylin staining response; RRL, relative root length; RRW, relative root weight. ' Within targeted gene pool, excluding reference genotypes; more recently developed germ plasms are listed

Reference Unruh and Whitney ( 1986) Carver el al. (1988) Zale and Briggs (1988) Briggs el al. (1989) Macfie ef a/. (1989) Bona and Carver ( 1992) Bona el al. (1992) Bona ef al. (1992) Scott ef al. (1992) Rengel and Jurkic (1993)

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parents originating in Mexico, Brazil, or Kenya. With those exceptions, no causal relationship exists between the frequency and level of A1 tolerance and breeding origin. Cultivars developed in regions where soil acidity is not corrected will often show A1 tolerance. Tolerance may even exist in genotypes selected under limeamended conditions, depending on their parentage. This phenomenon has invited speculation that A1 tolerance is not uniquely regulated but is instead pleiotropically related to other biological functions. Briggs et al. (1989) suggested that selection for grain yield under high soil fertility may favor genotypes with high nitrogen-use efficiency. They cited examples in wheat and other crops where differences in nitrogen uptake relate to differences in A1 tolerance. This premise deserves further investigation with the benefit of near-isogenic material.

C. SCREENINGSTRATEGIES Genetic improvement in acid soil tolerance has been accelerated by the availability of screening criteria for detecting tolerance. Again, greater attention has been focused on A1 tolerance where symptoms of intolerance might be more obvious and the duration of A1 exposure is much shorter than screening for Mn tolerance. Laboratory- and greenhouse-based techniques are widely employed with rapidity and a high degree of accuracy, are usually nondestructive, and can be applied in early developmental stages before pollination. Field-based techniques in comparison are more laborious and do not have the same reputation for success, but are nevertheless critical to complete the transition from A1 tolerance to acid soil tolerance. Choice of a particular screening test is influenced by the kind of material under selection, i.e., germ plasm collections for identifying suitable parents, large segregating populations, or advanced breeding lines under consideration for release. 1. Cell and Tissue Culture

The application of cell or tissue culture offers a unique opportunity for improving tolerance to mineral toxicity, provided mineral composition of the culture medium approaches that of the targeted acid soil. That mechanisms of tolerance are expressed at the cellular level and provide agronomic benefit at the whole plant level are no less critical. Unfortunately, this opportunity has not been successfully explored for A1 tolerance in wheat, possibly due to the technical difficulty of culturing cells in a low pH, Al-toxic medium. Low pH (<4.5) inhibits cell growth even in the absence of A1 and also inhibits agar solidification. The latter problem has been overcome in applications with other plant species by the addition of a solidifying agent to the callus growth medium (Parrot and Bouton, 1990) and the use of sponges soaked in a liquid medium to support callus (Meredith et al.,

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1988). Cell and tissue culture can serve at least two functions in a breeding program. Callus assays may be developed to survey preexisting variability for Al tolerance (Parrot and Bouton, 1990) or selection pressure can be applied in cell culture to recover mutant Al-tolerant cells. For cell culture to play a significant role in selection, it must be shown that selected variants are truly A1 tolerant and not tolerant to other nutritional disorders inherent to the low pH culture medium. That prerequisite has been met with some success in species other than wheat, but not without modifying the traditional inorganic composition of the culture medium by reducing calcium and phosphate concentrations and by using unchelated iron at pH 4.0 (Conner and Meredith, 1985b). To facilitate selection of Altolerant variants of Nicotiana plumbaginifolia Viv., Conner and Meredith ( 1985~) controlled cell density and aggregate size in plated cells and inoculum size in callus cultures. They found total growth inhibition of plated cells, callus cultures, and in vim-grown shoots at 20.60 mM Al supplied as AI2(S0,),18H2O. Subsequent characterization of cloned variants indicated no relationship between Al tolerance and callus growth in the absence of A1 (Conner and Meredith, 1985a). Tolerance was inherited as a single dominant mutation. In other applications of tissue culture, Waskom et al. (1990) relied on somaclonal variation among sorghum plants regenerated from tissue culture to identify lines with improved field tolerance to acid soil stress. In this case, tissue culture did not serve as the screening medium, but instead as a novel source of genetic variation for subsequent field screening. While tissue culture can supply useful genetic variation for agronomic traits in wheat (Carver and Johnson, 1989), its application to acid soil tolerance has not been explored.

2. Nutrient Solution Culture By far the most common screening medium for Al and Mn tolerance is solution culture, which provides easy access to root systems, tight control over nutrient availability and pH, and nondestructive measurements of tolerance. This subject is already extensively reviewed for wheat and other cereal crops (see Konzak et al., 1977; Little, 1988; Scott and Fisher, 1989) and will receive proportionately less attention here. Wheat researchers have relied on three general criteria for tolerance: ( I ) degree of root and/or shoot damage following continuous exposure to A1 or Mn (Polle et al., 1978; Aniol and Kaczkowski, 19791, ( 2 ) the degree of recovery following pulse exposure to Al (Aniol, 1984b), and (3) concentration required for irreversible inhibition of primary root growth (Kerridge et al., 1971). Most of the research devoted to quantifying and characterizing genetic variation of tolerance (cited in Sections 111 and 1V.B) has used those criteria in solution culture. The third criteria has received less attention than the other two. Several factors may affect the degree of root damage and therefore the minimum concentration required for irreversible inhibition. Variation in temperature in the greenhouse or growth chamber and minor fluctuations in pH and P concentration of the

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nutrient solution, as it affects effective Al concentration, can reduce repeatability of results (Moore et al., 1977). They considered tolerance not i n absolute terms but relative to the conditions which it was assessed. Nutrient solution experiments are designed around four variables with infinite permutations within a given breeding objective (A1 or Mn tolerance), but major differences between the two objectives. The concentration and duration of exposure are varied inversely. A long continuous exposure to Al for 3 to 4 weeks requires much lower concentrations (usually <0.3 mM) than brief exposure for about 24 hr (usually c 0.9 mM). These concentrations change depending on the general tolerance level of the germ plasm screened and whether complete growth inhibition is desired. Shuman et al. (1993b) found effective screening of wheat cultivars for Al tolerance using very low solution Al levels (<0.01 mM) and short exposure (4 days). Their method was designed to minimize A1 precipitation and to more closely represent actual environmental stresses compared to traditional short-term experiments with much higher Al concentrations. Aluminum is usually supplied as AIC1,.6H,O, but AlK(SO,),. 12H20is used to avoid chloride toxicity. Longer exposures (several weeks) are used to screen for Mn tolerance in the range of 0.5- I .OmM Mn. This longer exposure makes solution culture technically more difficult, requiring constant adjustments for water and nutrient loss. Manganese is often supplied as MnSO,.H,O or MnC1,.4H,O. Other variables to consider in solution-based screening experiments are nutrient composition and the standards for measuring tolerance. Changes in nutrient composition can change the intensity of Al or Mn stress at a given concentration (Little, 1988; Scott and Fisher, 1989; Foy et al., 1988). High concentrations of phosphorus may lead to Al-phosphate precipitates in Al solutions and protect plants against Al toxicity. Hence, phosphorus is often avoided in nutrient solutions, particularly in short-term Al exposures when phosphorus needs are satisfied by seed reserves. Huang and Grunes (1992) demonstrated the importance of the K/Mg concentration ratio when selecting for Al tolerance in nutrient solutions. The Al tolerance of Scout 66, normally considered highly susceptible, increased as the proportion of Mg in solution increased. The proper use of solution culture does not so much specify an optimum concentration range of mineral nutrients; rather, genotypic differences in tolerance are described relative to the chosen nutrient composition. Standards for measuring tolerance generally focus on root growth, i.e., length and weight for Al tolerance and root weight for Mn tolerance. Tedious manual measurements of root length may be substituted with automated methods of digital image analysis (Harris and Campbell, 1989), which are effective for measuring the Al tolerance of sorghum plants after 4 days in nutrient solutions (Shurnan et al., 1993a). Less expensive systems featuring a hand-held scanner are also effective (Kirchhof, 1992). Measurements of shoot growth as total dry weight also provide good separation of genotypes for Mn tolerance (Foy et al., 1973; Macfie et al. 1989) but not for A1 tolerance of wheat seedlings, in which the indirect

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effects of Al toxicity on shoot growth are not yet manifested (Ruiz-Torres and Carver, 1992). An extremely powerful screen for A1 tolerance in solution culture is the hematoxylin staining method, which provides visualization of tolerance without laborious quantitative measurements. This method, as originally described by Polle et al. (1978), has stood the test of time, with applications to genotypic classification (Takagi et al., 1981; Carver et al., 1988), genetic characterization (RuizTorres and Carver, 1992; Bona et al., 1994), and selection (Fisher and Scott, 1987; Carver et al., 1993). Our protocol has been to imbibe seeds for 1 day, transfer the seeds to trays suspended above aerated deionized water for 2 days, and to nutrient solution for the fourth and fifth days. Aluminum is added to the nutrient solution for the fifth day. Seedling roots soaked in a sodium iodate-hematoxylin (NaI0,-C , h H , 4 0 0solution ) are stained along the vertical axis with increased intensity in sensitive genotypes, particularly in the meristematic region. The hema-

ACID SOIL TOLERANCE IN WHEAT

1.5.5

Figure 4. Staining patterns for wheat seedling roots exposed to 0.18 mM Al for 24 hr at the end of a 4-day period in nutrient solution culture and stained with a sodium iodate-hematoxylin (NaI0,-C,,H,,O,) solution for 15 min. Greater stain intensity of the root tip and lower root represent greater susceptiblity to Al; the four genotypes represent decreasing tolerance to Al (proceeding left to right). Stain ratings range from no detectable stain (A), to partial staining of the lower root with a wider intermittent stain-free region in B than in C, to complete staining (D)of the lower root. Genotypes which show further separation for partial staining can be identified but are not shown. Experimental protocols are described in more detail by Carver et al. (1988). as modified from the original procedure by Polle er al. (1978).

toxylin dye apparently binds to tissue A1 immobilized by extracellular phosphate on or immediately below the root surface (Ownby, 1993). Polle et al. (1978) recommended a qualitative scale to rate genotypes as completely, partially, or not stained for each of three A1 concentrations (0.18, 0.36, and 0.72 mM). An intermittent stain-free region immediately above the stained root tip (a partial stain rating) can vary in length depending on genotype and actually produce a continuous scale of stain phenotypes (Fig. 4). These minor dif-

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ferences are genetically stable but are usually ignored in classification. While the three seminal roots of 5-day-old wheat seedlings have a consistent stain pattern, some genotypes show a differential staining pattern (none vs. complete) among roots on the same plant, making genotypic classification difficult. The hard winter wheat cultivar Chisholm produces this phenotype at lower A1 concentrations (e.g., 0.18 mM), as do progenies having Chisholm as one parent. The repeatability and simplicity of the hematoxylin staining method is reinforced by its agronomic relevance. Genotypes classified as tolerant based on the staining method often show improved agronomic performance under acid soil stress. Two sister lines with opposite hematoxylin stain patterns differed in grain yield by 14% under acidic field conditions (Ruiz-Torres et al., 1992). Isolines selected for divergent staining patterns differed in grain yield by as much as 68% depending on recurrent parent background (Carver er al., 1993). The hematoxylin staining method identified Australian wheat cultivars with superior yield performance in acid soils, although cultivars were further differentiated within staining classes based on their responses in soil solution culture (Scott ef al., 1992). More than one-half of the genotypic variation in total dry matter resided within hematoxylin staining classes. The discrepancy is not so wide that a genotype rated as very susceptible in staining pattern would show exceptional tolerance under field conditions (authors’ unpublished data). Compared to RRL measurements in nutrient solution culture, Zale and Briggs (1988) found a moderate relationship with hematoxylin ratings among Canadian cultivars. The classification of genotypes was the most consistent between methods for the most tolerant genotypes. RuizTorres and Carver (1992) used a more precise scale to define stain classes and found a high correlation with RRL, but the narrower genetic base in their experiment might account for the higher correlation. The hematoxylin assay appears highly suited for mild selection in single plant populations, but it should be followed by selection of progenies in a soil medium indicative of the targeted production area. Tolerance mechanisms having less significance in the hematoxylin assay could then be identified in the soil bioassay. This tandem breeding approach is necessary to combine internal tolerance mechanisms with those operating at the soil-plant interface and to combine seedling tolerance with adult plant tolerance.

3. Soil Bioassays The use of soil media has traditionally received less attention than solution media in wheat-breeding applications. Foy (1976) provided practical guidelines for effective screening in acid soils and pointed to some of the complications when the objective is to create a soil environment, like a nutrient solution environment, with a specific type (A1 vs. Mn) and amount (for maximum separation of genotypes) of phytotoxicity. Foy ( 1976) discusses the conditions necessary to screen

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plants specifically for A1 tolerance or for Mn tolerance. Mitigating effects of other nutrients (e.g., Ca, P, or Mg) or organic matter must be considered to properly match a certain soil with a breeding objective. Other factors to consider are variability at the soil collection site, time of collection, and soil storage conditions (Scott and Fisher, 1989). Results from soil bioassays may lack consistency when the same soil is used repeatedly due to the effects of continuous wetting and drying on soil chemistry. Soil media offer the distinct advantage over nutrient solution media for screening genotypes to varying levels of A1 and Mn stress. This is especially important when genetic response is determined by soil-dependent external tolerance mechanisms. Ring er al. (1993) found that for various pasture grasses and legumes, tolerance to A1 in soils may be influenced by the capacity of a particular soil to reduce Mn in the rhizosphere. Their cultivars showed improved tolerance to <0.075 mM A1 (lower range of toxicity) when grown in Mn0,-rich soil. Recent attention has shifted to the development of rapid soil bioassays in screening for acid soil tolerance. This effort grew out of the realization that the same bioassay used to rapidly screen a collection of soils for A1 toxicity with an indicator genotype(s) can also be used to screen a collection of genotypes for A1 tolerance with an indicator soil(s). Fundamental to such an assay is that reduction in length of the primary root is the first visual indicator of A1 sensitivity and that reduction in primary root length only 2 days after germination under dark conditions is equally effective in discriminating genotypes as root lengths measured later under light conditions (Ahlrichs et al., 1990). Early root growth under dark conditions is primarily supported by seed reserves without possible confounding effects of nutrient uptake. Performing a similar bioassay in extracted soil solution may alleviate some of the complications and ambiguity in results from solid-phase soil bioassays (Aitken et al., 1990). Soil bioassays in wheat do not traditionally involve soils from the targeted production area but are chosen to provide genotypic separation for a specific phytotoxicity (Bona et al., 1991). Screening in soils representative of the targeted production area where soil acidity is yield limiting would, however, provide a critical intermediate step in selection of tolerant genotypes-after preliminary screening in nutrient solutions but before more tedious and costly screening under natural field conditions.

4. Field Evaluation The ultimate, and certainly most direct, screen for acid soil tolerance is the measurement of economic yield (forage or grain) under field conditions. The result is an integrated measurement of tolerance expressed throughout development. The general procedure is to conduct duplicate tests in unamended (naturally acidic) and lime-amended blocks to allow a direct measurement of tolerance (yield depression caused by acid soil stress) and to ensure that acid soil tolerance is not

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related to low yield potential in the absence of stress. The latter is evidently not of practical concern in wheat cultivars developed to date (Briggs er al., 1989; Scott and Fisher, 1989; Ruiz-Torres er al., 1992). In Oklahoma, a target pH of at least 5.5 in lime-amended blocks has been used (Ruiz-Torres et al., 1992). The target pH may vary in other locations depending on soil type and the potential for increased root-rot incidence at higher pH (further discussed below). Soil management practices are otherwise equal between blocks. The data from these field experiments are often reported as the ratio of grain yield in the unamended block to that in the lime-amended block to adjust for differences in yield potential without acid soil stress. However, Scott and Fisher (1989) show that equally responsive genotypes may have equal absolute tolerances (yield differential between blocks) but appear quite different in their yield ratios. Thus, the ratios should be interpreted with caution, or at least not reported alone. A long-term field experiment was initiated in 1987 on a critically acid site in Oklahoma to monitor acid soil tolerance of hard winter wheat cultivars with commercial importance to the southern Great Plains (Westerman et al., 1992). Of the initial 20 genotypes studied, over one-half were replaced from year to year to include new, improved cultivars. Lime was applied at the buffer-index recommended rate (to raise pH to approx. 6.8) on a Grant silt loam (fine-silty, mixed, thermic Udic Argiustoll). Yearly soil test data indicated adequate P and K, calling only for N applications. Seven cultivars were tested in 5 consecutive years ( 1987- 1992) to measure long-term performance (hematoxylin stain ratings in parentheses): ‘2 157’ (very susceptible), Century (very susceptible), ‘Newton’ (very susceptible),Chisholm (moderately susceptible), ‘Mesa’ (moderately susceptible), ‘TAM W-101’ (intermediate), and ‘TAM 200’ (tolerant). Average grain yield in the acid soil ranged from 1480 (Newton) to 2670 kg ha I (Mesa), almost a twofold difference.With this limited sample, a maximum differentiation in grain yield occurred between the very susceptible and moderately susceptible groups, as was previously observed among lines selected solely for different hematoxylin stain ratings (Ruiz-Torres er al., 1992). These experiments uncovered moderate field tolerance in 2157 that the hematoxylin stain method obviously did not. More recent cultivars entered in the test show even higher levels of tolerance than this common set, such as ‘2180’ and ‘2163’ (Boman er al., 1993). Scott et al. (1992) also detected wide differences in productivity based on field trials on acid soil sites of southeastern Australia. The field experiments conducted in Oklahoma unfortunately did not allow comparisons between unamended and limed blocks in any year. The expected increase in yields with liming were reversed by take-all disease, caused by Gaeumannomyces graminis var. tritici Walker. This soilborne disease is not unique to wheat production in Oklahoma; infection is often favored by application of lime to low pH soils in other wheat production areas in the United States (Taylor et al., 1983) and Australia (Murray el al., 1987). The fungal pathogen is often observed in ~

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combination with other soilborne pathogens, such as Fusarium spp., Rhizoctonia spp., and Pythium spp. Infection may be visible by premature ripening of the spikes (“white heads”), while yield may be reduced without obvious aerial symptoms. This disease seriously hinders field screening in acid soils because the amount of yield depression, or tolerance to acid soil stress, cannot be determined without reference to yield potential in the absence of acid soil stress. The narrow pH range which reduces both acid soil stress and take-all incidence is difficult to project across environments and forces the adoption of control measures disruptive to breeding efforts (crop rotation) or with inconsistent results (application of chloride fertilizers or fungicide seed treatment). Another hindrance to field screening for acid soil tolerance is the inherent spatial variability for pH or plant nutrients (e.g., P) which influence A1 and/or Mn stress severity. Spatial variability can greatly bias the interpretation of field trials (Ball et al., 1993) and, in turn, lead to an inefficient selection response if not considered in the experimental design or in the statistical analysis by use of a covariate or nearest-neighbor method. Spatial variability of pH in the surface and subsurface layers was found in a continuously cropped wheat field with a history of acid soil stress (Guertal, 1993). On the othcr hand, direction and degree of spatial continuity of soil P are more sensitive to cropping practices and, therefore, do not show a strong spatial relationship at any depth. Spherical models were fit to semivariograms for pH with a range of 60 m (samples within that range were spatially related). Spatial dependence in soil pH could induce spatial patterns in forage or grain yield, which might be removed using the nearest-neighbor analysis to base selection on adjusted yields (Ball et al., 1993). Adjustments must also be made in field data, either statistically or intuitively, for a variable response to other environmental factors, such as disease or insect pressures and water supply. Waterlogged pockets in the field could influence expression of Mn tolerance, drought-prone areas may accentuate A1 toxicity, and irregular infection of diseases could obscure tolerance to both A1 or Mn. These same factors may also explain year-to-year fluctuation in response to acid soil stress. For example, expression of Al tolerance may be obscured in years where soil moisture is not yield limiting.

D. BREEDING APPROACHES The simplest breeding method for improving acid soil tolerance is to backcross genes with relatively large effects from locally unadapted tolerant parents to adapted susceptible parents. The dominance expression of A1 tolerance of seedlings in solution culture lends itself to this method, as does the availability of rapid bioassays. Aluminum tolerance genes are easily tracked through consecutive backcross (BC) generations by hematoxylin staining. Selected BCF, seedlings are subsequently crossed to the recurrent parent. The backcross method was employed

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by Fisher and Scott (1987) to transfer a major gene for A1 tolerance from Carazinho to the susceptible Australian cultivar Egret. Selection for hematoxylin staining pattern resulted in the partial transfer of acid soil tolerance from the donor parent, implying that the gene(s) undetected by the stain also contributed to field tolerance of Carazinho. More than one gene appeared to control segregation of the hematoxylin staining pattern when backcrossing A1 tolerance from Atlas 66 to Chisholm and Century (Carver et al., 1993). While the backcross method produces quick and profound results, genetic gains are limited to single gene increments and may bypass other sources of genetic variation not so simply expressed. Further improvements in tolerance may be realized by combining additional genes with minor effects from different genetic backgrounds (Takagi et al., 1983). Recurrent selection procedures have been proposed to increase the frequency of nonallelic genes with additive effects (perhaps through different physiological mechanisms), but this effort may not be economically justified where soil acidity is less severe (Carver et al., 1988; Minella and Sorrells, 1992). The CIMMYT wheat improvement program employs a shuttle breeding approach to improve tolerance to the acid soil “complex,” which not only includes A1 and Mn toxicities but also deficiencies of Ca, Mg, P, and Mo (Rajaram et al., 1986). Successive generations are grown in alternating locations between Mexico, where selection is primarily aimed at agronomic type and disease resistance, and Brazil, where selection is focused on acid soil tolerance. Promising segregating populations are first identified in Mexico using the hematoxylin stain method. Selections derived from the CIMMYT program provide a valuable parent base for other wheat-breeding programs, as in New South Wales, Australia (Fisher and Scott, 1993a). Their breeding approach is to delay selection for acid soil tolerance until lines are first evaluated for yield potential and grain quality. Hematoxylin staining identifies potentially tolerant or moderately tolerant lines for subsequent field testing and more intense selection on acid soils. Genetic improvement of acid soil tolerance has not been constricted by a narrow genetic base in hexaploid wheat. Thus, relatively little attention has been given to selection in interspecific crosses. Some exotic sources of A1 tolerance have already been identified in wild Triticae species, which show promise not so much for elevated tolerance per se but for their apparently unique physiological mechanisms (Berzonsky and Kimber, 1986). Their research suggests that tolerance genes located on the Un genome should be combined with tolerance genes of the A and D genomes to achieve even higher levels of tolerance than currently present in hexaploid wheat. This prospect could be compromised, however, by intergenomic interactions in phenotypic expression, as discussed previously for wheat X rye hybrids in Section 111. Molecular biology has yet to tangibly contribute to breeding strategies for acid soil tolerance, although recent advances have been made in characterizing the molecular response of wheat roots to A1 (Snowden and Gardner, 1993). Efforts are underway to identify and isolate either the

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genes controlling tolerance or DNA markers closely linked to such genes (Putterill et al., 1991). The recent advent of near-isogenic lines like those used by Ryan and Kochian (1993) should allow identification of gene differences with greater resolution.

E. CULTIVAR DEVELOPMENT EFFORTS Several wheat-breeding programs now feature acid soil tolerance as a specific breeding objective that was once limited to programs in the tropical regions. Two Australian programs breed wheat cultivars for acid soils, one at Wagga Wagga and the other in Western Australia (Blarney et al., 1987; Scott and Fisher, 1989). ‘Dollarbird’ was released by the New South Wales Department of Agriculture in 1987 and shows improved adaptation to acid soils (Fisher and Scott, 1993b). Cultivars selected and released in the United States with acid soil tolerance are ‘Edwall,’ a soft white spring type adapted to eastern Washington (Morrison et al., 1988), and Cardinal, a soft red winter type adapted to northeastern Ohio (Lafever, 1988). The development of cultivars with acid soil tolerance is underway in several programs located in the Great Plains, including the Kansas and Oklahoma Agricultural Experiment Stations and Agripro Biosciences, Inc., in Colorado. Tolerant germ plasms have been released in Oklahoma and registered in two hard red winter wheat backgrounds (Carver et al., 1993). This activity has escalated rapidly in only the last decade. With the decline in lime application to acidic wheatland soils in NW Canada, the breeding program at the University of Alberta has also significantly increased germ plasm and cultivar development activities in the last decade. Canadian wheat cultivars have been extensively classified for A1 tolerance, nearisogenic materials have been developed in several genetic backgrounds for characterization of physiological mechanisms, and high levels of A1 tolerance have been developed in better adapted cultivars like PT741 and ‘Cutler’ (Briggs and Taylor, 1994).

V. SUSTATNABLE PRODUCTION IN ACID SOILS The greater emphasis placed on sound production practices and sustainable agriculture has created concerns both real and perceived for an acidifying wheat production system. Maintenance of soil fertility (e.g., pH and essential nutrients) is paramount to sustainable production; if the rate of depletion of soil fertility, such as loss of basic nutrients like Ca and Mg, exceeds the rate of addition, then sustainability is obviously threatened (Dalal et al., 1991). Continuous wheat cultivation may be misperceived as causing soil acidification, but in reality it accel-

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erates what is already a natural process (Helyar and Porter, 1989). Certain wheatland soils in the Great Plains were acidic when first cultivated a century ago and have acidified further with nitrogen fertilization and crop removal. Two critical questions emerge in the development of an economically viable wheat production system: To what degree should corrective additions of lime or other basic materials be used to ameliorate the effects of acidification, and to what degree should genetic alteration be used to overcome the limitations of reduced fertility in acid soils? Answers to these questions are certainly not mutually exclusive, nor should either be expected to provide the sole solution. Scott and Fisher (1989) argue with supporting evidence that tolerant cultivars are just as critical to managing acid wheatland soils as lime application. Grain yields can be sustained at lower pH and, therefore, with lower lime inputs by choosing tolerant cultivars. Obviously, there are two parts (management and tolerance) to the equation for achieving sustainable production in acid soils, and they must be properly balanced as inputs change and genotypes improve. Other factors enter into the equation because lime application is not the only means for correcting acidification. Helyar and Porter ( 1989) prescribe more effective management of the nitrogen and carbon cycles as environmentally sound alternatives. For example, Fisher and Scott ( l993b) recommend earlier fall planting of wheat in acid soils of southern NSW to minimize acidification caused by the loss of added nitrate. Earlier planting also allows the wheat crop a “jump-start” before waterlogging conditions occur in the winter. Researchers are also investigating other management alternatives which ameliorate the effects of acidification in the highly sensitive seedling stages. Phosphorus precipitates in an unavailable form in acid soils as it reacts with Al. The flip side to this reaction is that Al also precipitates in the seedling root zone, which may explain why wheat yields can be sustained when phosphate fertilizer is applied with the seed at planting (Boman et al., 1991). Phosphorus provides more protection from Al if banded in the seedling root zone as opposed to broadcast application, even when the soil test index indicates adequate P for wheat production. Boman el al. (1992) found rates of 34 and 67 kg ha - I of P20ssufficient to sustain normal grain and forage yields from liming. Returns on investment make banded P applications highly economical for producers who lease land on a yearly basis. Still the most economical approach to sustained yields in acid soils over several years is to apply lime with adequate P and to plant tolerant cultivars.

VI. CONCLUSIONS Tolerance to acid soils bears at least some concern in almost every agronomic plant grown throughout the world. Wheat is no exception and has actually cata-

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lyzed global concerns of the phytotoxic elements associated with acid soils. Awareness of soil acidity has increased dramatically in wheat production areas historically regarded as pH safe. Locations undergoing increasing acidification include the wheat belts of the United States, Canada, Australia, and South Africa. This increased awareness has led to advances in our knowledge of the biochemical and genetic basis of adaptations to acidic wheatland soils. For example, a significant component of both Al and Mn tolerance now appears to be chelation of these phytotoxic metals with organic acids. In the case of Al this probably occurs in the apoplast or rhizosphere as part of an Al exclusion mechanism. There is also circumstantial evidence that constituents of the cell wall and plasmalemma are directly involved in exclusion; however, the details are not yet known. Technology now exists to isolate and characterize proteins encoded by genes involved in A1 tolerance. In the next decade we expect that the nature of some of these proteins and their functions will be revealed. Although less is known about Mn tolerance mechanisms, the ability of the plant to achieve a uniform distribution of leaf Mn, in sites apart from cell metabolism, seems to be critical. Silicon and organic acids, including oxalate, are likely to play important roles in tolerance. Manganese-tolerant and Mn-sensitive spring wheat lines have been identified, and current assays should permit assessment of Mn tolerance among winter wheats. The next step is to survey these cultivars to determine which biochemical and physiological parameters correlate with Mn tolerance in wheat. Wheat researchers now have a better appreciation for the complexity of genetic control of A1 and/or Mn tolerance. Recent work in locating Al tolerance genes to chromosomes demonstrates that several genes can influence phenotypic expression and with different magnitude. These genes appear to be concentrated in the A and D genomes. The specific genetic model used to describe inheritance of A1 tolerance in one series of progeny may not apply to another series, or even the same series when challenged at a different level of stress. Gene expression may be altered in different backgrounds, or possibly different genes are expressed at different levels of stress. One common thread to genetic control of Al tolerance in wheat is dominance expression in heterozygous materials; still, exceptions to that trend are found, and the possible role epistasis plays in nonadditive gene action is not well explored. While significant improvements have been made, particularly for AI tolerance, by selection of major genes with easily identifiable effects, further advances will require manipulation of polygenes with much smaller effects, perhaps too small to detect without the aid of markers. Further advances will also be realized with due consideration to other yield-limiting factors inherent in an acidic production system, such as improvement in phosphorus-use efficiency and water-use efficiency of wheat. Improved understanding of acid soil tolerance has not come without voids of information. For example, linkage of physiological mechanisms of tolerance with

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genetic mechanisms are lacking in wheat, but can be addressed with the recent availability of near-isogenic stocks. The physiological characterization of tolerance genes from alien species is needed to verify uniqueness of control and to warrant interspecific gene transfer. Gene location in other backgrounds exceeding the tolerance level of Chinese Spring will provide evidence of allelic variation vs. nonallelic variation for A1 tolerance. Genetic variation has not been described well in wheat regarding allelic or nonallelic forms. Geneticists might focus on the B genome to identify sources of tolerance to complement genes already present in the A and D genomes; use of wheat-rye translocations offers one possible mechanism. Finally, greater priority has been given to the genetic improvement of A1 tolerance without systematically weighing the agronomic and economic benefits of A1 vs. Mn tolerance, at least in U.S. wheatland soils. These information gaps provide incentive to further advance an already active but fragmented research area, one that beseeches cooperation of specialists in soil fertility, stress physiology, wheat breeding, and cellular and molecular biology.

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Krizek, D. T., and Foy, C. D. 1988. Role of water stress in differential aluminum tolerance of two barley cultivars grown in an acid soil. J . Plant Nutr. 11, 35 1-367. Lafever, H. N. 1988. Registration of ‘Cardinal’ wheat. Crop Sci. 28,377. Lafever, H. N., and Campbell, L. G. 1978. Inheritance of aluminum tolerance in wheat. Can. J. Genet. Cvtol. 20,355-364. Lafever, H. N., Campbell, L. G., and Foy, C. D. 1977. Differential response of wheat cultivars to Al. Agrun. J. 69,563-568. Larkin, P. J. 1987. Calmodulin levels are not responsible for aluminium tolerance in wheat. Aust. J . Plant Physiol. 14, 377-385. Lindberg, S. 1990. Aluminium interactions with K + (RhRb+) and V a 2 +fluxes in three cultivars of sugar beet (Beta vulgaris). Physiol. Plant. 79,275-282. Lindberg, S., and Griffiths, 0.1993. Aluminium effects on ATPase activity and lipid composition of plasma membranes in sugar beet roots. J . Exp. Bot. 44, 1543- 1550. Little, R. 1988. Plant soil interactions at low pH: Problem solving-the genetic approach. Commun. Soil Sci. Plant Anal. 19, 1239- 1257. Loper, M., Brauer, D., Patterson, D., and Tu, S.-I. 1993. Aluminum inhibition of NADH-linked electron transfer by corn root plasma membranes. J. Planr Nutr. 16,507-514. Maan, S. S. 1987. Interspecific and intergeneric hybridization in wheat. In “Wheat and Wheat Improvement” (E. G. Heyne, ed.). 2nd Ed., pp. 453-461. Agron. Monogr. 13, ASA, CSSA, and SSSA, Madison. Macfie, S. M., Cossins, E. A,, and Taylor, G. J. 1994. Effects of excess manganese on production of organic acids in Mn-tolerant and Mn-sensitive cultivars of Triticum aestivum L. (wheat). J. Plant Physiol. 143, 135- 144. Macfie, S. M., and Taylor, G. J. 1992. The effects of excess manganese on photosynthetic rate and concentration of chlorophyll in Triticum aestivum grown in solution culture. Physiol. Plant. 85, 467-475, Macfie, S. M., Taylor, G. J., Briggs, K. G., and Hoddinott, J. 1989. Differential tolerance of manganese among cultivars of Triticum aestivum. Can. J. Bot. 67, 1305- 1308. Marienfeld, S., and Stelzer, R. 1993. X-ray microanalyses in roots of Al-treated Avena sativu plants. J. Plant Physiol. 141,569-573. Marion, G. M., Hendricks, D. M., Dutt, G. R., and Fuller, W. H. 1976. Aluminum and silica solubility in soils. Soil Sci. 121, 76-85. Marschner, H. 1986. “Mineral Nutrition of Higher Plants.” Academic Press, New York. Matsumoto, H., and Yamaya, T. 1986. inhibition of potassium uptake and regulation of membraneassociated Mg”-ATPase activity of pea roots by aluminium. Soil Sci. Planr Nurr. 32, 179- 188. Meharg, A. A. 1993. The role of the plasmalemma in metal tolerance in angiosperms. Physiol. Planr. 88,191-198. Memon, A. R., and Yatazawa, M. 1984. Nature of manganese complexes in the manganese accumulator plant Acanthopanux sciadophylloides. J. Plant Nutr. 7,96 1-964. Mengel, K., and Kirkby, E. A. 1982. “Principles of Plant Nutrition,” 3rd Ed. Intl. Potash Inst., Bern, Switzerland. Meredith, C. P., Connor, A. J., and Schettini, T. M. 1988. The use of cell selection to obtain novel plant genotypes resistant to mineral stresses. lowa Stare J. Res. 62,523-535. Mesdag, J., and Slootmaker, L. A. J. 1969. Classifying wheat varieties for tolerance to high soil acidity. Euphytica 18, 36-42. Minella, E., and Sorrells, M. E. 1992. Aluminum tolerance in barley: Genetic relationships among genotypes of diverse origin. Crop Sci. 32,593-598. Miyasaka, S. C., Buta, J. G., Howell, R. K., and Foy, C. D. 1991. Mechanism of aluminum tolerance in snapbeans: Root exudation of citric acid. Plant Physiol. 96, 737-743. Moore, D. P, Kronstad, W. E., and Metzger, R. I. 1977. Screening wheat for aluminium tolerance. In

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“Plant Adaptation to Mineral Stress in Problem Soils” (M. J. Wright and S. A. Ferrari, eds.), pp. 287-295. Special Publ., Cornell Univ. Agr. Exp. Sta., Ithaca, New York. Morgan, P. W., Taylor, D. M., and Joham, H. E. 1976. Manipulations of IAA oxidase activity and auxin deficiency symptoms in intact cotton plants with manganese nutrition. Physiol. Plant. 37, 149- 156. Morrison, K. J., Konzak, C. F., Reisenauer, P., Davis, M., and Rubenthaler, G. 1988. Edwall spring wheat. Extension Bulletin 1483. Coop. Ext. and College of Agric. and Home Econ., Washington State Univ., Pullman. Mukhopadhyay, M. J., and Sharma, A. 199 I . Manganese in cell metabolism of higher plants. Bot. Rev. 57, 117- 149. Murray, G. M., Scott, B. J., Hochman, 2.. and Butler, B. J. 1987. Failure of liming to increase grain yield of wheat and triticale in acid soils may be due to the associated increase in incidence of take-all (Gmcumannomycesgraminis var. tritici). Aust. J. Exp. Agric. 27,411-417. Nable, R. 0..Houtz, R. L., and Cheniae, G. M. 1988. Early inhibition of photosynthesis during development of Mn toxicity in tobacco. Plant Physiol. 86, 1136- 1 142. Naidoo, G., Stewart, J. M., and Lewis, R. J. 1978. Accumulation sites of Al in snap beans and cotton roots. Agron. J. 70,489-492. Neenan, M. 1960. The effects of soil acidity on the growth of cereals with particular reference to the differential reaction thereto. Plant Soil 12,324-338. Nichol, B. E., Oliveira, L., Glass, A. D. M., and Siddiqi, M. Y. 1991. The effects of short and long term aluminium treatment on potassium fluxes in roots of an aluminium sensitive cultivar of barley. In “Plant-Soil Interactions at Low pH” (R. J. Wright, V. C. Baligar, and R. P. Murrmann, eds.), pp. 741 -746. Kluwer Academic Publishers, Dordrecht, The Netherlands. Norvell, W. A,, Welch, R. M., Adams, M. L., and Kochian, L. V. 1993. Reduction of Fe(lII), Mn(III), and Cu(1I) chelates by roots of pea (Pisum srrtivum L) or soybean (Glycine max). In “Plant Nutrition: From Genetic Engineering to Field Practice” (N. J. Barrow, ed.), pp. I 15- 118. Kluwer Academic Publishers, Dordrecht, The Netherlands. Ohki, K. 1984. Manganese deficiency and toxicity effects on growth, development, and nutrient composition in wheat. Agron. J . 76,213-218. Ohki, K. 1985. Manganese deficiency and toxicity effects on photosynthesis, chlorophyll, and transpiration in wheat. CropSci. 25, 187-191. Ohki, K. 1986. Photosynthesis, chlorophyll, and transpiration responses in aluminum stressed wheat and sorghum. Crop Sci. 26,572-575. Ownby, J. D. 1993. Mechanisms of reaction of hematoxylin with aluminium-treated wheat roots. Physiol. Plant. 87,37 1-380. Ownby, J. D., and Popham, H. R. 1989. Citrate reverses the inhibition of wheat roct growth caused by aluminum. J . Plant Physiol. 135,588-59 I . Parker, D. R. 1995. Root growth analysis: An underutilized approach to understanding aluminum rhizotoxicity. In “Soil-Plant Interactions at Low pH-1993” (R. A. Date, N. J. Grundon, G . E. Rayment, and M. E. Probert, eds.). Kluwer Academic Publishers, Dordrecht, The Netherlands (in press). Parker, D. R., Kinraide, T. B., and Zelazny, L. W. 1988. Aluminum speciation and phytotoxicity in dilute hydroxy-aluminum solutions. Soil Sci. Soc. Am. J. 52,438-444. Parker, D. R., Kinraide, T. B., and Zelazny, L. W. 1989. On the phytotoxicity of polynuclear hydroxyaluminum complexes. Soil Sci. Soc. Am. J. 53,789-796. Parrot, W. A., and Bouton, J. H. 1990. Aluminum tolerance in alfalfa as expressed in tissue culture. Crop Sci. 30,387-389. Penney, D. C., Nyborg, M., Hoyt, P. B.. Rice, W. A,, Siemens, B., and Laverty, D. H. 1977. An assessment of the soil acidity problem in Alberta and northeastern British Columbia. Can. J. Soil Sci. 57, 157-164.

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MICROBIAL REDUCTIONOF IRON, MANGANESE, AND OTHER METALS Derek R.Lovley Water Resources Division United States Geological Survey Reston, Virginia 22092

I. Introduction II. Fe(1II) and M n o Reduction A. F e w ) - and Mno-Reducing Microorganisms B. Electron Transport to Fe(III) and Mn(W C. Enumeration and Isolation D. Monitoring Activity E. Mechanisms for Environmental Fe(III) and M n O Reduction F. Interaction of F e w ) and M n o Reduction with Other Microbially Catalyzed Redox Processes G. Electron Flow to Fe(II1) and Mn(W in Anoxic Soils and Sediments H. Degradation of Organic Contaminants I. Dissolution and Formation of Iron and Manganese Minerals J. Effects on Soil Properties K. Effects on Plant Growth 111. Uranium reduction A. U(VI)-Reducing Microorganisms B. Enzymatic Mechanisms for U O Reduction C. Enzymatic versus Nonenzymatic U(VI) Reduction D. Bioremediation of Uranium-Contaminated Soils and Water IV Selenium Reduction A. Microorganisms That Reduce Selenium B. Enzymatic Mechanisms for Selenate Reduction C. Microbial Reduction of Selenium in Soils and Bioremediation V. Chromate Reduction A. Cr(VI)-Reducing Microorganisms B. Mechanisms for Cr(VI) Reduction C. Microbial Reduction of C r O in Soils and Bioremediation VI. Microbial Reduction of Other Metals VII. Conclusions References

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I. INTRODUCTION Microbial reduction of metals and metalloids greatly influences the geochemistry of these materials and affects a variety of other soil properties. In soils and sediments, the reduction of other inorganic electron acceptors such as nitrate, sulfate, and carbon dioxide is a well-known enzymatically catalyzed redox process that is carried out by microorganisms that have specifically evolved to use these compounds (Zehnder and Stumm, 1988).In contrast, metal reduction is often regarded as a nonenzymatic process. Even when microorganisms are thought to be involved in metal reduction, their role is often considered to be nonspecific. A typical example is this statement from a review on the biogeochemistry of iron in soil (Aristovskaya and Zavarzin, 1971): “Microorganisms taking part in iron reduction do not belong to any specific, physiological groups. Reduction of iron is not a vital process for these bacteria; the presence of ferric iron cannot be considered a necessary condition for their development.” However, recent studies have demonstrated that there are specific physiological groups of microorganisms that conserve energy to support growth from metal and metalloid reduction. This form of enzymatic metal reduction, as well as the enzymatic reduction of toxic metals by microorganisms that use metal reduction as a detoxification mechanism, may account for much of the metal reduction in the soils and sediments. This chapter summarizes the recent literature on these forms of dissimilatory metal reduction with the hope that it might provide further insight into factors controlling the fate and mobility of metals in the environment.

II. Fe(III) AND M n o REDUCTION The most important geochemical change that takes place in many submerged soils and aquatic sediments is the reduction of Fe(II1) to Fe(I1) (Ponnamperuma, 1972). As discussed in detail in the next section, the reduction of Fe(II1) and Mn(1V) not only greatly influences iron and manganese geochemistry but it can also have a dramatic influence on a host of other important soil properties.

A. Fe(I1I)- AND M ~ O - R E D U C I NMICROORGANISMS G 1. Oxidation of Organic Matter and H, Many microorganisms are capable of Fe(II1) and Mn(IV) reduction. As previously reviewed (Lovley, 199 11, early studies on Fe(II1) and Mn(IV) reduction

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dealt with microorganisms that metabolize fermentable sugars and amino acids. A detailed list of such organisms has been published (Lovley, 1987). None of these organisms are able to conserve energy from Fe(II1) or Mn(IV) reduction and all of them can grow in the absence of Fe(II1) or Mn(1V). Fe(II1) and Mn(IV) merely serve as minor electron sinks for these organisms so that there is less production of reduced organic compounds or H, when Fe(II1) and Mn(IV) are present. Organisms that can completely oxidize fermentable sugars or amino acids to carbon dioxide with Fe(II1) or Mn(IV) serving as the electron acceptor have never been isolated and may not exist. Studies on the metabolism of glucose in several types of sediments in which Fe(II1) reduction was the terminal electron-accepting process indicated that glucose was first fermented to organic acids (primarily acetate) with the subsequent oxidation of the fatty acids to carbon dioxide. Thermodynamic considerations suggest that if organisms attempted to completely oxidize glucose to carbon dioxide with Fe(II1) as the electron acceptor, they could be at a competitive disadvantage with organisms that merely ferment glucose (Lovley and Phillips, 1989). This is because the amount of energy released per mole of electrons transferred is higher for fermentation than it is for glucose oxidation coupled to Fe(II1) reduction. Within the last decade, studies have indicated that there are many microorganisms which can conserve energy to support growth by coupling the oxidation of organic compounds or H, to the reduction of Fe(II1) or Mn(1V). The first example of this (Balashova and Zavarzin, 1980) was a Pseudomonas sp. that grew by oxidizing H2 with the reduction of Fe(II1) (reaction I , Table I). This organism did not reduce Mn(IV) under the conditions evaluated. Shewanella (previously Alteromonas) putrefuciens, which has been isolated from a wide variety of environments (Obuekwe et al., 1981; Semple and Westlake, 1987; Myers and Nealson, 1988a; Nealson et al., 1991; DiChristina and DeLong, 1993), can use Mn(IV) (reaction 2, Table I) as well as Fe(II1) as an electron acceptor for H, oxidation (Lovley et al., 1989b). Another organism that can conserve energy to support growth from H2 oxidation coupled to Fe(II1) reduction is the estuarine organism known as strain BrY (Caccavo et al., 1992). A number of microorganisms, most notably Desulfovibrio sp. which grow with sulfate as the terminal electron acceptor, can also couple H, oxidation to Fe(II1) reduction (Coleman et al., 1993; Lovley et al., 1993b). In contrast to the other H2-oxidizing, Fe(II1) reducers, none of these organisms conserve energy to support growth from this metabolism. Despite this, Desulfovibrio may still be important catalysts for Fe(II1) reduction in some environments (see the following discussion). Until recently, it was considered that HI-oxidizing, Fe(II1)-reducing microorganisms were limited in their ability to oxidize organic compounds with Fe(II1). S. putrefaciens oxidizes formate with the reduction of Fe(II1) and Mn(IV) (Lovley

D. R. LOVLEY

I78

Table I Metal Reduction Reactions Reaction number

Product

Reactan t H,

+ 2Fe(III)

2H

H,

+ Mn(lV)

2H

+

+ 2Fe(II)

1 +

+ Mn(l1)

2

+ 2Fe(III) + H,O

HC0,-

+ 2Fe(II) + 2H'

Formate-

+ Mn(1V) + H,O

HC0,-

+ Mn(I1) + 2H'

Lactate-

+ 4Fe(III) + 2 H z 0

Acetate

Lactate -

+ 2Mn(IV) + 2H,O

Acetate

Formate

~

3 4 ~

+ HC0,- + 4Fe(II) + 5H

+

5 ~

+ H C 0 , - + 2Mn(II) + 5H

+

6 Pyruvate-

+ 2Fe(IlI) + 2H,O

Acetate-

+ HC0,- + 2Fe(II) + 3H '

Pyruvate

+ Mn(1V) + 2H,O

Acetate-

+ HC0,- + Mn(I1) + 3H'

2HC0,-

+ SFe(I1) + 9H '

3HC0,-

+ I4Fe(II) + 16H'

4HC0,-

+ 20Fe(II) + 23H'

+ 5H20

2HC0,-

+ 12Fe(II) + 14H'

Benzoate- + 3OFe(III) + 19H,O Phenol + 28Fe(III) + 17H,O

7HC0,-

+ 30Fe(II) + 36H'

6HC0,-

+ 28Fe(II) + 34H

7 ~

8

+ 8Fe(III) + 4H,O

Acetate9

Propionate + I4Fe(III) + 7H20 Butyrate + 20Fe(III) + IOH,O ~

10

~

I1

Ethanol +12Fe(III) 12 13

+

14 p-Cresol

+ 34Fe(III) + 2 0 H z 0

7HC0,-

+ 34Fe(II) + 41H'

Toluene

+ 36Fe(11I) + 21H,O

7HC0, -

+ 36Fe(lI) + 43H

15 +

16 S'

+ 6Fe(III) + 4H,O

SO,-,

+ 6Fe(II) + 8H '

S'

+ 3Mn0, + 4H

SO,-2

+ 3Mn(II) + 2 H 2 0

17 +

18

Mn(I1) + MnO,

+ 4H

+

2Mn(III) + 2H,O

19

Cysteine 20

+ IOFe(II1) + 7H,O

3HC01-

+ 9Fe(II) + lFeS + NH,' + 14H'

179

MICROBIAL REDUCTION Table I-Continuid Reaction number

Reactant

Product

U ( I V ) + 2Fe(III)

U(V1)

+ 2Fe(II)

2Fe(II) + Mn(1V)

2Fe(III) + Mn(I1)

21 22 3 Acetate

~

+ 4Se0,-'

6HC0,-

+ 4%' + 4 H 2 0

2HC0,-

+ 4Se0,-* + H '

23 Acetate

~

+ 4Se0;'

24

3H2 + 2Cr(VI)

6H

+

+ 2Cr(III)

25

et al., 1989b) (reactions 3 and 4, Table I). However, the only multicarbon compounds it is known to oxidize with Fe(II1) as the electron acceptor are lactate and pyruvate (Lovley et al., 1989b) and these are only incompletely oxidized to acetate and carbon dioxide (reactions 5-8, Table I). Other H1-oxidizing, Fe(II1)- and Mn( 1V)-reducing microorganisms such as strain BrY and D. desulfuricans also carry out these reactions. Clostridium pasteuranium can couple formate oxidation to Fe(II1) reduction (Eisen, 1985; Lovley et al., 1991b), but does not conserve energy to support growth from this metabolism (D. R. Lovley and E. J. P. Phillips, unpublished data). Prior to 1987, no organisms that could completely oxidize organic compounds to carbon dioxide with Fe(II1) or Mn(IV) serving as the electron acceptor had been described. Now it is apparent that there is a large diversity of such organisms (Coates et al., 1994). The first of these to be described was Geobacter metallireducens, a freshwater organism isolated from surficial river sediments (Lovley et al., 1987, 1993a; Lovley and Phillips, 1988a). G. metallireducens oxidizes a number of organic compounds completely to carbon dioxide with the reduction of Fe( 111) or Mn(1V). Electron donors include acetate, propionate, butyrate, benzoate, ethanol, phenol, p-cresol, and toluene (reactions 9- 16, Table I). To date, other organisms that can couple the complete oxidation of organic compounds to the reduction of Fe(II1) or Mn(IV) have been found to be closely related to G. metallireducens. Desulfuromonus is the genus in the delta proteobacteria that is phyologenetically closest to Geobacter, and the marine organism Desulfuromonas acetoxidans as well as the freshwater strain, D. actexigens, can grow by coupling the oxidation of acetate to the reduction of Fe(II1) (Roden and Lovley, 1993a). Preliminary analysis of a partial sequence of the 16s rRNA of strain PCA, an acetate-oxidizing organism isolated from ditch sediments, indi-

D. R. LOVLEY

180

!ex

Sugars Aminn

*

H2, Acetate and Fermentative Bacteria Other Short-Chain Fattv Acids)

\*bc'

SCFAand H2 Oxidizin Fe(lii) Reducers

and - + COZ H20

/

LongChain Fatty AcidOxidizing Fe(lii) Reducers

Figure 1. Model for microbial oxidation of organic matter in soils and sediments with Fe(II1) serving as the electron acceptor.

cates that it is closely related to Geobacter and Desulfuromonas (Caccavo et al., 1994), as is strain H-2, an acetate-oxidizing organism isolated from a contaminated aquifer (Coates et al., 1994). An interesting finding with PCA and H-2 is that they represent the first examples of organisms that use both H2 and acetate as electron donors for Fe(II1) reduction. From the known metabolism of the microorganisms that are available in pure culture, as well as studies with purified enrichment cultures (Lonergan and Lovley, 199 1; Lovley, 199 1 ) and studies with natural communities of microorganisms living in sediments (Lovley and Phillips, 1989), it is possible to construct a model for how complex organic matter can be oxidized to carbon dioxide with Fe(II1) [and presumably by analogy, Mn(IV)] as the sole electron acceptor (Fig. I). In this model, complex organic matter is first attacked by hydrolytic enzymes which release monomeric compounds such as sugars and amino acids, long chain fatty acids, and monoaromatics. Fermentative microorganisms metabolize the sugars and amino acids, possibly reducing small amounts of Fe(III), but primarily fermenting the sugars and amino acids to short-chain fatty acids and H,. These fermentation products are then oxidized with the reduction of Fe(1II). It was previously considered that the Fe(II1)-reducing microorganisms catalyzing the oxidation of the fermentation products conserved energy to support growth from this metabolism, but organisms such as Desulfovibrio species which actively reduce Fe(II1) but cannot grow by this reaction may be important in some environments (Coleman et al., 1993). Furthermore, it was previously considered that the organisms which could completely oxidize the fermentation acids to carbon dioxide

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were a distinct community from the H,-oxidizing organisms (Lovley, 199 1, I993), but the discovery of Fe(II1) reducers that can metabolize both short-chain fatty acids and H, indicates that this is not necessarily the case. Long-chain fatty acids can be oxidized to carbon dioxide with the reduction of Fe(III), but there is no organism in pure culture that can serve as a model for this metabolism. G. metallireducens is the only pure culture model for the direct oxidation of aromatic compounds to carbon dioxide with the reduction of Fe(II1). However, as with the other types of metabolism involved with organic matter oxidation coupled to Fe(II1) reduction, there are certain to be a wide diversity of as yet to be isolated organisms that can carry out these reactions.

2. So-OxidizingFe(II1) and Mn(IV) Reducers Under acidic conditions, Thiobucillus thiooxiduns, iT: ferrooxidans, and Sulfolobus acidocaldarius can oxidize S' to sulfate with Fe(II1) serving as the electron acceptor (reaction 17, Table I). Early studies indicated that energy to support growth was not conserved from this metabolism (Kino and Usami, 1982; Sugio et al., 1988; Sand, 1989). However, an electron transport chain inhibitor, HQNO (2-n-heptyl-4-hydroxyquinolineN-oxide), inhibited Fe(II1) reduction with S' by 7:ferrooxidans, suggesting that Fe(II1) reduction was linked to electron transport (Corbett and Ingledew, 1987). More direct studies have demonstrated that S' oxidation coupled to Fe(II1) reduction can provide energy to support active transport and growth (Pronk et al., 199 I , 1992). Studies with aquatic sediments and soils have provided evidence suggesting that Mn(IV) can also be reduced with S' as the electron donor (reaction 18, Table I) (Tisdale and Bertranison, 1950; Vavra and Frederick, 1952; Aller and Rude, 1988; King, 1990). In some instances this may be an indirect nonenzymatic reaction in which Fe(II), sulfite, or other reductants produced during S' oxidation chemically react with Mn(IV) (Vavra and Frederick, 1952; Sugio er al., 1988). However, S oxidation coupled to Mn(IV) reduction in aquatic sediments appears to be biologically mediated (Aller and Rude, 1988; King, 1990). Sulfate is produced when Mn(IV) is added to anoxic, sulfide-containing sediments, but additions of Fe(II1) do not result in sulfate production (Aller and Rude, 1988; King, 1990). Both Mn(1V) and Fe(II1) nonenzymatically oxidize sulfide, but only to the redox level of S' (Goldhaber and Kaplan, 1974; Burdige and Nealson, 1986). This suggests that the production of sulfate in the presence of Mn(IV) is microbially catalyzed. This conclusion is supported by the finding that metabolic inhibitors inhibited the Mn(IV)-dependent sulfate production (Aller and Rude, 1988). It has been suggested that the microorganisms responsible for S' oxidation coupled to Mn(IV) reduction might be sulfate reducers and related microorganisms

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D. R. LOVLEY

(Lovley and Phillips, 1994b). Sulfate reducers catalyzing this reaction in cell suspensions include D. desulfuricans, Desulfomicrobium baculatum, and Desulfobacterium autotrophicum. The S *- and/or Fe(II1)-reducing microorganisms D. acetoxidans and G. metallireducens also carry out this reaction. None of these organisms could be grown with S' as the sole electron donor and Mn(IV) as the electron acceptor. However, many of these organisms are likely to be already growing in sediments via different mechanisms, and thus will often be present in large enough numbers to catalyze S' oxidation as a side reaction (Lovley and Phillips, 1994b). In concurrence with the previous results with marine sediments, these organisms produced little or no sulfate from S o with Fe(II1) as a potential electron acceptor. It has been suggested that large rod-shaped magnetotactic bacteria that can be observed in freshwater sediments might be capable of oxidizing sulfide with the reduction of Fe(II1) (Spring et al., 1993). This speculation was based primarily on the observation of the accumulation of sulfur and iron-containing magnetic particles in this organism. However, there is no direct proof for this metabolism and the organism has yet to be grown under laboratory conditions or isolated in pure culture.

3. Fe(II1) Reduction by Magnetotactic Bacteria Magnetotactic microorganisms contain single domain magnetic particles which permit the organisms to orient themselves in the Earth's geomagnetic field (Blakemore, 1982; Mann et al., 1990a). Magnetite is the magnetic mineral found in many magnetotactic bacteria, but some, living in anoxic environments, may contain iron sulfides (Farina et ul., 1990; Mann etal., 1990b).An increasing diversity of magnetotactic bacteria have been discovered (Moench, 1988; DeLong et al., 1993; Sakaguchi et al., 1993; Spring et al., 1993). Furthermore, although magnetotactic bacteria were originally thought of as sediment organisms, they also inhabit soils (Fassbinder et al., 1990) and water (Mann et al., 1987; Stolz, 1993). The reduction of Fe(II1) that is part of the formation of magnetite inside magnetotactic bacteria (Mann et al., 1990a) may play an important role in the iron cycle in some environments. This Fe(II1) reduction, which can take place either aerobically (Matsunaga et al., 1991) or under microaerophilic (Mann et al., 1984) or anoxic conditions (Bazylinski et al., 1988), may have an important influence on the iron geochemistry and magnetic properties of soils (Fassbinder et al., 1990), sediments (Stolz et al., 1990), and water (Stolz, 1993). It has been suggested that the most highly studied magnetotactic bacterium, Aquaspirillum magnetotacticurn, can also act as a dissimilatory Fe(II1)-reducing microorganism and reduce Fe(II1) with the release of extracellular Fe(I1) under anaerobic conditions (Guerin and Blakemore, 1992). However, the importance of this metabolism in natural environments is not clear. The rates of Fe(II1) reduction

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by A . magnetoracricum were relatively slow in comparison with other dissimilatory Fe(II1) reducers and it could not be conclusively demonstrated whether energy to support growth was conserved from Fe(lI1) reduction (Guerin and Blakemore, 1992).

B. ELECTRON TRANSPORT TO Fe(III) AND Mn(n3 As previously discussed in detail (Lovley, 1991), numerous early studies, most notably those of Ottow and co-workers (Ottow, 1968, 1970b; Ottow and von Klopotek, 1969; Ottow and Munch, 1977; Munch and Ottow, 1983) (and references therein), demonstrated that Fe(II1) and Mn(1V) reduction in the presence of dissimilatory Fe(II1)- and Mn(1V)-reducing microorganisms was the result of an enzymatic process. However, studies on enzymatic mechanisms for Fe(1II) and Mn(1V) reduction in these organisms and many others (Bromfield, 1954a; Dailey and Lascelles, 1977; Lascelles and Burke, 1978; Lundgren et al., 1983; De Vrind er al., 1986; Short and Blakemore, 1986; Karavaiko er al., 1987; Ghiorse, 1988) focused on organisms in which Fe(II1) or Mn(1V) reduction was not a primary process for organic matter oxidation and growth. Thus, these studies may have little relevance in explaining most of the Fe(II1) and Mn(1V) reduction in soils and sediments. A major question has been the mechanisms by which electrons are transported to the insoluble Fe(II1) and Mn(IV) oxides outside the cell. Numerous studies have suggested that direct cell-oxide contact is required for Fe(II1) and Mn(1V) reduction (Munch and Ottow, 1983; Arnold er al., 1988; Lovley and Phillips, 1988a; Lovley et al., 1991b; Caccavo et al., 1992). In G. metallireducens (Gorby and Lovley, 199 1 ) and S. putrefaciens (Myers and Myers, 1993a) the Fe(II1)-citrate reductase activity is localized in the membrane fraction. Growth under anoxic conditions induces the production of c-type cytochromes in the outer membrane of S. putrefuciens (Myers and Myers, 1992). This demonstrates a potential for enhanced capacity for electron transport to metal oxides in contact with the outer cell surface. However, it has yet to be demonstrated whether the c-type cytochromes themselves or another electron carrier which accepts electrons from the c-type cytochrome actually donates electrons to the metal oxides. The periplasmic c ? cytochrome in Desulfovibrio vulgaris can function as a Fe(II1) oxide reductase (Lovley, 1993). Whether this is the physiological Fe(1II) reductase has yet to be demonstrated. Two Fe(II1) reductases have been purified from Thiobacillus ferrooxidans. A periplasmic enzyme oxidizes sulfide to sulfite with the reduction of Fe(II1) (Sugio et al., 1987, 1989) and a membrane-bound Fe(II1) reductase oxidizes sulfite to sulfate (Sugio et al., 1988). No Fe(II1) reductases have been purified from dissimilatory Fe(II1) reducers that conserve energy to support growth from Fe(II1) reduction at neutral pH. However, investiga-

184

D. R. LOVLEY

tions have indicated that the Fe(II1) reductases in such organisms are distinct from the nitrate reductase (Myers and Nealson, 1990; Gorby and Lovley, 1991; DiChristina, 1992; Myers and Myers, 1993a). It has been proposed that the electron carrier that transfers electrons from the outer cell surface to the metal oxides may be metal adsorbed onto the outer surface of the cells (Ehrlich, 1993). In the Mn(1V)-reducing microorganism, strain BIII 88, there is a strong correlation between the manganese content of the cell envelopes and the capacity for the organism to reduce Mn(IV) (Ehrlich, 1993). This has led to the suggestion that manganese in the outer membrane may be directly involved in electron transport across the cell envelope/MnO, interface (Ehrlich, 1993). In this model, Mn(I1) on the cell surface enters into a disproportionation reaction with the MnO,, generating Mn(II1) (reaction 19, Table I). The Mn(II1) then accepts electrons from an as yet unspecified electron donor in the electron transport chain and is reduced back to Mn(I1). An extension of this model is the hypothesis that strain BIII 88 can reduce Mn(1V) aerobically as well as anaerobically because Mn(I1) is not readily oxidized by 0, at neutral pH. It is speculated that other organisms such as G. metallireducens and S. putrefuciens, which can only reduce Mn(IV) in the absence of O,, use Fe(I1) rather than Mn(I1) as an electron shuttle (Ehrlich, 1993). Fe(I1) cannot be effectively used as an electron shuttle in the presence of 0, because it is rapidly chemically oxidized. This model is attractive but requires further verification. For organisms that conserve energy to support growth from Fe(II1) reduction, studies have implicated b- or c-type cytochromes (Obuekwe er al., 1981; Obuekwe and Westlake, 1982; Arnold et a/., 1986; DiChristina et al., 1988; Myers and Myers, 1992; Lovley et al., 1993a; Naik et a/., 1993; Roden and Lovley, 1993a) and menaquinone (Lovley el al., 1993a; Myers and Myers, 1993b) as involved in electron transport to Fe(II1). However, detailed models for the electron transport in these organisms have yet to be elucidated.

C. ENUMERATION AND ISOLATION Although not providing direct information about rates of Fe(II1) and Mn( IV) reduction, determinations of the numbers and types of Fe(II1)- and Mn(1V)reducing microorganisms can be helpful in evaluating this process. Many of the techniques for culturing Fe(II1)- and Mn(1V)-reducing microorganisms in liquid and solid media have already been reviewed (Lovley, 1991 ). The insoluble nature of Fe(II1) and Mn(IV) oxides is often a limitation in these methods, especially for culturing on solid medium where it is difficult to provide enough Fe(II1) or Mn(1V) in contact with the organisms to permit colony development (Lovley, 1991). This problem can be overcome through the use of soluble Fe(II1) forms.

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Fe(II1) citrate has been used as a soluble Fe(II1) form but is not always appropriate as citrate degraders that are present in most soil and sediment samples consume the citrate with the precipitation of the Fe(II1). Furthermore, citrate may inhibit Fe(II1) reduction in some organisms (Lovley et al., 1993b; Roden and Lovley, 1993a). Fe(II1) chelated with nitrilotriacetic acid (NTA) is a much better source of soluble Fe(II1) than Fe(II1) citrate because the NTA is resistant to anaerobic degradation and does not inhibit Fe(II1) reduction in a wide range of Fe(II1)reducing microorganisms (Lovley et al., 1993b; Roden and Lovley, 1993a). When the goal is merely isolation rather than enumeration, another potential approach is to use an electron acceptor other than Fe(II1). For example, many of the acetate-oxidizing Fe(II1)-reducing microorganisms can use fumarate or nitrate as an electron acceptor. Thus, isolation of acetate-oxidizing fumarate or nitratereducing colonies may yield organisms that are also Fe(II1) reducers. This technique was used to obtain G. metallireducens in pure culture (Lovley and Phillips, 1988a) and several other acetate-oxidizing Fe(II1)-reducing microorganisms (J. D. Coates, unpublished data). Fe(II1)-reducing microorganisms have even been purified from enrichments using standard aerobic culturing and isolation techniques (Caccavo et al., 1992). Techniques for enumerating Fe(II1)- and Mn(1V)-reducing microorganisms which do not require isolation of the organisms from the environment are also emerging. An oligonucleotide probe for the 16s rRNA of S. putrefaciens was successfully employed for detecting S. putrefaciens in freshwater aquatic sediments (DiChristina and DeLong, 1993). The finding that acetate-oxidizing Fe(II1)and Mn(1V)-reducing microorganisms cluster in a tight phylogenetic group in the delta proteobacteria (see previous discussion) suggests that the 16s rRNA probe approach may prove useful in detecting organisms in this important metabolic group. In a similar manner, the study of the ecology of magnetotactic bacteria has been limited by the difficulty in culturing these organisms (Mann et al., 1990a). However, recent advancements in phylogenetic studies of the magnetotactic bacteria have made it possible to develop 16s rRNA probes which might be used to study populations of unculturable magnetotactic bacteria in soils and sediments (Eden etal., 1991; Schleifer etal., 1991; Spring et al., 1992, 1993; Burgess et al., 1993; DeLong et nl., 1993). Fe(II1)-reducing microorganisms can also potentially be monitored through analysis of lipids in soils and sediments. For example, G. metallireducens is the only bacterium known to contain lipopolysaccharide hydroxy fatty acids of 16 carbons with the hydroxyl on the 9th, loth, or I I th carbon (Lovley e f al., 1993a). However, these midchain hydroxy fatty acids have been observed in sediments and may serve as a marker for G. metallireducens and related organisms. The Fe(111)-reducing microorganism S. putrefaciens has a lipopolysaccharide

186

D. R. LOVLEY

structure that is different than that of most terrestrial Gram-negative bacteria (Pickard et al., 1993). The overall lipid profile of this organism is also highly unusual when compared with other Gram-negative bacteria (Moule and Wilkinson, 1987). These might be useful characteristics for screening for the potential presence of S. putrefaciens.

D. MONITORING ACTMTY Techniques for studying the activity of Fe(II1) and Mn(1V) reducers are similar to those that are used for ecological investigations of other anaerobic organisms. Although the following discussion focuses on Fe(II1) reduction, the study of Mn(IV) reduction is similar. Environmental rates of Fe(II1) reduction can be estimated with laboratory incubations of sediments or soils under anaerobic conditions and following the production of Fe(I1) and/or the loss of Fe(II1) over time (Kamura et al., 1963; Sorensen, 1982; Jones et al., 1984; Lovley and Phillips, 1986a,b, 1988a,b; Ellis-Evans and Lemon, 1989; Canfield et al., 1993a). As previously reviewed (Lovley, 1991), most of the Fe(I1) that is produced during Fe(II1) reduction remains in solid phases. Therefore, for quantitative estimates it is important to use a technique such as HCl extraction (Lovley and Phillips, 1986a,b) which accounts for this solid-phase Fe(I1). The limitation of such anoxic laboratory incubations is that they may distort in situ conditions (Lovley and Phillips, 1986a,b; Canfield et al., 1993a). For other processes, most notably sulfate reduction (Jorgensen, 1978), this problem has been significantly overcome by injecting radiolabeled tracers into undisturbed sediment cores. However, this approach is not feasible for Fe(II1) reduction because there is rapid isotope exchange between the Fe(II1) and Fe(I1) pools (Roden and Lovley, 1993b). In some instances it may be possible through geochemical modeling of solid and dissolved iron and manganese phases to estimates rates of Fe(II1) and Mn(IV) reduction (Aller, 1980; Burdige and Gieskes, 1983; Aller, 1990; Baedecker et al., 1993). However, this approach can require significant assumptions about the equilibrium solubility of the metals for which there is often little supporting data. A particularly novel approach for estimating rates of Fe(II1) and Mn(IV) reduction was developed by Canfield and co-workers (1993a,b) for continental margin sediments. At depths in the sediments where 0, and nitrate were depleted it could be assumed that organic matter oxidation could be attributed to Mn(IV) reduction, Fe(II1) reduction, or sulfate reduction. Overall rates of organic matter oxidation in these zones were estimated from rates of ammonia production and in one instance were further confirmed with estimates of inorganic carbon production. Subtraction of the contribution of sulfate reduction as measured with the 35SOd-tra~er

MICROBIAL REDUCTION

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technique gave an estimate of the amount of organic matter oxidation coupled to Fe(II1) and Mn(1V) reduction. Proportioning electron flow between Fe(II1) reduction and Mn(1V) reduction was based on results of sediment incubation studies and geochemical considerations.

E.

MECHANISMS FOR ENVIRONMENTAL Fe(I1I) AND Mn(IV) REDUCTION

The fact that microorganisms can reduce Fe(II1) and Mn(1V) has been known for a century (Adeney, 1894; Harder, 1919; Allison and Scarseth, 1942). However, as previously discussed in detail (Lovley et al., 1991b), it has only become apparent within the last decade that enzymatic reduction of Fe(II1) is the major mechanism for Fe(II1) reduction in most soils and sediments. Although the relative importance of enzymatic and nonenzymatic mechanisms for Mn(1V) reduction have not been studied in as much detail, there are instances in which enzymatic Mn(1V) reduction must predominate (discussed next). The relative lack of significance of nonenzymatic Fe(II1) reduction in sediments can be clearly seen by the lack of Fe(II1) reduction in sterilized sediments and by the fact that Fe(II1) reduction has temperature optima characteristic of an enzymatically catalyzed reaction (Bromfield, 1954b; Kamura et al., 1963; Aristovskaya and Zavarzin, I97 1; Sorensen, 1982; Jones et al., 1983; Lovley and Goodwin, 1988; Canfield, 1989). Furthermore, model organic compounds that were oxidized to carbon dioxide in live Fe(lI1)-reducing sediments were not oxidized when the microorganisms were killed with heat (Lovley et al., 1991b). These findings indicate that the bulk of the organic matter in sediments cannot directly reduce Fe(II1). In fact, testing of a wide variety of organics revealed that very few can nonenzymatically reduce Fe(III), especially at the circumneutral pH typical of most submerged soils and aquatic sediments (Lovley et al., 1991b). This includes the common microbial metabolites that are produced during anaerobic metabolism. In contrast, all of these organics could be oxidized to carbon dioxide with Fe(II1) serving as the electron acceptor when the appropriate Fe(II1)-reducing microorganisms were present. Furthermore, even for those few organic compounds that can enzymatically reduce Fe(III), the extent of Fe(II1) reduction and oxidation of the organic compound is trivial compared to oxidation of the same compounds coupled to microbial Fe(II1) reduction. For example, although most monoaromatic compounds do not nonenzymatically reduce Fe(III), a few can (LaKind and Stone, 1989; Lovley et al., 199 1b). However, the nonenzymatic reaction is typically only a two electron transfer, it does not result in any carbon dioxide production, and it leaves an intact ring structure. In contrast, in the presence of the appropriate Fe(I1I)-reducing mi-

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D. R. LOVLEY

croorganisms, monoaromatic compounds can be completely oxidized to carbon dioxide. Depending on the aromatic compound, this is typically a 20-40 electron transfer. In other words, even for the few aromatic compounds that can nonenzymatically reduce Fe(III), enzymatic oxidation coupled to Fe(II1) reduction must be a much more significant Fe(II1)-reducing process than the nonenzymatic reaction. Organics containing a sulfhydryl group are often cited as another important example of how organics nonenzymatically reduce Fe(111). However, the sulfhydry1 group can only nonenzymatically transfer one electron to Fe(II1). In contrast, Fe(II1)-reducing microorganisms can completely oxidize the organic containing the sulfhydryl. For example, in the absence of microorganisms, the nonenzymatic reduction of Fe(II1) by cysteine is only a one electron transfer as cysteine is oxidized to cystine (Cornell et al., 1989). However, with an enrichment culture that could oxidize cysteine with Fe(II1) as the sole electron acceptor (Lovley et al., 1991b), 10 mol of Fe(II1) was reduced per mole of cysteine provided, which is consistent with complete oxidation of cysteine to carbon dioxide with Fe(II1) serving as the sole electron acceptor (reaction 20, Table I). Mn(1V) is more susceptible than Fe(II1) to nonenzymatic reduction by organics. For example, simple organic acids such as oxalate and pyruvate, as well as some dihydroxy aromatic acids and reducing sugars which do not react with Fe(III), can reduce Mn(1V) at circumneutral pH (Troshanov, 1969; Stone and Morgan, 1984; Lovley et al., 1989b). However, these compounds are the exception rather than the rule (Lovley, 1992). Most of the readily metabolizable organic matter in soils and sediment cannot nonenzymatically reduce Mn(1V). Furthermore, the nonenzymatic reactions between organics and Mn(IV) are similar to those discussed for Fe(II1) in that the oxidation of the organics in the nonenzymatic reaction is trivial in comparison with the complete oxidation of the organics that is possible when the appropriate Mn(1V)-reducing microorganisms enzymatically couple the oxidation of the organics to Mn(1V) reduction. Another potential mechanism for nonenzymatic reduction of Fe(II1) reduction that is particularly relevant for environments which contain high concentrations of sulfate is reduction of Fe(II1) by sulfide to form elemental sulfur (Goldhaber and Kaplan, 1974; Pyzik and Sommer, 198I ) . However, synthetic poorly crystalline Fe(II1) oxides (King, 1990) or natural sediment Fe(II1) oxides (Sorensen, 1982)do not oxidize 3sS2-in marine sediments, and selective inhibition of sulfate reduction has no effect on rates of Fe(II1) reduction (Sorensen, 1982; Canfield, 1989; Canfield er al., 1993a). Furthermore, as previously reviewed (Lovley, 199 I ) , extensive literature indicates that in many marine sediments, Fe(II1) reduction takes place in zones in which there is little sulfide production from sulfate reduction. Sulfide reduction of Fe(II1) [or Mn(IV)] cannot explain the Fe(II1) and Mn(IV) reduction in environments such as Toolik Lake in which sulfate concen-

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trations are too low to support significant sulfate reduction (Cornwell and Kipphut, 1992). The potential for nonenzymatic reduction by inorganics is also greater for Mn(IV) than for Fe(II1). In addition to possible reduction by sulfide (Burdige and Nealson, 1986), Mn(IV) can also be reduced by Fe(II), nitrite, and hydrogen peroxide (Lovley, 1991). Reduction of Mn(IV) by Fe(I1) and sulfides is definitely an important mechanism for Mn(1V) reduction in some marine sediments, but in others these processes can be ruled out (Canfield et al., 1993a). In the latter type of sediments, the addition of molybdate to inhibit sulfide production via sulfate reduction and trapping Fe(I1) with ferrozine did not inhibit rates of Mn(I1) production. These results suggest that direct microbial Mn(IV) reduction can be an important process in some marine sediments (Canfield et al.. 1993b). Evidence that Mn(1V) can be a direct respiratory process rather than indirect via microbial reduction of Fe(II1) or sulfate followed by nonenzymatic reduction of Mn(1V) with Fe(I1) or sulfide is the finding that H, concentrations in Mn(1V)amended freshwater sediments are significantly lower than they are in sediments in which Fe(II1) reduction or sulfate reduction is the terminal electron-accepting process (Lovley and Goodwin, 1988). Since H 2 concentrations in the sediments are controlled by the H,-consuming population (Lovley and Goodwin, 1988; Lovley er al., 1994a), this suggests that Mn(1V)-reducing microorganisms directly catalyze H2 uptake. The finding that zones of sulfide or Fe(I1) production are often segregated from the zone of Mn(IV) reduction (Froelich et al., 1979; Reeburgh, 1983; Nealson et al., 1991) further suggests that Mn(1V) is enzymatically catalyzed in some instances. Another reductant that could potentially bring about nonenzymatic reduction of Fe(II1) and Mn(1V) in anoxic environments is U(1V). U(IV) produced during microbial U(V1) reduction nonenzymatically reduced Fe(II1) (reaction 2 l , Table I) (J. C. Woodward and D. R. Lovley, unpublished data). The U(V1) produced could then be enzymatically reduced by microorganisms back to U(IV) bringing about more nonenzymatic Fe(II1) reduction. In cell suspensions of organisms capable of reducing both U(V1) and Fe(III), the rate of insoluble Fe(II1) oxide reduction was much faster in the presence of uranium than in its absence, presumably due to the cycling of uranium. However, it has not been determined whether such an electron-shuttling mechanism is important in sediments where uranium is only available at low concentrations. A less explicit mechanism for nonenzymatic Fe(II1) reduction is the common assumption that Fe(II1) is nonenzymatically converted to Fe(I1) under conditions of a low redox potential (Starkey and Halvorson, 1927; Hem, 1972; Zehnder and Stumm, 1988). However, numerous studies have demonstrated that the development of a low redox potential is an insufficient condition to bring about Fe(II1) reduction, both in defined laboratory cultures and in sediments (Ottow and

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Munch, 1977; Munch and Ottow, 1983; Lovley and Goodwin, 1988; Lovley et al., 1991b). The microorganisms with the appropriate enzymes to catalyze Fe(II1) reduction must be present and, even when present, the enzymes must be able to contact the Fe(II1).

F.

INTERACTION OF Fe(I1I) AND Mnov) REDUCTION WITH OTHER MICROBIALLY CATALYZED REDOX PROCESSES

Fe(II1) and Mn(1V) reduction are generally only geochemically significant processes in anoxic environments. One reason is that many (Lovley, 1991), but not all (Trimble and Ehrlich, 1968; De Castro and Ehrlich, 1970), Fe(II1) and Mn(1V) reducers do not reduce Fe(II1) and Mn(1V) in the presence of 0,. 0, inhibits strict anaerobes such as G. metallireducens and D. acetoxidans. Facultative anaerobes such as 5’. putrefaciens and strain BrY preferentially divert electron flow to O2 when it is available (Arnold et al., 1990). Furthermore, any Fe(I1) and Mn(I1) that might be produced in aerobic environments will be reoxidized to Fe(II1) and Mn(1V) so that no significant net Fe(II1) and Mn(1V) reduction is likely in such environments. Many aerobic soils often contain anaerobic microzones in which Fe(II1) and Mn(1V) reduction could presumably take place. However, this has yet to be investigated. Nitrate inhibits Fe(II1) (Yuan and Ponnamperuma, 1966; Sorensen, 1982; Jones et al., 1983; Ellis-Evans and Lemon, 1989) but generally not Mn(IV) (Froelich et a f . . 1979; Klinkhammer, 1980; Billen, 1982) reduction in sediments and submerged soils. Concurrent Mn(1V) and nitrate reduction is also observed in cultures (Troshanov, 1969; Munch and Ottow, 1983; Burdige and Nealson, 1985). Nitrate inhibition of Fe(II1) reduction can be explained by a combination of factors. In organisms that can reduce both nitrate and Fe(III), preferential nitrate reduction may inhibit Fe(II1) reduction, but this does not explain inhibition of Fe(II1) reduction by microorganisms that do not reduce nitrate (Hammann and Ottow, 1974; Obuekwe et al., 1981; Jones et al., 1983; Munch and Ottow, 1983; Ellis-Evans and Lemon, 1989). Nitrite produced during nitrate reduction can prevent net Fe(II1) reduction by oxidizing Fe(I1) (Obuekwe et al., 1981), but this appears to be a minor effect in sediments (Sorensen, 1982). The ability of nitrate reducers to outcompete Fe(II1) reducers for H2and simple organics as well as the low production of the fermentation products that are electron donors for most of the Fe(II1) reduction in sediments are probably the ultimate factors limiting most of the Fe(II1) reduction in the presence of nitrate (Lovley, 1991). Fe(II1)- and Mn(IV)-reducing microorganisms can reduce both simultaneously (Lovley and Phillips, 1988b; Myers and Nealson, 1988b). However, the zones of net Fe(II1) and Mn(1V) reduction are generally segregated in time or space in part because when Mn(1V) is available, Fe(I1) is rapidly oxidized back to Fe(II1)

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(reaction 22, Table I) through a strictly chemical reaction (Postma, 1985; Krishnamurti and Huang, 1987; Lovley and Phillips, 198%; Myers and Nealson, 1988b). Furthermore, concentrations of electron donors such as H, and acetate may be maintained at concentrations too low to support Fe(II1) reduction when Mn(1V) is available (Lovley, 1991). Both sulfate reduction and methane production are generally inhibited when Fe(II1) oxides are readily available (Ponnamperuma, 1972; Froelich et al., 1979). The inhibition of sulfate reduction markedly influences the sulfur chemistry of anoxic environments. Furthermore, the inhibition of methane production may have an impact on the extent to which this important greenhouse gas is produced and released, especially in shallow freshwater environments such as swamps, marshes, and rice paddies (Lovley, 1991). The inhibition of methane production and sulfate reduction in the presence of Fe(II1) is the result of preferential electron flow to Fe(II1) (Lovley and Phillips, 1987b; Lovley, 1991; Coleman et al., 1993; Lovley et al., 1993b). Hz- and acetate-oxidizing Fe(II1) reducers have a higher affinity for these electron donors than sulfate reducers and methanogens. H, and acetate are the principal electron donors for sulfate reduction and methane production in most natural environments (Lovley and Klug, 1986). Thus, if Fe(II1) reducers outcompete sulfate reducers and methanogens for H2 and acetate, this will greatly inhibit sulfate reduction and methane production. Laboratory studies with sediments and pure cultures have indicated that when Fe(II1) is not limiting, Fe(II1) reducers are able to maintain the concentration of H, and acetate too low for sulfate reducers and methanogens to metabolize (Lovley and Phillips, 1987b; Lovley ef al., 1989b; Caccavo et al., 1992). The competition between sulfate reduction and Fe(II1) reduction for H, (and possibly other electron donors) is not necessarily always between separate populations. As discussed earlier, some H,-oxidizing sulfate reducers have the capacity to reduce Fe(II1).These organisms can metabolize H, to lower concentrations with Fe(II1) as the electron acceptor than they can with sulfate (Coleman et al., 1993). This finding and the finding that the c3 cytochrome in the H,-oxidizing, Fe(II1)reducing microorganism D.vulgaris can function as an Fe(II1) reductase have led to the suggestion that at the low concentrations of H, typically found in anoxic environments, H,-oxidizing Fe(II1) reducers may preferentially reduce Fe(II1) when it is available by diverting electron flow to Fe(II1) at the level of the c1 cytochrome (Lovley et al., 1993b). Field evidence for the inhibition of sulfate reduction as the result of Fe(II1) reducers outcompeting sulfate reducers and/or sulfate reducers preferentially reducing Fe(II1) was provided in a study on microbial metabolism in the Middendorf aquifer, a deep pristine aquifer in the Atlantic Coastal Plain of the United States (Chapelle and Lovley, 1992). Geochemical evidence indicated that Fe(II1) reduction was the terminal electron-accepting process in an up gradient portion of the

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aquifer whereas sulfate reduction predominated down gradient. Concentrations of H2,formate, and acetate were significantly lower within the Fe(II1) reduction zone than in the sulfate reduction zone. Thus, although sulfate-reducing microorganisms could be recovered from the sediments within the Fe(II1) reduction zone, they presumably could not reduce sulfate because electron donor concentrations were too low to support sulfate reduction. Preferential electron flow to Fe(II1) reduction does not always result in complete exclusion of sulfate reduction and methane production during Fe(II1) reduction. When the availability of Fe(II1) is limited either by low Fe(II1) concentrations or by the inability of Fe(II1) reducers to access Fe(II1) oxides, then Fe(II1) reduction may proceed concurrently with either sulfate reduction or methane production (Lovley and Phillips, 1987b). For example, when sediments in which sulfate reduction or methane production is the predominant terminal electron-accepting process were amended with highly crystalline Fe(II1) oxides, the extent of inhibition of sulfate reduction or methane production was much less than when less crystalline, readily reducible Fe(II1) oxides were added (Lovley and Phillips, 1986, 1987b; Aller and Rude, 1988). Another factor that might limit the accumulation of methane in rice paddy soils is the oxidation of methane coupled to Fe(II1) reduction (Miura et al., 1992). However, there is as of yet no direct evidence that such metabolism is possible. For example, additions of Fe(II1) did not stimulate methane oxidation in sediments any more than did additions of Fe(I1) (Zehnder and Brock, 1980). Mn(IV) oxide additions also stimulated methane oxidation (Zehnder and Brock, 1980), but the mechanisms for the stimulation were not clear.

G. ELECTRON FLOWTO Fe(I1I) AND M n O m ANOXICSOILSAND SEDIMENTS Most studies on Fe(II1) and Mn(IV) reduction have been conducted on submerged soils such as rice paddies (see the following discussion) or permanently water-saturated environments such as aquatic sediments and aquifers. Geological evidence suggests that Fe(II1) reduction has been an important process for organic matter oxidation since early in the Earth’s biotic history (Perry et al., 1973; Walker, 1984, 1987; Baur et al., 1985). The strong correlation in the occurrence of isotopically light carbonates and magnetite in the pre-Cambrian banded iron formations indicates that organic matter oxidation was coupled to Fe(II1) reduction in this environment. The fine-scale variability of the carbonate isotope signatures suggests that the organic matter oxidation was an early diagenetic process which would be consistent with the activity of dissimilatory Fe(II1)-reducing microorganisms (Walker, 1984; Baur et al., 1985). It has been suggested that iron may have been central to the overall carbon flow in these pre-Cambrian environments

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(Walker, 1987) because Fe(I1) may also have served as the electron donor for photosynthesis (Hartman, 1984; Widdel et al., 1993). In this model, the organic matter and Fe(II1) that were produced as the result of photosynthesis in the water column would then settle out where dissimilatory Fe(II1)-reducing microorganisms could oxidize the organic matter with the reduction of Fe(II1). This would not only produce magnetite, but also regenerate dissolved Fe(I1) for further photosynthesis. This Archean biosphere would be “upside down” in comparison with present day environments because the primary oxidant for organic matter, Fe(III), would be concentrated in the sediments and the atmosphere would be relatively reducing, whereas in modern environments the atmosphere is oxidizing and sediments are highly reduced (Walker, 1987). Similar isotopic evidence has implicated organic matter oxidation coupled to Mn(1V) reduction as an important process in the marine sediments that led to the development of the Molango manganese ore deposit that was formed during the Jurassic period (Okita et al., 1988). Isotopically light carbonates are strongly correlated with the presence of manganese carbonate in this extensive manganese carbonate deposit. The geochemical evidence indicated that there was little sulfate or Fe(II1) reduction during the manganese carbonate deposition which suggests that the bulk of the manganese reduction could have been microbially catalyzed. Until recently, Fe(II1) and Mn(1V) reduction have generally been considered to be minor pathways for organic matter decomposition in most modern soils and sediments. This is despite the fact that Fe(II1) is often the most abundant potential electron acceptor for organic matter oxidation in many soils and sediments (Takai and Kamura, 1966; Bostrom, 1967; Reeburgh, 1983; Van Breeman, 1988). Mn(1V) oxides are generally less abundant than Fe(II1) oxides but can also be quantitatively significant electron acceptors for organic matter oxidation in some environments (see the following discussion). A major factor leading to the underestimation of the significance of Fe(II1) and Mn(IV) reduction in organic matter decomposition has been the practice of estimating the rates of these processes from the accumulation of dissolved Fe(I1) and Mn(I1). As previously reviewed in detail (Lovley, 1991), most of the Fe(I1) that is produced as the result of microbial Fe(II1) reduction is found in solid or adsorbed phases. In a typical freshwater aquatic sediment, over 98% of the Fe(I1) produced during Fe(II1) reduction was not dissolved (Lovley and Phillips, 1988b). In a similar manner, most of the Mn(I1) produced from Mn(1V) reduction in marine sediments was in solid phases (Canfield et al., 1993a). Fe(I1) and Mn(I1) may be absorbed onto various minerals or organic matter or may be in the form of minerals such as vivianite (ferrous phosphate), siderite (ferrous carbonate), ferrous hydroxides, magnetite, rhodochrosite (manganese carbonate), or sulfides (Lovley, 199 1; Burdige, 1993). The importance of considering solid-phase Fe(I1) and Mn(I1) forms is apparent in recent studies which have demonstrated the importance of Fe(II1) and Mn(1V)

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reduction in organic matter decomposition in some marine sediments. Until recently it was considered that Fe(II1) and Mn(IV) reduction were minor processes for organic matter decomposition in these environments (Canfield et a[., 1993a). However, this conclusion was primarily based on the analysis of pore water chemistry. More detailed analyses, including solid-phase data and incubation studies, have demonstrated that Fe(II1) and Mn(IV) reduction can be major processes for organic matter decomposition in the marine environment. This was evident at three sites off the coast of Denmark where Fe(II1) and/or Mn(IV) reduction accounted for 21 - 100% of the anaerobic decomposition of organic matter in the upper 10 cm (Canfield et al., 1993a). Subsequent analyses have demonstrated that oxygen and nitrate respiration are minor mechanisms for organic matter oxidation in these sediments and that Fe(II1) and Mn(IV) reductions account for ca. 30-90% of the carbon oxidation (Canfield et al., 1993b). These rapid rates of Fe(II1) and Mn(IV) reduction are supported by active bioturbation which permits iron and manganese to proceed through hundreds of cyclings between oxidized and reduced states prior to being permanently buried. The activity of infaunal macrobenthos also contributes to the importance of Mn(IV) reduction as a process for organic matter decomposition in hemipelagic deposits in the eastern equatorial Pacific (Aller, 1990). In this area, high primary productivity contributes organic matter and hydrothermal production of manganese contributes relatively high concentrations of manganese to the sediments. The benthic macroorganisms mix the bottom sediments to depths greater than 30 cm. In these environments, geochemical evidence suggests that there is no Fe(II1) or sulfate reduction and that O2 diffusing into the sediments is consumed for the reoxidation of Mn(I1). Mn(IV) reduction may account for up to 100% of the organic matter oxidation at these sites. In addition to bioturbation, the physical mixing of sediments can also favor the establishment of extensive Fe(II1) and Mn(IV) reduction zones in sediments. For example, the mixing of continental shelf sediments off the mouth of the Amazon River periodically recycles Fe(I1) back to Fe(II1) (Aller er al., 1986). In combination with relatively low concentrations of organic matter, this results in broad sediment zones of a meter or more in which Fe(II1) reduction is the dominant process for organic matter decomposition. Another example of physical mixing of iron-rich sediments and organic matter generating extensive Fe(II1) reduction zones in sediments was observed when a dam break resulted in the sediments burying herbaceous vegetation at the mouth of the Roaring River in Colorado (Litaor and Keigley, 1991). After burial there was a significant loss of carbon from the sediments associated with the reduction and dissolution of Fe(II1) oxides. Other studies have presented evidence for significant zones in which organic matter oxidation is coupled to Fe(II1) and/or Mn(1V) reduction in marine (Sorensen and JBrgensen, 1987; Hines er al., 1991), estuarine (Lovley and Phillips, 1986a,b), and Antarctic lake (Ellis-Evans and Lemon, 1989) sediments. Bio-

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chemical studies have indirectly estimated that Fe(II1) or Mn(IV) reduction could account for a significant portion of the anaerobic decomposition of organic matter in Toolike Lake, an oligotrophic lake located in Alaska (Cornwell and Kipphut, 1992). As previously reviewed (Lovley, 1991), studies on the importance of Fe(II1) and Mn(IV) reduction in temperate lakes have often concluded that Fe(II1) and Mn(IV) reductions are of minor importance in organic matter decomposition, but such studies are likely to have greatly underestimated the extent of Fe(II1) and Mn(1V) reduction because only the accumulation of dissolved Fe(I1) and Mn(I1) was measured. Microbial Fe(II1) and Mn(IV) reduction are also significant processes for organic matter decomposition in pristine aquifers that serve as important drinking water supplies (Gottfreund et al., 1985a,b; Jaudon et al., 1989; Di-Ruggiero and Gounot, 1990; Lovley et al., 1990; Chapelle and Lovley, 1992). The production of dissolved Fe(I1) and Mn(I1) as the result of this activity is a significant worldwide problem that is expensive to remediate.

H. DEGRADATION OF ORGANIC CONTAMINANTS Microbial Fe(II1) reduction can be an important mechanism for the degradation of organic contaminants in anoxic environments. Microbial metabolism often quickly depletes the O2 from water-saturated soils that have been contaminated with organic compounds (Baedecker and Back, 1979; Suflita et al., 1988; Lyngkilde and Christensen, 1992b; Salanitro, 1993). Fe(II1) is generally available in quantities significant enough for it to be an important electron acceptor for contaminant oxidation under such anoxic conditions. As discussed earlier, G. metallireducens can oxidize a number of monoaromatic contaminants to carbon dioxide with Fe(II1) serving as the electron acceptor. Highly purified enrichment cultures which can oxidize other monoaromatic contaminants with Fe(II1) serving as the sole electron acceptor have also been reported (Lonergan and Lovley, 1991). Studies with sediments from a petroleum-contaminated aquifer have suggested that even benzene, which is notoriously difficult to degrade under anoxic conditions, can be oxidized to carbon dioxide under Fe(II1)-reducing conditions (Lovley et al., 1994b). S. putrefaciens can reductively dechlorinate carbon tetrachloride to chloroform under anoxic conditions (Picardal et al., 1993). Dechlorination activity is related to the presence of c-type cytochromes. These cytochromes appear to be similar to the ones that may be involved in electron transport to Fe(II1) reduction (see earlier discussion). Environmental studies have demonstrated that Fe(II1) reduction is an important process for the degradation of soluble organic contaminants in aquifers that have been polluted with petroleum products and landfill leachate. In an aquifer located in Bemidji, Minnesota, that was contaminated by a break in an oil pipeline, ap-

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parent oxidation of aromatic hydrocarbon contaminants in the groundwater was accompanied by an accumulation of dissolved Fe(I1) and a depletion of Fe(II1) from the sediments (Lovley et al., 1989a). Fe(II1) reduction may account for half of the degradation of monoaromatic hydrocarbons in the highly contaminated zone immediately downgradient from the petroleum contamination (Baedecker et al., 1993). Fe(II1) reduction also takes place in an aquifer located in Hanahan, South Carolina, that was contaminated with aviation fuels (Lovley et al., 1994a,b). Sediments from the Fe(II1)-reducing zone consumed toluene in laboratory incubations and the rate of toluene oxidation could be enhanced by stimulating the activity of Fe(II1)-reducing microorganisms in the sediments (Lovley et al., 1994b). However, the importance of Fe(II1) reduction in the overall degradation of aromatic hydrocarbons in this aquifer has yet to be determined. In an aquifer contaminated with landfill leachate, there were narrow zones in which methane production and sulfate reduction were the terminal electronaccepting processes immediately downgradient from the source of contamination (Lyngkilde and Christensen, 1992b). This was followed by a broad zone in which geochemical evidence suggested that Fe(II1) reduction was the terminal electronaccepting process. Geochemical data indicated that there was little, if any, degradation of important contaminants such as aromatic hydrocarbons and chlorinated aromatics within the methanogenic and sulfate-reducing zones, but these compounds were removed from the groundwater within the Fe(II1)-reducing zone (Lyngkilde and Christensen, 1992a). These results suggest that Fe(II1)-reducing microorganisms degraded the contaminants. Numerous other studies have noted accumulations of Fe(I1) within contaminated aquifers (Schwille, 1976; Humenick and Mattox, 1978; Baedecker and Back, 1979; Ehrlich et al., 1983; Nicholson et al., 1983; Wilson et al., 1990; Williams et al., 1992). This provides circumstantial evidence that Fe(II1) reduction is involved in contaminant oxidation in many polluted sites. The finding that Fe(II1) reduction naturally functions to remove organic contaminants from polluted environments has led to investigations of whether Fe(II1) reducers might be manipulated to enhance contaminant degradation. A study with aquifer material collected from a shallow sand and gravel aquifer that had been contaminated with aviation fuel indicated that the rates of Fe(II1) reduction could be greatly stimulated by adding Fe(II1) chelators such as NTA and ethylenediaminetetraaectic acid (EDTA) (Lovley et al., 1994b). Stimulation of Fe(II1) reduction greatly accelerated the loss of toluene degradation after a brief lag of several days. Furthermore, benzene, which was never degraded under natural Fe(II1)reducing conditions in the aquifer material, was degraded in the presence of NTA after an extended lag period (ca. 100 days). Once a population adapted to benzene was established, it could be used to inoculate new sediments that would then degrade benzene in the presence of NTA without a lag.

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Studies with IT-labeled toluene and benzene indicated that they were completely oxidized to carbon dioxide. The stoichiometry of benzene oxidation and Fe(II1) reduction was consistent with Fe(II1) serving as the sole electron acceptor. The rates of toluene and benzene degradation in the NTA-amended Fe(II1)reducing sediments were comparable to rates that had previously been reported for aerobic aquifer material (Salanitro, 1993). These studies indicate that the ability of Fe(II1) reducers to access Fe(II1) oxides in aquifer material and the lack of Fe(II1) chelators in groundwater can be important factors limiting the rate of organic contaminant degradation in contaminated aquifers (Lovley ef al., 1994b). This suggests that the addition of Fe(II1) chelators is a potential strategy for stimulating microbial removal of organics from aquifers in which Fe(II1) oxides are present. Furthermore, Fe(II1) oxides need not necessarily be present because addition of soluble-chelated Fe(II1) can stimulate aromatic hydrocarbon degradation in aquifer material that is depleted of Fe(II1) oxides (Lovley et al., 1994b). Fe(II1)-reducingmicroorganisms can also indirectly contribute to contaminant removal. As is discussed next, microbial Fe(II1) reduction can lead to the formation of ultrafine-grained magnetite (Lovley ef al., 1987; Lovley, 1991). Fe(I1) in microbially produced magnetite can nonenzymatically reduce 4-chloronitrobenzene to 4-chloroaniline (Heijman et al., 1993). This nonenzymatic process may account for the general nitroreductive capacity of anoxic soils and sediments (Heijman et al., 1993).

I. DISSOLUTION AND FORMATION OF IRON ANDMANGANESEMINERALS A diversity of Fe(II1) and Mn(1V) oxide forms are potentially available for microbial reduction in soils and sediments (Dixon and Skinner, 1992; Schwertmann and Fitzpatrick, 1992). A large number of studies with pure cultures have demonstrated that Fe(II1)- and Mn(1V)-reducing microorganisms preferentially reduce the less crystalline oxide minerals (Lovley, 1991; Burdige et al., 1992). Geochemical studies have suggested that the forms of Fe(II1) that are most readily microbially reduced in soils are poorly crystalline Fe(II1) oxides (Back and Barnes, 1965; Ponnamperuma et al., 1967; Van Breeman, 1969; Brannon et al., 1984). The distribution of Fe(II1)-reducingactivity is related to the distribution of poorly crystalline Fe(II1) oxides (Lovley and Phillips, 1987a). The more crystalline Fe(II1) oxide forms in sediments are resistant to rapid microbial reduction (Lovley and Phillips, 1986a,b; Phillips et al., 1993). Further evidence that Fe(II1) reducers only slowly reduce highly crystalline forms such as hematite and goethite in sediments is the finding that whereas additions of poorly crystalline Fe(II1) oxides to sediments permit Fe(111) reducers to outcompete sulfate reducers and

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methanogens, the addition of crystalline Fe(II1) forms does not (Lovley and Phillips, 1986a,b; Aller and Rude, 1988). As discussed earlier, some of the Fe(I1) and Mn(I1) that is produced as the result of microbial reduction of insoluble Fe(II1) and Mn(IV) oxides is released as soluble Fe(I1) and Mn(II), but much of it remains in solid phases. Formation of reduced minerals such as magnetite (Fe,O,), siderite (FeCO,), vivianite (Fe,PO4.8H,O), and rhodochrosite (MnCO,), as well as Fe(I1) adsorbed onto Fe(111) oxides, has been observed in cultures of Fe(II1)- and Mn(1V)-reducing microorganisms (Adeney, 1894; Pfanneberg and Fischer, 1984; Lovley et al., 1987; 1989b; Fischer, 1988; Lovley and Phillips, 1988a) and could contribute to the formation of these minerals in natural environments (Sokolova-Dubina and Deryugina, 1967; Ellwood et a/., 1988; Coleman et al., 1993). An example of microbial Fe(II1) reduction leading to siderite formation is the rapid formation of large siderite concretions in a salt marsh (Coleman et a/., 1993). Geochemical modeling suggested that the siderite concretions formed primarily as the result of H, oxidation coupled to Fe(II1) reduction. Further support for this model was the fact that lipid analysis suggested that the concretions were enriched with Desulfovibrio-like organisms. As discussed earlier, several Desulfovibrio species act as H,-oxidizing Fe(II1) reducers. Mechanisms for the formation of magnetite are of interest for understanding such phenomena as the magnetization of soils and sediments, the formation of magnetic anomalies around hydrocarbon deposits, and the magnetite accumulations in the banded iron formations. Dissimilatory Fe(II1) reduction can lead to accumulations of large quantities of ultrafine-grained magnetite (Lovley et al., 1987, 1993b; Lovley, 1990). Magnetite formation has been observed during Fe(II1) reduction under the appropriate conditions with all of the known dissimilatory Fe(II1)-reducing microorganisms, even sulfate reducers (Lovley et a/., 1993b). With the exception of the Fe(II1) reduction step, the process of magnetite formation during dissimilatory Fe(II1) reduction does not appear to be an enzymatically catalyzed process, yet actively metabolizing cells are required (Lovley, 1990). It is hypothesized that this is because the metabolism generates localized conditions that favor magnetite formation. In contrast to magnetotactic bacteria which produce well-defined crystals of intracellular magnetite (Blakemore, 1982; Mann et al., 1990a; Stolz et a/., 1990), the magnetite produced during dissimilatory Fe(II1) reduction is extracellular and nonuniform (Lovley et al., 1987; Sparks et a/., 1990). Whereas the magnetite crystals of magnetotactic bacteria are single domain magnets, only a portion of the magnetite produced during dissimilatory Fe(II1) reduction is of the single domain variety (Lovley et al., 1987; Moskowitz eta/., 1989). Some of the ultrafine-grained magnetite that contributes to the magnetization of aquatic sediments has the unique morphology that is characteristic of the magnetotactic bacteria (Mann et al., 1990a; Stolz et a/., 1990). However, many sedi-

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ments also contain other, less morphologically distinct, magnetites that could result from magnetite formed by abiological mechanisms (Maher and Taylor, 1988) or dissimilatory Fe(II1) reduction. Further evidence suggestive of the potential contribution of dissimilatory Fe(II1) reduction to magnetite formation in marine sediments was the finding that magnetite appears to form in a sediment layer in which geochemical evidence indicates that organic matter is oxidized with the reduction of Fe(II1) (Karlin et al., 1987). As discussed earlier, geological evidence suggests that organic matter oxidation coupled to dissimilatory Fe(II1) reduction was the mechanism for magnetite accumulation in the banded iron formations. Magnetotactic bacteria are unlikely to have significantly contributed to this magnetite formation since they are not major contributors to anoxic organic matter oxidation coupled to Fe(II1) reduction (Lovley, 1991). In a similar manner the accumulation of magnetite around hydrocarbon seeps which appears to be related to hydrocarbon degradation (Elmore et al., 1987; McCabe et al., 1987) may be modeled by the formation of magnetite that results when G. metallireducens oxidizes the aromatic hydrocarbon toluene with the reduction of Fe(II1) oxide (Lovley, 1990). Magnetic studies of soils can provide useful information on soil-forming processes (Mullins, 1977; Maher, 1986). Magnetite appears to be produced in soils (Maher and Taylor, 1988). This magnetite is morphologically similar to the magnetite produced by dissimilatory Fe(II1) reducers. Another possibility is that the magnetites are formed nonenzymatically as the result of abiotic Fe(I1) oxidation (Maher and Taylor, 1988). However, this abiotic process would require the activity of Fe(II1) reducers in order to generate the Fe(I1). Based on the available evidence, it is not possible to distinguish which of these two mechanisms predominates (Lovley and Stolz, 1989; Lovley, 1990). Magnetotactic bacteria have been recovered from soil and may contribute some soil magnetite (Fassbinder et al., 1990), but the magnetites recovered from soils generally do not have the morphology of magnetite produced by magnetotactic bacteria (Maher and Taylor, 1988). The finding of large accumulations of ultrafine-grained magnetite in granitic rock at depths of 5.5-6.7 km in Sweden has been proposed as evidence of the activity of dissimilatory Fe(II1)-reducing microorganisms at great depths (Gold, 1992). However, thermophilic dissimilatory Fe(II1) reducers that could account for this magnetite production have yet to be isolated.

J. EFFECTSON SOILPROPERTIES Microbial reduction of Fe(II1) and Mn(IV) can affect the physical and chemical nature of soils in various ways. Five to 50% of the Fe(II1) in soil may be reduced within several weeks following the development of anoxic conditions (Ponnamperuma, 1984). One of the most visually obvious effects is the phenomenon of

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soil gleying in which the red-yellows of Fe(II1) oxides in the soil are converted to the gray-bleached appearance associated with the accumulation of Fe(I1) compounds. Fe(II1)-reducing microorganisms have been implicated in soil gleying in that some Fe(I1I) reducers have been isolated from gleyed soils and soils can be artificially gleyed in the laboratory using cultures of Fe(II1) reducers (Allison and Scarseth, 1942; Bloomfield, 1950; Bromfield, 1954a,b; Ottow, 1970a, I97 1 ; Ottow and Glathe, 1971). It has been suggested that a similar bleaching of the red Fe(II1) color associated with the late depositional formation of variegated redbeds and reduction spots (Picard, 1965; Thompson, 1970; McBride, 1974; Hoffman, 1990) is the result of the activity of dissimilatory Fe(I1I)-reducing microorganisms (Lovley er al., 1990). The reduction of Fe(II1) to Fe(I1) is generally the most significant redox change that takes place with the development of anoxic conditions in soils (Ponnamperuma, 1972). The low measured redox potential in such freshwater environments is usually defined by iron redox couples (Ponnamperuma, 1972, 1984). Fe(II1) and Mn(IV) reduction is associated with other phenomena such as an increase in pH; the release of trace metals, sulfate, and phosphate adsorbed onto Fe(II1) oxides; and an increase in the ionic strength of the soil solution, as well as the displacement of sodium, potasium, calcium, and magnesium into solution (Ponnamperuma, 1972, 1984). All of these factors can affect soil fertility. The release of phosphate (Jansson, 1987) and trace metals (Ehrlich et al., 1973; Francis and Dodge, 1989, 1990; Landa et al., 1992) as the result of Fe(II1) and Mn(IV) reduction has been demonstrated under defined conditions with cultures of Fe(II1)- and Mn(1V)reducing microorganisms. Reduction of structural Fe(II1) in clays as well as Fe(II1) associated with clays can greatly influence such soil characteristics as aggregate stability, permeability, friability, porosity, and hydraulic conductivity, as well as clay swelling and mobility (Stucki et al., 1987; Wu et al., 1988; Goldberg, 1989; Ryan and Gschwend, 1990). Fe(II1) reducers in aquatic sediments can readily reduce Fe(lI1) oxide coatings on clays (Lovley and Phillips, 1986, 1987b). Fe(II1) reducers have also been exploited to remove Fe(II1) impurities from kaolins used in the production of ceramics (Hintz et al., 1977). Several studies have indicated that microorganisms can reduce the structural Fe(II1) in clays (Stucki et al., 1987; Wu et al., 1988). However, the conclusions of these studies have been questioned (Lovley, 1991), in part because the organisms that were used in the studies were not Fe(II1) reducers. Furthermore, abiological controls were not conducted in one study (Wu et al., 1988). In the other study (Stucki et al., 1987), the difference in the extent of Fe(II1) reduction in the abiological and biological treatments could potentially be explained by microbial metabolism maintaining low O 2 concentrations and preventing the oxidation of Fe(I1) rather than by direct microbial Fe(II1) reduction (Lovley, 1991).

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K. EFFECTSON PLANTGROWTH The Fe(I1) that accumulates in soils as the result of Fe(II1) reduction is a major chemical sink for 0, that diffuses into the soils and thus can limit soil reaeration (Howeler and Bouldin, 1971; Ponnamperuma, 1984). For most plants the lack of 0,supply to the root zone can limit plant growth (Ponnamperuma, 1984). However, the release of phosphate and trace metals associated with the reduction of Fe(II1) may favor plant growth. The reduction of Fe(II1) in rice paddy soils has been studied intensively because of the important effects it can have on rice cultivation. With the flooding of rice fields, anoxic conditions are established, promoting Fe(II1) reduction. Fe(II1) reduction is an important process for organic matter oxidation during the initial period of flooding (Kamura et al., 1963; Takai and Kamura, 1966; Ponnamperuma, 1972; Yoshida, 1975; Lovley, 1987). The Fe(I1) produced is beneficial to rice growth at low levels, especially if other nutrients are available in appropriate amounts, but Fe(I1) is toxic at high levels (Ponnamperuma et al., 1955; Ponnamperuma, 1972; Yoshida, 1975; Van Breeman, 1988; Jacq et al., 1991). Inhibition of Fe(II1) reduction by the addition of Mn(1V) or nitrate can prevent the toxicity associated with Fe(II1) reduction (Yuan and Ponnamperuma, 1966). Although Fe(I1I) reduction in rice paddies has been intensively investigated from a geochemical standpoint, there have been few microbiologically oriented studies on this topic until one by Jacq and co-workers (1991). In that study, it was found that Fe(II1)-reducing bacteria could readily be isolated from rice paddy soils. Enterobacter sp. was the most dominant, but species of Bacillus, Pseudornonas, and Clostridia capable of Fe(II1) reduction were also isolated. Efforts to isolate sulfate- and S '-reducing microorganisms yielded, among others, D. acetoxiduns, a known dissimilatory Fe(II1) reducer (Roden and Lovley, 1993a). Furthermore, a wide variety of Desulfovibrio were recovered, and many Desulfovibrio species have the capacity to enzymatically reduce Fe(II1) (Coleman et al., 1993; Lovley et al., 1993b). The populations of Fe(II1)-reducing microorganisms greatly increased during the reproductive growth phase of the rice (Jacq et al., 1991). The concentrations of dissolved Fe(I1) in the planted soils were significantly higher than in unplanted soils, further suggesting that the rice plants stimulated microbial Fe(II1) reduction, presumably through the release of dissolved organic compounds from the roots. In cases in which the rice plants had phosphate or potassium deficiencies, the production of high concentrations of Fe(I1) damaged the roots (Jacq et al., 199 I ). Roots of young plants were also damaged under conditions of rapid Fe(II1) reduction.

202

D. R. LOVLEY

111. URANIUM REDUCTION The reduction of U(V1) to U(IV) under anoxic conditions greatly affects uranium mobility in water-saturated soils and sediments. The predominant natural forms of uranium are U(V1) and U(IV) (Langmuir, 1978). U(VI), which readily forms strong complexes with dissolved inorganic carbon, is much more soluble than U(IV). Reductive precipitation of uranium sequesters U(IV) in anoxic marine sediments and is the most globally significant sink for dissolved uranium (Veeh, 1967; Cochran et al., 1986; Klinkhammer and Palmer, 1991). U(V1) reduction in subsurface environments can lead to the deposition of uranium ores (Jensen, 1958; Hostetler and Carrels, 1962; Taylor, 1979; Maynard, 1983). U(V1) reduction is also the likely explanation for the concentration of uranium in the reduction spots of rocks (Hoffman, 1990). Furthermore, the ability of bottom sediments of waste treatment ponds to remove dissolved uranium from uranium mine wastewaters is probably due to reductive precipitation of uranium (Brierley and Brierley, 1980).

A. U(VI)-REDUCINGMICROORGANISMS The first microorganisms found to enzymatically reduce U(V1) were the dissimilatory Fe(II1)-reducing microorganisms, G. metallireducens and S. putrefaciens. These organisms can use U(V1) in place of Fe(II1) as an electron acceptor to support their growth (Lovley e t a / . , 1991a). Despite the fact that uranium is a toxic heavy metal, both these organisms grew on high concentrations (ca. 8 mM) of uranium and grew at rates comparable to those observed with Fe(II1) serving as the electron acceptor. The capacity for growth with U(V1) as an electron acceptor is somewhat surprising in that the concentrations of U(V1) in most natural waters are so low (ca. 12 nM) (Durrance, 1986) that U(V1) reduction cannot be an important mode of growth in most instances. The estuarine Fe(II1)-reducing microorganism, strain BrY, which is closely related to S. putrefaciens can also reduce U(V1) (Caccavo et a/., 1992), but studies to determine whether strain BrY can grow via U(V1) reduction have not been conducted. Several sulfate-reducing microorganisms are able to reduce U(VI), including D. desulfuricans, D. vulgaris, and D. baculatum (Lovley and Phillips, 1992a; Lovley et al., 1993b). Attempts to grow the U(V1)-reducing sulfate reducers with U(V1) as the sole electron acceptor have been unsuccessful (Lovley and Phillips, 1992a; Lovley e f al., 1993b). This is in agreement with their inability to grow with Fe(II1) as the electron acceptor. The ability to reduce Fe(II1) does not necessarily confer the ability to reduce U(V1) as active Fe(II1) reducers such as the sulfate reducers, Desulfobulbus propionicus and Desulfobacterium autotrophi-

203

MICROBIAL REDUCTION

cum, as well since the S' reducer Desulfuromonas acetoxidans do not reduce U(V1) (Lovley e t a / . , 1993b; Roden and Lovley, 1993a).

B. ENZYMATIC MECHANISMS FOR

REDUCTION

Attempts to elucidate the mechanisms for U(V1) reduction in G. merallireducens and S. putrefaciens have been stymied by the fact that it has, as yet, not been possible to maintain U(V1)-reducing activity in cell-free extracts. However, as with Fe(II1) reduction, it appears that c-type cytochromes are involved in electron transport to U(V1) in G. metallireducens (Lovley et al., 1993a). In D. vdgaris, the c 7cytochrome functions as a U(V1) reductase (Lovley e t a / . , 1 9 9 3 ~ )Most . (ca. 95%) of the H,-dependent U(V1) reductase activity is localized in the soluble fraction of broken cells. When the c3 cytochrome is selectively removed from the soluble fraction, the capacity for U(V1) reduction is lost. U(V1) reductase activity is restored when pure c 7cytochrome is added back. U(V1) oxidized reduced c 7 cytochrome, demonstrating that the c1 cytochrome can act as a U(V1) reductase. When cytochrome c 3 is combined with pure periplasmic hydrogenase, its physiological electron donor, U(VI), is reduced. The hydrogenase alone has no U(V1)-reducing capacity. This simple two-component electron transport chain from H, to U(V1) suggests that it should be feasible to construct cellfree U(V1)-reducing enzymatic systems for bioremediation purposes (see Section 1II.D). Furthermore, since the gene for the c 3cytochrome of D. vulgaris has been cloned and successfully expressed in other organisms (Voordouw et al., 1990; Cannac et al., 1991), it may be possible to genetically engineer microorganisms with enhanced capacity for U(V1) reduction.

C. ENZYMATIC VERSUSNONENZYMATIC U(VI)REDUCTION Until recently, microorganisms were generally considered to have an indirect role in U(V1) reduction. It was thought that microorganisms produced reduced end products such as sulfide and H, which then nonenzymatically reduced U(V1) (Jensen, 1958; Hostetler and Garrels, 1962; Langmuir, 1978; Taylor, 1979; Maynard, 1983). For example, several microbiological studies with the sulfatereducing microorganism D. desulfuricans suggest that sulfide produced as the result of sulfate reduction could reduce U(V1) (Viragh and Szolnoki, 1970; Mohagheghi et al., 1985). However, more recent studies have indicated that the reduction of U(V1) in the presence of D. desulfuricans is an enzymatically catalyzed reaction (Lovley and Phillips, 1992a; Lovley et al., 1993b). Sulfide and H2 are, in fact, poor reductants for U(V1) under the circumneutral pH and low temperatures

2 04

D. R. LOVLEY

in the microbial cultures and in most sedimentary environments (Lovley et al., 199 la; Lovley and Phillips, 1992a). Environmental observations also indicate that sulfide does not reduce U(V1) (Anderson, 1987; Anderson et al., 1989a,b). A series of studies with cultures and sediments suggest that enzymatic reduction is the likely mechanism for U(V1) reduction in many sedimentary environments (Lovley et al., 1991a, 1993b; Lovley and Phillips, 1992a; Barnes and Cochran, 1993). The relative role of the various types of U(V1)-reducing microorganisms in precipitating uranium in anoxic environments is unknown. Numerous studies (for references see Cochran et al., 1986; Lovley et al., 1991a) have indicated that deposition of U(IV) in marine sediments takes place within the Fe(II1) reduction zone. Dissimilatory Fe(II1)-reducing microorganisms which conserve energy to support growth from Fe(II1) reduction might be expected to predominate over Desulfovibrin-like organisms in such sediments and may be responsible for most of the U(V1) reduction. However, studies with estuarine sediments have suggested that U(V1) reduction takes place in the sulfate reduction zone, after Fe(II1) oxides have been reduced (Barnes and Cochran, 1993). In such sediments the U(V1)reducing sulfate reducers might be more important. Sulfate reducers may also be important for U(V1) reduction in the formation of roll-type uranium deposits in which uranium is precipitated under sulfate-reducing conditions (Lovley and Phillips, 1992a).

D. BIOREMEDIATION OF URANIUM-CONTAMINATED SOILSAND WATER U(V1)-reducing microorganisms or their U(V1)-reducing enzymes can be used to remove uranium from contaminated waters and soils. The mining and processing of uranium can lead to uranium contamination of soils and waters (Macaskie, 1991). Irrigation practices may result in waters with undesirably high concentrations of uranium (Bradford et al., 1990). Microbial or enzymatic U(V1) reduction can effectively remove uranium from contaminated waters by converting the U(V1) to U(IV) which then precipitates as the mineral uraninite, UO, (Gorby and Lovley, 1992; Lovley and Phillips, 1992a,b; Lovley et al., 1 9 9 3 ~ )In . whole cell suspensions, all the uraninite precipitate is extracellular. Depending on conditions there is a lag of several hours (whole cells) to days (enzymes) between the reduction of U(V1) to U(IV) and the formation of uraninite particles large enough to be filtered out of solution or to settle by gravity (Lovley and Phillips, 1992b; Lovley et al., 1 9 9 3 ~ )This . means that it is possible to separate the process of U(V1) reduction and U(IV) precipitation so that the precipitate does not accumulate on the cells or enzymes. Whole cells and cell-free enzyme preparations have been used successfully on the bench scale to remove uranium from contaminated groundwater from a Department of Energy site and contaminated surface water

MICROBIAL REDUCTION

205

from an abandoned mining operation on Department of Interior lands (Lovley and Phillips, 1992b; Lovley er al., I993c). Uranium-contaminated soils can be treated by first extracting the uranium with bicarbonate which solubilizes the U(V1) as a carbonate complex. The U(V1) can be precipitated with microbial U(V1) reduction and then the bicarbonate can be reused for further soil extraction (Phillips et ul., 1994). The potential for this technique to be used in the remediation of a variety of soils has been evaluated at the bench scale, but this technique has yet to be tried on the large scales required in environmental remediation.

IV. SELENIUM REDUCTION There is intense interest in microbial reduction of selenium because although this metalloid is a minor element in most environments, it may accumulate to toxic levels in some soils and waters (Oremland, 1994). Selenium contamination is associated with metal refining (Nriagu and Wong, 1983), fly ash waste (Adriano et al., 1980),and agricultural drainage waters in the western United States, most notably the highly publicized Kesterson National Wildlife Refuge (Presser and Barnes, 1984; Weres et al., 1989). The predominant forms of selenium in natural environments are Se(V1) (selenate, SeO,->), Se(1V) (selenite, Se0,-2), Se(0) (elemental selenium, Se'), and Se( - 11) (selenide) (Doran, 1982). All except selenide can serve as electron acceptors for microbial metabolism. Selenium is in some aspects chemically similar to sulfur and, until recently, it was generally regarded that many biogeochemical transformations of selenium were nonspecific reactions catalyzed by enzymes involved in sulfur biogeochemistry (Heider and Bock, 1993). However, it is now clear that some microorganisms have evolved biochemical mechanisms unrelated to sulfur metabolism for using selenate, the predominant form of oxidized selenium in most environments, as a terminal electron acceptor.

A. MICROORGANISMS THAT REDUCESELENIUM A wide diversity of microorganisms are capable of selenate reduction. However, mechanisms for selenate reduction and the role of selenate reduction in energy conservation in many of these selenate reducers are not well documented. Over 20% of the bacteria, 80% of the actinomycetes, and 19% of the fungi isolated from a silty clay loam reduced selenate (Bautista and Alexander, 1972). The yeast, Pichiu guillermondii, and a Micrococcus sp. reduced selenate to red elemental selenium on aerobic agar plates of heterotrophic medium (Bautista and Alexander,

206

D. R. LOVLEY

1972). Selenate was also reduced in cell suspensions but not in cell-free extracts. Microorganisms which appeared to be Clostridium species reduced selenate when grown on agar plates under anaerobic conditions, but this metabolism was not further investigated (Kauffman et al., 1986). Organisms in the genera Citrobacter, Flavobacterium, and Pseudomonas that reduced selenate to Se' on agar plates were recovered from sediments of Kesterson reservoir (Burton et al., 1987). A Gram-negative,facultatively anaerobic rod capable of reducing selenate with lactate as the electron donor was also isolated from Kesterson Reservoir (Maiers et al., 1988). Selenate was first reduced to selenite, some of which was subsequently reduced to an unidentified insoluble selenium compound. It was assumed that some of the selenate was completely reduced to volatile selenide as 10- 18% of the selenium added as selenate was not recovered in soluble or insoluble forms. The physiological significance of selenate reduction by this organism is not clear. Although selenate reduction was associated with growth, the cultures were incubated under air and there was no evidence for selenate-dependent growth. Pseudomonas stutzeri reduced selenate to Se' when grown under aerobic conditions in an organic-rich medium (Lortie et ul., 1992). Optical density increases were greater in the presence of selenate but this was due to the production of Se' particles rather than enhanced growth. Wolinella succinogenes, which was inhibited by selenate or selenite, was adapted to grow in the presence of these compounds (Tomei et al., 1992). In the adapted culture, both selenate and selenite were reduced to Se', but the culture would not grow with either of these compounds as the sole terminal electron acceptor (Tomei et al., 1992). An interesting feature of selenate reduction in M! succinogenes is that some of the Se' that is produced is deposited intracellularly. Sulfate reducers can reduce selenate to selenite when selenate is present at low concentrations (Zehr and Oremland, 1987). The selenate is presumably reduced by the sulfate reduction pathway (Zehr and Oremland, 1987). However, sulfate inhibits selenate reduction and thus this type of metabolism is probably not an important pathway for selenate reduction in agricultural drainage waters which are typically high in sulfate (Zehr and Oremland, 1987). A selenate-reducing culture known as strain SeS was enriched from intertidal sediments of San Francisco Bay (Oremland et al., 1989). However, it was not certain that the culture contained only one organism (Oremland et al., 1989). SeS was a Gram-negative cocci which grew rapidly in medium with acetate as the sole electron donor and nitrate or trimethylamine-N-oxide as the electron acceptor. Growth on selenate was slow, but after 6 weeks the culture densities were comparable to cultures grown with nitrate or trimethylamine-N-oxide as the electron acceptor. Selenate was reduced to elemental selenium. As selenate concentrations in the medium were increased to 20 mM, there was a corresponding increase in the production of I4CO2from [2-I4C]acetate.It was proposed (Oremland et al., 1989) that SeS obtained energy to support growth by acetate oxidation coupled to

MICROBIAL REDUCTION

207

selenate reduction (reaction 23, Table I). Unfortunately, the SeS culture has been lost and is not available for further study (Steinberg er al., 1992). Another selenate-reducing microorganism, designated SES-3, grew in defined medium with lactate as the electron donor and selenate as the electron acceptor (Steinberg el al., 1992; Oremland, 1994). Selenate was reduced to selenite and Se'. Nitrate, trimethylamine, and Mn(IV) can also serve as electron acceptors with nitrate being reduced to ammonia (Oremland, 1994). The selenate-reducing microorganism in pure culture that has been studied the most intensively is Thauera selenatis. This organism was isolated from seleniumcontaminated drainage water in the San Joaquin Valley of California (Macy et al., 1989, 1993b). It conserves energy to support growth by coupling the oxidation of acetate to the reduction of selenate to primarily selenite (reaction 24, Table I). However, there is also some slight reduction of selenate to Se' (DeMoll-Decker and Macy, 1993). In addition to selenate, nitrate and 0, can serve as electron acceptors to support growth. When grown in the presence of both selenate and nitrate, the selenite produced from selenate reduction is further reduced to Se' (Rech and Macy, 1992; DeMoll-Decker and Macy, 1993). With 0, as the electron acceptor, 'I: selenatis can use a wide range of electron donors, including benzoate, several sugars, and amino acids (Macy et al., 1993b). Whether these compounds can serve as electron donors for selenate reduction was not reported. Many organisms reduce selenite to Se', but this metabolism is probably only a detoxification mechanism as it has never been shown to be involved in energy conservation (Doran, 1982; Oremland, 1994). Reduction of Se' to selenide has been reported for cell suspensions of Thiobacillusferrooxidans (Bacon and Ingledew, 1989).

B. ENZYMATIC MECHANISMS FOR SELENATE REDUCTION Enzymatic mechanisms for selenate reduction have been investigated in Tlzauera selenatis (Rech and Macy, 1992). Several lines of evidence suggested that the selenate reductase was distinct from the nitrate reductase. For example, the pH optima for selenate and nitrate reductase activities were different; nitrate did not inhibit selenate reductase activity; selenate reductase activity was periplasmic whereas nitrate reductase activity was in the cytoplasmic membrane; and mutants which lacked nitrate reductase activity continued to reduce selenate. Evidence also ruled out that nitrite reductase was the selenate reductase. Studies on cell yields during growth with selenate as the electron acceptor suggested that 'I: selenaris must conserve energy to support growth by linking electron transport from acetate to selenate with an electron transport chain capable of generating a proton gradient for ATP production (Macy and Lawson, 1993). Although the selenate reductase and the nitrate reductase in 7: selenatis are

208

D. R. LOVLEY

separate enzymes, one enzyme may be responsible for both selenite and nitrite reduction (DeMoll-Decker and Macy, 1993). Evidence for this includes the findings that mutants which lack periplasmic nitrite reductase activity do not reduce selenite and mutants with enhanced nitrite reductase activity reduce selenite faster than the wild type.

C. MICROBIAL REDUCTION OF SELENIUM INSOILSAND BIOREMEDIATION Selenium reduction in soils has been well-documented. Under anaerobic conditions, a red precipitate that looked like red amorphous Se' accumulated in soil amended with selenite, and soil amended with selenate, selenite, or selenocystine produced selenide (Doran and Alexander, 1977). There was no activity in heatkilled soil. Selenate and selenite were effectively removed from mine water that was passed through soil from the bottom of a mine pond (Kauffman er al., 1986). Removal of dissolved selenium was associated with the accumulation of insoluble red Se' near the column inlet. The soil's capacity for selenium removal was lost if the soil was steam-sterilized. The ability to remove selenium could be restored by inoculating the sterilized soil with a slurry of nonsterilized soil. Sediments and soils collected near Kesterson Reservoir had the potential to reduce selenate to a red precipitate that was not Se' but was more reduced than selenite (Maiers et al., 1988). Detailed studies on selenate reduction were conducted with slurries of San Francisco Bay sediments amended with selenate (Oremland et al., 1989). H,, acetate, and lactate stimulated selenate disappearance. Autoclaving partially (ca. 66%) inhibited the selenate loss. In the presence of the radiolabeled tracer 7sSe04-'and 0.5 mM Se04-,, loss of 7sSefrom solution was accounted for by an accumulation of 75Sein the sediments. There was no loss of 75Sefrom solution in autoclaved controls or when the sediments were incubated under aerobic conditions. The endproduct of selenate reduction was insoluble elemental selenium. A microorganism that served as a model for selenate reduction in these sediments was recovered (see earlier). All of these results suggested that selenate reduction was an enzymatically catalyzed process. Subsequent studies demonstrated that the potential for microbially catalyzed selenate reduction in aquatic sediments was widespread (Steinberg and Oremland, 1990). Although sulfate-reducing microorganisms can reduce selenate under some conditions (Zehr and Oremland, 1987), studies in sediments have suggested that other organisms, with metabolic properties more akin to the respiratory selenate reducers described earlier, are responsible for selenate reduction in sediments. For example, in an agricultural evaporation pond in the San Joaquin Valley of California, it was apparent that selenate and selenite were reduced in sediment layers that

MICROBIAL REDUCTION

209

were shallower than the zone of sulfate reduction (Oremland et al., 1989, 1990). Furthermore, selenate was reduced at pore-water sulfate concentrations which inhibit selenate reduction by sulfate reducers (Zehr and Oremland, 1987). Molybdate, a specific inhibitor of sulfate reduction, did not inhibit selenate reduction (Oremland et af., 1989). The similar distribution of the denitrification and selenate reduction potentials in sediments (Oremland et al., 1990; Steinberg and Oremland, 1990) suggests that organisms such as 7: selenatis and SES-3 that have both nitrate- and selenate-reductase activities might be responsible for selenate reduction. Microbial reduction of selenate and selenite to insoluble Se' can be an effective mechanism for removing contaminated selenium from water. For example, rates of selenate reduction in the sediments of an evaporation pond built to collect agricultural drainage water indicated that microbial selenate reduction could naturally remove all of the selenate in the overlying water within several months (Oremland et al., 1990). Most of the dissolved selenium in exposed surface soil at Kesterson Reservoir could be immobilized by flooding the soil with low selenium water to promote the development of an anoxic soil (Long et al., 1990). Over a 2-year period such flooding decreased the concentrations of selenium in pond water, vegetation, and invertebrates by 95, 85, and 75%, respectively (Weres et ul., 1989). However, such selenium immobilization may only transiently solve the selenium contamination problem as the selenium only remains immobilized as long as the soils are kept anoxic (Alemi et al., 1988) and bottom-feeding organisms may reintroduce selenium into the food chain (Luoma etal., 1992). Several microbial treatment techniques for ex situ- processing of seleniumcontaminated water have been proposed (Kauffman et al., 1986; Oremland et al., 1990; Altringer etal., 1991; Gerhardt etal., 1991; Macy et al., 1993a). In each of these, the water is passed through a system containing selenate-reducing microorganisms which then immobilize the selenium in the elemental state. Although such systems do not appear to be in widespread use, pilot studies have indicated that they could potentially be economically feasible (Altringer et al., 1991). The most detailed microbiological study of the use of selenate-reducing bioreactions was with a bench-scale system inoculated with 7: selenatis (Macy et al., 1993a). Selenate-contaminated drainage water from the San Joaquin Valley was passed through a sludge blanket reactor and a fluidized bed reactor connected in series. Acetate was added to the drainage water in order to provide an electron donor and additions of ammonium were required as a source of fixed nitrogen for 7: selenatis. 7: selenatis persisted in the reactor and was the dominant selenatereducing microorganism that could be isolated from reactor samples. Selenate was reduced primarily to elemental selenium which precipitated out in the reactor. It was presumed that 7: selenatis reduced selenate to selenite and that the selenite was further reduced to elemental selenium by the nitrite reductases of 7: selenatis and other denitrifying microorganisms that were abundant in the reactor. The re-

210

D. R. LOVLEY

actor system was very effective in removing nitrate as well as selenate as the concentrations of both were reduced by 98%. It was concluded from these benchscale trials that T selenaris would be suitable for use in large-scale reactor systems.

V. CHROMATE REDUCTION Most of the chromium in the environment is in the form of Cr(II1). High concentrations of Cr(V1) are usually the result of pollution (Bartlett, 1991; Palmer and Wittbrodt, 1991). Cr(V1) is highly soluble, toxic, and a carcinogen (Richard and Bourg, 1991; Baruthio, 1992). Cr(II1) forms insoluble oxides and hydroxides in most natural waters (Palmer and Wittbrodt, 1991; Richard and Bourg, 1991) and is less toxic than Cr(V1) (Petrilli and Flora, 1977). The high midpoint potential of the Cr(VI)/Cr(III) couple [ca. 1.3 V (Palmer and Wittbrodt, 1991)] means that all known physiological electron donors for microbial metabolism can potentially serve as electron donors for Cr(V1) reduction.

A.

Cr(w)-REDUCING MICROORGANISMS

Numerous microorganisms are capable of reducing Cr(V1) to Cr(II1) (Table 11). In those instances in which the mechanisms for Cr(V1) have been investigated, it is clear that the Cr(V1) reduction in the presence of the Cr(V1)-reducing microorganisms is an enzymatically catalyzed reaction (Horitsu et al., 1987; Bopp and Ehrlich, 1988; Komori et al., 1989; Wang et al., 1989, 1990; Shen and Wang, 1993). Some microorganisms reduce Cr(V1) under either aerobic or anaerobic conditions whereas others only reduce it under one or the other (Table 11). In general, Cr(V1) reduction has been documented analytically as a loss in Cr(V1) which is often associated with the accumulation of insoluble Cr(II1) precipitates, presumably Cr(OH,). Only with the development of novel liquid chromatographic techniques which permit quantification of Cr(II1) have true stoichiometric conversions of Cr(V1) to Cr(II1) been documented (Llovera et al., 1993; Shen and Wang, 1993). Furthermore, there have been very few instances when the actual electron donors for Cr(V1) reduction in whole cells have been documented. The recent study of H2 oxidation coupled to Cr(V1) reduction (reaction 25, Table I) by D. vulgaris (Lovley and Phillips, 1994a) is a rare example. In general, Cr(II1) accumulates outside the cell, and although Cr(II1) is less soluble than Cr(VI), the Cr(II1) often stays in solution (Shen and Wang, 1993). The reason that some microorganisms have developed a capacity for Cr(V1) reduction has not yet been adequately explained. It has been suggested that Cr(V1)

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211

Table I1 Examples of Chromate-Reducing Microorganisms Chromate reduction Organism

Aerobic Anaerobic

+ +

Electron donor

Bcicillus sirbtilis

+

Glucose Endogenous electron donors Glucose

Desulfovibrio wlgciris

+

H,

Achromobacter eurvdice Agrobacterium radiobricrer

Enterobacrer c l o m i e Escherichin coli Micrococcus roseus Pseudomonus neruginosri

P nmbigun G- I

+

-t

+

4-

+ + +

Amino acids Glucose and peptone Glucose Glucose Nutrient broth Primarily organic acids

P. chromcitophila

+

P. dechromnricnns

4-

Unspecified, grown in complex medium

-t

Glucose (aerobic) Acetate (anaerobic) Glucose Endogenous electron donors Glucose

P. fluorescens

+

+ + Streptotnyces sp

+

Reference Gvozdyak er al. ( 1987) Llovera et al. (1993) Gvozdyak et nl. ( 1 987) Lovley and Phillips ( 1994a) Wang et nl. ( 1989) Shen and Wang (1993) Gvozdyak el of. (1987) Gvozdyak et nl. (1987) Horitsu er 01. (1987) Lebedeva and Lyalikova (1 979) Romanenko and Koren’kov (1977) Bopp and Ehrlich (1988) Blake eral. (1993) Ishibashi et nl. (1990) Das and Chandra (1990)

reduction may be a mechanism for chromate resistance (Horitsu el al., 1987; Suzuki er al., 1992; Shen and Wang, 1993), but this has never been directly demonstrated (Cervantes and Silver, 1992). The resistance hypothesis is supported by the finding that chromate-sensitive mutants of P. ambigua reduced Cr(V1) much slower than the chromate-resistant wild type (Horitsu er al., 1987). However, studies with P. jhorescens indicated that chromate-resistant and the chromatesensitive strains had similar capacities for Cr(V1) reduction (Bopp and Ehrlich, 1988). Cr(V1) reduction may just be a fortuitous reaction carried out by enzymes that have other physiological substrates since the Cr(V1) reductase is often constitutive (Bopp and Ehrlich, 1988; Das and Chandra, 1990; Ishibashi et al., 1990; Cervantes, 1991). This could be true, for example, of the c j cytochrome of

D. R. LOVLEY

212

D. vulgaris which functions as a Cr(V1) reductase (see Section V.B) but has other physiological functions in this organism (LeGall and Fauque, 1988). There have been several suggestions that Cr(V1) reduction may provide energy to support the growth of some organisms (Romanenko and Koren’Ken, 1977; Lebedeva and Lyalikova, 1979; Bopp and Ehrlich, 1988), but the data available are not yet sufficient to adequately support this claim. For example, Pseudomonas chromatophilia incorporated I4CO, as it reduced Cr(V1) in a complex medium containing acetate and meat-peptone broth (Romanenko and Koren’Ken, 1977). I4CO, incorporation stopped as Cr(V1) was depleted. No anaerobic incorporation of ‘ T O , in the “control” was found but whether this means a control without Cr(V1) is not clear. P. chromatophilia grows via Cr(V1) reduction with several electron donors, including acetate, a compound which presumably can only be oxidized with a respiratory process (Lebedeva and Lyalikova, 1979). Yet, the data presented indicated that most of the Cr(V1) was reduced after growth was complete and there were no data that demonstrated that Cr(V1) was required to support anaerobic growth. In a similar manner, the bulk of Cr(V1) reduction by Aeromonas dechromatica appeared to be after most of the growth had taken place (Kvasnikov et al., 1985). P. JIuorescens LB300 grew in an anaerobic chamber under N, on agar medium in which acetate was provided as the electron donor and Cr(V1) was provided as a potential electron acceptor (Bopp and Ehrlich, 1988). However, acetate oxidation or Cr(V1) reduction was not measured and P. Puorescens could not be grown under strict anaerobic conditions in liquid acetate-Cr(V1) medium. Enterobacter cloacae stain HO 1 reduced Cr(V1) when grown anaerobically in a medium that contained acetate and Casamino acids as potential electron donors (Wang et al., 1989), but there was no evidence that Cr(V1) reduction provided energy to support growth. In fact, analysis of the growth data (Ohtake et al., 1990a) suggests that growth and Cr(V1) reduction were not coupled (Lovley, 1993). Amino acids were the preferred electron donors for Cr(V1) reduction (Ohtake et al., 1990~).Metabolism of the electron donors was not investigated, but E. cloacae could probably grow via amino acid fermentation since there was good growth in the absence of Cr(V1) (Wang et al., 1989). In most of the other studies listed in Table I1 there was no attempt to establish a link between Cr(V1) reduction and growth, and Cr(V1) reduction was most often studied with cell suspensions.

B.

MECHANISMS FOR Cr(m)REDUCTION

In most instances, Cr(V1) reductase activity has been found primarily in the soluble cell fraction. This was true, for example, in P. ambigua G-1 (Horitsu et al., 1987). NADH was required as an electron donor for the soluble Cr(V1) reductase in this organism (Horitsu et al., 1987).A protein purified from the soluble fraction

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reduced Cr(V1) to Cr(II1)with the oxidation of 3 mol of NADH per mole of Cr(V1) reduced (Suzuki et al., 1992). NADH could nonenzymatically reduce Cr(V1) to Cr(V) in the absence of the enzyme. Cr(V) was also formed as an intermediate in the enzymatic reduction of Cr(VI), and the extent of Cr(V) formation was 10 times greater in the enzymatic reaction. The organism contained at least one other Cr(V1)-reducing enzyme which has yet to be completely purified (Suzuki et al., 1992). Chromate reductase activity has also been located in the soluble fraction of Pseudomonas putida (Ishibashi et al., 1990). NADH or NADPH is required for activity. Cr(V1) reductase activity was not inhibited by oxyanions such as sulfate, sulfite, molybdate, vanadate, phosphate, or nitrate. Cr(II1) did not inhibit enzyme activity but Hg(I1) and Ag(1) were strong inhibitors. No Cr(V1)-reducing enzyme could be purified as the enzyme activity did not elute in a distinct band with the chromatographic conditions employed. Cross inhibition studies failed to suggest another potential substrate for the Cr(V1)-reducingenzyme. NADH-dependent Cr(V1) reductase activity was also found in the soluble fraction of E. coli (Shen and Wang, 1993). Membrane fractions did not reduce Cr(V1). Cr(V1) was reduced under both aerobic and anaerobic conditions. The capacity for Cr(V1) reduction was heat labile. The respiratory inhibitors cyanide, azide, and rotenone did not inhibit Cr(V1) reduction in whole cells. The uncoupler 2,4,dinitrophenol stimulated Cr(V1) reduction under both aerobic and anaerobic conditions. Cr(V1) did not oxidize either the b or d cytochromes in pure membrane fraction, but the cytochromes were oxidized in the presence of a combination of membrane and soluble fractions. These results suggest that the membrane-bound cytochromes might be involved in electron transport to a soluble Cr(V1) reductase (Shen and Wang, 1993). The c? cytochrome from D. vulgaris functions as a Cr(V1) reductase (Lovley and Phillips, 1994a). Experiments similar to those described earlier on the U(V1)reducing capability of the c3cytochrome indicated that it is also likely to account for the ability of D. vulgaris to reduce Cr(V1). Cr(V1) reduction in the presence of E. cloacae is also clearly an enzymatically catalyzed reaction (Komori et al., 1989; Wang et al., 1989; Ohtake et al., 1990a). However, unlike the organisms discussed earlier, the Cr(V1) reductase activity is located in the membrane fraction (Wang et al., 1990). Cr(V1) oxidizes both c- and b- type cytochromes in the membrane fraction, suggesting that these cytochromes are involved in electron transport to Cr(V1) (Wang e f ul., 1991). There was a higher ratio of c- to b-type cytochromes oxidized with Cr(V1) as compared with 0,. 0, also oxidized cytochrome d,,,, but Cr(V1) did not. Multiple b- and c-type cytochromes could be distinguished, but it appeared that one of the c-types, c C j x , might be specifically involved in Cr(V1) reduction, serving as a branch point between Cr(V1) and O 2reduction. A membrane protein(s) also appears to be important for Cr(V1) reduction by

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P. juorescens as removal of the membrane fraction with centrifugation removed the capacity for Cr(V1) reduction (Bopp and Ehrlich, 1988). NADH could serve as an electron donor for Cr(V1) reduction in crude extracts as could glucose. KCN ( l o - * M ) or NaN, ( l o - ? M ) inhibited Cr(V1) reduction. The NAD(P)Hdependent Cr(V1) reductase activity in Pseudomonas maltophilia was also membrane bound (Blake et al., 1993). A membrane fraction of a Streptomyces sp. reduced Cr(V1) with NADH as the electron donor (Das and Chandra, 1990). EPR spectra indicated that Cr(II1) was the product of the Cr(V1) reduction. The capacity for Cr(V1) reduction in cell suspensions of Agrobacterium radiobacter was related to the redox potential of the cell suspension as measured with a platinum electrode and the presence of intracellular polyglucose (Llovera et al., 1993). The more electronegative the measured redox potential, the greater the subsequent rate of Cr(V1) reduction. In most, but not all, instances the lower redox potential was associated with a greater accumulation of polyglucose in the cells. Addition of gramicidine, which can depolarize cell membranes, inhibited Cr(V1) reduction, suggesting that Cr(V1) reduction required an energized membrane.

C. MICROBIAL REDUCTIONOF Cr(VI) INSOILSAND BIOREMEDIATION Typical constituents of anoxic environments such as Fe(II), sulfides, and some organic compounds can nonenzymatically reduce Cr(V1) (Schroeder and Lee, 1975; Saleh etal., 1989; Bartlett, 1991; Palmer and Wittbrodt, 1991; Richard and Bourg, 1991). Thus, it seems unlikely that enzymatic Cr(V1) reduction by Cr(V1)reducing microorganisms is an important process for chromate reduction in such environments (Masscheleyn et al., 1992). Microbial Cr(V1) reduction could potentially contribute to Cr(V1) reduction in aerobic environments, but this does not appear to have been evaluated. The reduction of Cr(V1) to the less toxic, less mobile Cr(II1) is likely to be a useful process for the remediation of contaminated waters and soils (Palmer and Wittbrodt, 1991). The solubility of Cr(II1) hydroxide maintains chromium concentrations below the drinking water limit (10 - 6 M )at pH values between 6 and 12 (Rai et al., 1989). Although previous focus appears to have been on nonenzymatic mechanisms for Cr(V1) reduction (Palmer and Wittbrodt, 1991), a number of microbiological studies have suggested that enzymatic processes may also be useful in remediation of chromate contamination, especially in ex situ treatment of contaminated waters and waste streams. Most studies on microbial Cr(V1) reduction have mentioned the potential use of this metabolism for removing chromate from contaminated environments, but detailed studies on actual chromate removal have only been conducted with E. cloacae stain H01. Initial studies demonstrated that the Cr(II1) produced during

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Cr(V1) reduction in an organic-rich medium (Wang et al., 1989) could not be readily removed from solution with simple centrifugation (Komori et al., 1990b). Similar results have been observed with E. coli cultures (Shen and Wang, 1993). Cr(II1) removal with E. colacae could be improved by separating E. cloacae from the bulk of the Cr(V1)-containing medium with an an anion-exchange membrane that permitted passage of Cr(V1) but not Cr(II1) (Komori et al., 1990a). Cr0,diffused into the chamber containing E. cloacae and was retained as Cr(II1). Although E. cloacae could effectively reduce Cr(V1) in culture medium, difficulties were encountered in trying to reduce Cr(V1) in real chromate-contaminated waste streams. Other heavy metal contaminants and sulfate in the industrial materials inhibited Cr(V1) reduction (Ohtake et ul., 1990b; Hardoyo and Ohtake, 1991). It has been suggested that D. vulgaris might be better than E. cloacae for treating such materials because Cr(V1) reduction in this organism is less sensitive to heavy metals and is not inhibited by the presence of sulfate (Lovley and Phillips, 1994a). However, no tests with D. vulgaris on industrial effluents were conducted. Aeromonas dechromatica was reported to reduce Cr(V1) from industrial wastewaters (Kvasnikov et al., 1985), but the data on this are in a difficult to obtain Russian document. Further investigation is required before it will be possible to determine whether microbial Cr(V1) reduction has the potential to be a useful bioremediation tool.

VI. MICROBIAL REDUCTION OF OTHER METALS As reviewed by Lovley ( 1 993), microorganisms are also able to catalyze the reduction of other metals such as technetium, vanadium, molybdenum, copper, gold, and silver. However, investigations into these processes have been rather preliminary with little information of their potential significance in natural environments. Probably the most intensively studied form of microbial metal reduction is the reduction of soluble Hg(I1) to volatile Hg(0) that is carried out by aerobic microorganisms as a detoxification mechanism. In-depth reviews of the microbiology, biochemistry, and genetics of this process are already available (Robinson and Tuovinen, 1984; Brown, 1985; Summers and Barkay, 1989; Schiering et al., 1991; Silver and Walderhaug, 1992; Summers, 1992) and thus this topic is not covered here. Identifying the specific genetic determinants for Hg(I1) reduction has made molecular techniques possible in helping to evaluate the potential role of microbial Hg(I1) reduction in affecting the fate and mobility of mercury in soils and aquatic environments (Goldstein etal., 1988; Barkay et al., 1989, 1990, 1991; Olson et al., 1991; Rochelle et al., 1991). Manipulation of the Hg(I1)-reducing population is a potential strategy to stimulate volatilization and removal of mer-

2 16

D. R. LOVLEY

cury from contaminated environments (Goldstein et al., 1988; Barkay et al., 199 1 ; Ogunseitan and Olson, 1991).

VII. CONCLUSIONS Microorganisms can enzymatically catalyze the reduction of a large number of metals. For abundant metals such as Fe(II1) and Mn(IV), microorganisms have evolved specific metabolic systems which permit them to conserve energy to support growth by coupling the oxidation of organic matter to metal reduction. Microbial Fe(II1) reduction accounts for most of the Fe(II1) reduction in many anoxic soils and aquatic sediments. Nonenzymatic processes such as the reduction of Fe(II1) by organic compounds and sulfide are generally of minor significance. Mn(1V) reduction is more susceptible to reduction by nonenzymatic processes, but enzymatic Mn(1V) reduction does predominate in some environments. Fe(II1) and Mn(IV) reduction are major processes for decomposition of naturally occurring organic matter in some soils and sediments and can play an important role in the degradation of organic contaminants. The microbial reduction of insoluble Fe(II1) and Mn(1V) oxides increases the solubility of these metals, releases nutrients and metals that were adsorbed on the oxides, leads to the formation of new Fe(I1)- and Mn(I1)-containing minerals, and alters a variety of soil properties. These changes can be both beneficial and detrimental to plant growth. Many Fe(II1)- and Mn(1V)-reducing microorganisms can also reduce highly soluble U(V1) to insoluble U(1V). Microbial U(V1) reduction provides a likely explanation for the reductive precipitation of uranium in anoxic soils and sediments. Furthermore, U(V1)-reducing microorganisms might be useful agents for the bioremediation of uranium-contaminated environments. In a similar manner, the microbial reduction of soluble selenate to insoluble Se' removes selenium from water and this metabolism can be employed to immobilize selenium in environments with high selenium concentrations. Microorganisms can reduce highly soluble and toxic Cr(V1) to Cr(III), which is less soluble and less toxic. However, the environmental significance of this metabolism and its utility as a bioremediation technique have yet to be clearly demonstrated. Some microorganisms can enzymatically reduce other metals such as mercury, vanadium, molybdenum, copper, gold, silver, and technetium. This metabolism may affect the fate and mobility of these metals. Although recent studies have demonstrated the importance of microbial metal reduction and have identified organisms which may serve as models for this metabolism, very little is known about the biochemistry of this process. There is little information on the organisms that are responsible for enzymatic metal reduction

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in natural environments. It is not clear whether the metal-reducing microorganisms that are available in pure culture are representative of the important metal reducers in soils and sediments. Further studies on the biochemistry and microbial ecology of metal reduction would enhance our understanding of the factors controlling the rate and extent of this important process.

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Chapelle, F. H., and Lovley, D. R. 1992. Competitive exclusion of sulfate reduction by Fe(I1I)reducing bacteria: A mechanism for producing discrete zones of high-iron ground water. Ground Water 30,29-36. Coates, J. D., Lovley, D. R., and Lonergan, D. J. 1994. Geobncter hydrogmophilia. a dissimilatory Fe(II1) reducer capable of oxidizing HZand formate as well as acetate. Submitted for publication. Cochran, J. K., Carey, A. E., Sholkovitz, E. R., and Surprenant, L. D. 1986. The geochemistry of uranium and thorium in coastal marine sediments and sediment pore waters. Geochinz. Costnochim. Actn 50,663-680. Coleman, M. L., Hedrick, D. B., Lovley, D. R., White, D. C., and Pye, K. 1993. Reduction of Fe(II1) in sediments by sulphate-reducing bacteria. Nriture 361,436-438.

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NITRIFICATION INHIBITORS FOR AGRICULTURE, HEALTH, AND THE ENVIRONMENT Rajendra Prasad and J. F. Power2 I Division of Agronomy Indian Agricultural Research Institute New Delhi, India *UnitedStates Department of Agriculme Agricultural Research Service University of Nebraska Lincoln, Nebraska 68583

I. Introduction II. Nitrification Inhibitors (NIs) A. Relative Effectiveness B. Soil Factors Affecting Effectiveness of NIs C. NIs and Nitrogen Losses and Immobilization m. NIs, NH,+/NO,- Ratios, and Plant Growth

IV.NIs and Crop Yields

A. Rice B. Corn C. Grain Sorghum D. Wheat E. Sugarcane F. Potato G. Cotton V. Phytotoxicity of NIs VI. Health and Nitrates A. Nitrates and Human Health B. Nitrates and Animal Health C. Nitrate Content of Drinking Water D. NIs and Nitrate Content in Vegetables VII. NIs and Environment A. NIs and Nitrate in Groundwater B. Ozone Layer Depletion C. Global Warming References

233 Aduances in Agranamny. Volume H Copyright Q 1995 by Academic Press,Inc. All righu of reproduction in any form reserved.

234

RAJENDRA PRASAD AND J. F. POWER

I. INTRODUCTION Nitrification inhibitors (NIs) emerged as a group of agrichemicals with the development of N-Serve [2-chloro-6(trichloromethyl)pyridine](Dow Elanco trade name for nitrapyrin) by Goring ( 1962ab), although inhibition of nitrification by a number of herbicides, insecticides, nematicides, and fungicides were known long before. Except for a few field experiments, research on nitrification inhibitors during the 1960s was mostly restricted to laboratory studies (Prasad el al., 1971). Intensive field investigations were carried out in the late 1960s and 197Os, and the American Society of Agronomy, the Crop Science Society of America, and the Soil Science Society of America jointly sponsored a symposium on December 6, 1978, at Chicago, Illinois, the proceedings of which were published in 1980 (Meisinger et al., 1980). Three years later a technical workshop on the nitrification inhibitor dicyandiamide (DCD) was held on December 4-5, 1981, in Muscle Shoals, Alabama; this workshop was jointly sponsored by the National Fertilizer Development Center, the Tennessee Valley Authority at Muscle Shoals, Alabama; the International Fertilizer Development Center, Muscle Shoals, Alabama; and SKW Trostberg AG, West Germany (Hauck and Behnke, 1981). A second workshop on DCD was held on December 4-5, 1987, at Atlanta, Georgia, and the proceedings were published as a special issue in Communications in Soil Science and Plant Analysis (Vol. 20, Nos 18 and 19, 1989) (Hauck et al., 1989). In addition to specific chemicals such as nitrapyrin or DCD, natural products like those from neem (Azadirachta indica Juss) are reported to have nitrificationinhibiting properties (Reddy and Prasad, 1975; Sahrawat and Parmar, 1975) and have been widely evaluated in India. Prasad et al. (1993) addressed the N use efficiency aspects of urea coated with neem cake and other neem products at the Neem World Conference held at Bangalore, India (February 24-28, 1993). An ideal nitrification inhibitor should be mobile, persistent, and, above all, economic in use (Hauck, 1972). It should also be nontoxic to other soil organisms, animals, and humans and should move with the fertilizer or nutrient solution. Compounds with high vapor pressure may move fast and compounds easily absorbed may not be very effective. An ideal NI should stay effective in soil for an adequate time period; at least for the growth period of a crop. Above all, the real testing ground is in the economics of use; most studies indicate that about a 0.3 to 0.5 mg ha - ' yield increase will pay for the cost. This one factor alone has stopped many nitrification inhibitors from reaching the farm level. The major goal in using a NI is to increase the efficiency of fertilizer N applied to agricultural/horticultural crops by reducing nitrate leaching losses as well as nitrification losses as N,O or N,. Thus ideal situations where NIs are likely to be the most effective are those where such losses predominate, such as rice paddies, areas receiving heavy precipitation, irrigated areas (especially furrow) because of leaching, and crops receiving high rates of N fertilization or manures.

NITRIFICATION INHIBITORS

235

During the 1980s there was considerable effort by ecologists, environmentalists, and some agriculturists to reduce fertilizer N use on the farm, mainly due to its likely role in increasing nitrate concentrations in groundwater and because N fertilizers are manufactured from a nonrenewable natural resource (natural gas). However, on a global scale this will neither be possible nor desirable if we are to feed the increasing world population. The available estimates indicate that 2422 Tg of cereals will be required in 2000 AD (Prasad, 1986) compared to the 1991 estimated production of 1884 Tg of cereals (FAO, 1991). Thus an additional 28.5% of cereals will have to be produced in the next decade, most of it in the developing countries. While this increase in cereal production can be achieved in most African and South American countries by bringing more land under cultivation, Asia has done it by increasing productivity per unit land per unit time. This calls for a sizable increase in the consumption of fertilizer, especially nitrogen. It is estimated by 2000 AD that 145.4 Tg of fertilizer N will be consumed annually (UNIDO, 1978), which is nearly double the 1990-1991 consumption of 77 Tg of fertilizer N (FAO, 1991). Furthermore, a large number of the developing countries, especially those in south and southeast Asia, grow rice as a principal crop, the crop for which fertilizer N losses are greatest (Prasad and De Datta, 1979; Fillery and Vlek, 1986; Reddy and Patrick, 1986; De Datta, 1986). In addition to the large amounts of fertilizer N needed in the developing countries, high costs involved in their production or purchase also need to be considered. Also the sustainability of synthetic fertilizer production from natural gas at some time in the future is a concern. Thus, efficient use of fertilizer N is necessary, suggesting that nitrification inhibitors have a role to play. This chapter provides an overview of the literature available on the use of nitrification inhibitors in relation to production and quality of agricultural and horticultural crops, human and animal health, and the environment.

11. NITRIFICATION INHIBITORS A fairly large number of chemicals have been reported as nitrification inhibitors: Nitrapyrin (abbreviated as NP in this chapter) or N-Serve [2-chloro-6-(trichloromethyl)pyridine] (Goring 1962ab); AM (2-amino-4-chloro-6-methylpyrimidine) (Toyo Koatsu Industries, 1965); DCD (Amberger and Guster, 1978); terrazole or Dwell or etridiazole (5-ethoxy-3-trichloromethyl- 1,2,bthiadiazole) (Olin Corp., I976ab); DCS [N-(2,5-dichlorophenyl)succinamicacid] (Namioka and Komaki, 1975ab); KN? (potassium azide) (Hughes and Welch, 1970); ATC (4-amino- 1,2,4-triazole) (Guthrie and Bomke, 1980); TU (thiourea); MBT (2mercaptobenzothiazole); 2-ethynyl pyridine (McCarty and Bremner, 1986); MPC (3-methyl-pyrazole-I-carboxamide) (McCarty and Bremner, 1990); ST (2-sulfanil-amido thiazole) (Mitsui Toatsu, 1968); CS, (Ashworth et ul., 1977); 2-mer-

236

RAJENDRA PRASAD AND J. F. POWER

capto- 1,2,4-triazole, sodium diethylthiocarbamate; 2,5-dichloroaniline; 4-amino1,2,4-triazole(Bundy and Bremner, 1973);C2H,(acetylene) (Hynes and Knowles, 1981; Berg et al., 1982); gaseous hydrocarbons such as C,H, (ethane), C,H, (ethylene), and CH, (methane) (McCarty and Bremner, 1991); ammonium thiosulfate (Goos, 1985); and thiophosphoryl triamide (Radel er al., 1992). Of these, only eight (NP, AM, DCD, ST, TU, Dwell, MBT, and C2H,) have been widely tested. In addition to specific chemicals, allelochemicals also have nitrification-inhibiting properties. For example, Rice ( 1984) postulated that because inhibition of nitrification results in conservation of both energy and nitrogen, vegetation in late succession or climax ecosystems contains plants that release allelochemicals that inhibit nitrification in soil. However, a critical appraisal of the available information does not lend support to such a hypothesis (Bremner and McCarty, 1993). As an example, terpenoids thought to be released by a ponderosa pine (Pinusponderosu Dougl.) and supposed to inhibit nitrification in soil had no such effects (Bremner and McCarty, 1988). However, some natural products are reported as nitrification inhibitors. These include “neem” (A. indica Juss.) cake or an acetone/alcohol extract of seed (Reddy and Prasad, 1975; Sahrawat and Parmar, 1975) and “karanj” (Pongarnia glabra Vent.) seed, bark, and leaves (Sahrawat et a!., 1974).

A.

RELATIVE EFFECTIVENESS OF M S

Rajale and Prasad (1970) found AM as effective as NP, while Bundy and Bremner (1973, 1974) found that AM was less effective than NP and DCD. Sommer (1970) compared a number of NIs and ranked them in the following order: Terrazole > NP > DCS > guanylthiourea > AM > MAST (2-amino-4-methyl6-trichloromethyltraizine) > ST. McCarty and Bremner (1989) compared 12 compounds and found 6 of them to be effective NIs: 2-ethynylpyridine > Dwell > NP > MPC > ATC > DCD (Table I). In a number of U.S. studies NP and DCD were found to be equally effective. In their studies in Illinois, Malzer et al. (1989) at Urbana, Monmouth (Typic Haplaquolls), and Dekalb (Aquic Arguidoll), showed that the disappearance of ammonium was similar between DCD ( 5 % DCD-N) and NP (0.5 kg ha ’). At Brownston (Mollic Albaqualf), however, ammonium disappearance was slower with DCD than with NP. Bronson et al. (1989) from Alabama reported that DCD in Norfolk loamy sand (Typic Paleudult) was equal to NP for up to 42 days, but was less effective than NP in Decatur silt loam (Rhodic Paleudult). Etridiazole and NP are equally effective in reducing nitrification of ammonium N in soils up to 160 days after application on silty loam soils (Typic Ochraqualfs and Aquic Hapludalfs) in Illinois (Shyilon et al., 1984). Blending of urea with neem cake inhibited nitrification by 70, 40, and 5% at ~

237

NITRIFICATION INHIBITORS Table I Effects of 5 mg kg-' Soil with Different Compounds on Nitrification of Ammonium in Soils" Soil Compound

Harps

2-Ethynylpyridine Etridiazole (Dwell) Nitrapyrin (N-Serve) 3-Methylpyrazole- I -carboxamide 4-Amino- I ,2,4-triazole Dicyandiamide Potassium azide N-(2,5-Dichlorophenyl)succinamide Sodium thiocarbonate Thiourea 2-Mercaptobenzothiazole Ammonium thiosulfate

79 61 45 43 41 8 0 0 0 0 0 0

Webster

Storden

g%, inhibition

of nitrification 80 I00 70 97 56 94 53 93 52 92 41 20 3 5 2 5 0 0 0 0 0 0 0 0

"Samples of soil (20 g) were incubated at 25°C for 25 days after treatment with 6 ml water containing 4 mg N as ammonium sulfate and 0 or 100 y g of the compound specified. Adapted from McCarty and Bremner (1989).

the end of 1,2, and 3 weeks of incubation, respectively; the corresponding figures for NP at 1% of N were 85,93, and 90% (Reddy and Prasad, 1975). Thomas and Prasad (1982) evaluated neem cake-coated urea on a number of soils (Entisols, Vertisols, Ultisols) and found it to be 50% as effective as NP. The active compounds in neem responsible for retardation of nitrification are thought to be meliacins (epinimbin, nimbin, desacetyl nimbin, salanin, desacetylsalanin, and azadirachtin) (Devkumar, 1986). Nitrification retardation after 2 weeks was 73.6,44.6, and 12.5% for NP, epinimbin, and desacetylnimbin, respectively (Devkumar and Goswami, 1992). Neem cake and DCD were evaluated for their efficiency in inhibiting nitrification of prilled urea-derived NH,+-N in a wheat field (Joseph and Prasad, 1993a,b). Prilled urea was blended with 10 and 20% DCD-N or with 10 and 20% neem cake and incorporated into the soil just before the wheat was sown. Both DCD and neem cake partially inhibited the nitrification of prilled urea-derived NH,; DCD was better than neem cake. The nitrification-inhibiting effects of DCD lasted for 45 days, while that of neem cake lasted for only 30 days. Most NIs inhibit nitrification by retarding the oxidation of NH,+-N to NO,--N by Nitrosomonas sp. Research with different strains of Nirrosomonas

RAJENDRA PRASAD AND J. F. POWER

238 40

30

-

1

-

I

0 0

3

6

9

1

2

0

3

6 9 1 D A Y S

2

0

3

6

9

1

2

Figure 1. Effect of dicyandiamide (DCD),nitrapyrin (NP), and thiourea (TU)on the activity of from Zacheri and Ainberger ( 1990).

Nitrosomonas eurupoeo in pure culture. Adapted

sp. showed remarkable differences in sensitivity to nitrapyrin (Belser and Schmidt, 1981), and it was concluded that NP does not retard the activity of the entire population of Nitrosomonas sp. Results of a study done by Zacheri and Amberger (1990) on the effect of three NIs are shown in Fig. 1. Growth of a pure culture N . europaea was completely suppressed by 10 ppm NP or 0.5 ppm TU; inhibition by 300 ppm DCD was 83%. Ammonium oxidation and respiration of Nitrosomonas cell suspensions were reduced by 93% with 10 ppm NP, 95% with 0.5 ppm TU, and 73% with 300 ppm DCD. When used at 1000 ppm, DCD had bacteriostatic effects. Enzymatic investigations revealed that hydroxylamine oxidoreductase was not affected by high concentrations of inhibitors (200 ppm DCD, 100 ppm TU). Cytochrome oxidase activity was increased 10% with 200 ppm DCD, was not affected by 100 ppm TU, and was inhibited by 52% with 100 ppm NP. These results suggest that different NIs probably have different modes of action.

B. SOILFACTORS AFFECTING EFFECTIVENESS OF N I S A number of studies have investigated the effect of different soil factors on the effectiveness of NIs, and this subject has been well reviewed by Slangen and Kerkhoff (1984). The main findings are summarized below.

1. Organic Matter Hendrickson and Keeney ( 1979b) found complete inhibition of nitrification with NP at 0.5 mg kg - I in a soil with 1% organic matter and none in the same soil when organic matter was raised to 5% by adding active carbon. Similar results were obtained by McClung and Wolf (1980) with NP and terrazole when they

239

NITRIFICATION INHIBITORS

added compost to the soil. The influence of organic matter is probably due to its effect on sorption and rate of decomposition of the chemical.

2. Temperature Most reports suggest that nitrification inhibitors are more effective at relatively low temperatures, i.e., below 20°C (Goring, 1962a; Bundy and Bremner, 1973). This is mainly due to the effect of temperature on degradation of a NI and the consequent persistence. Herlihy and Quirke (1975) found that the half-life of NP was 43 to 77 days at 10"C and 9 to 16 days at 20°C. Touchton et al. (1979) found the half-life of NP to be 22 days at 4" C and less than 13 days at 2 1" C for a loamy soil with pH 6.8 and an organic matter content of 2%. In a soil with pH 5.5 and an organic matter content of 5%,the half-life of NP was 92,44, and 22 days at 4, 13, and 2 I " C, respectively. Touchton ef a/. ( 1979) reported that the half-life of NP in a Cisne silt loam was 7 days at 2 1"C and 22 days at 8" C. DCD is highly sensitive to temperature. Vilsmeier (1980) reported that after 60 days, 0.67 mg DCD-N 100 g I soil degraded to 0.60 mg at 8" C, 0.4 at 14" C, and 0.1 mg at 20" C in a sandy silt loam soil of Germany with a pH of 6.2. Bronson et a/. ( 1989) found that the half-life of DCD decreased from 52.2 days at 8" C to 22 days at 22°C in Norfolk loamy sand and from 25.8 days at 8°C to 7.4 days at 22" C in Decatur soils. Data of McCarty and Bremner (1989) for Iowa soils showed that in 28 days inhibition of nitrification decreased from 72% at 15"C to 19% at 30°C in Harps silty clay soil when DCD was added at 10 mg kg - I soil (Table 11). At 30°C the inhibitory effect at 10 mg kg - ' of soil with etridiazole exceeded that at 100 mg kg soil with DCD and the inhibitory effect of 10 mg ~

~

Table I1 Influence of Soil Temperature on Effectiveness of Dicyandiamide (DCD) for Inhibition of Nitrification of Ammonium in Soils"

Soil

Amount of DCD added (mg kg ' soil) ~

Soil temperature ("C) 15

20

25

30

% inhibition of nitrification

Harps

10

50 Webster

10

50 Storden

10

50

72 83 78 85 90 97

60 82 65 84 75 94

48 72 51 13 53 89

19 49 25 62 23 81

"Samples of soil (20 g) were incubated at 15, 20, or 30°C for 28 days after treatment with 6 ml water containing 4 mg N as ammonium sulfate and 0.0.2, or I .O mg of DCD. Adapted from McCarty and Bremner (1989).

2 40

RAJENDRA PRASAD AND J. F. POWER

k g - ' soil with NP exceeded that at 50 mg kg - I soil with DCD (McCarty and Bremner, 1989). In Illinois, DCD and NP were equally effective on Drummer silty clay loam at 7.2"C. but DCD was more effective than NP at 15.5"C (Sawyer, 1985).

3. pH The influence of soil pH on the persistence of NP is reported to be minimal (Hendrickson and Keeney, 1979a). This can be expected since a number of genes of nitrifying organisms are involved in nitrification, each with different pH optima (Bhuija and Walker, 1977).

4. Soil Water Hydrolysis of NP is enhanced in water-saturated soils (Hendrickson and Keeney, 1979a) as compared to aerobic conditions in soils at field capacity (0.01 to 0.033 M Pa). Volatilization of NP is more pronounced in wet than in dry soils (McCall and Swann, 1978). In addition to these factors, method and time of fertilizer application and source of N used can affect the effectiveness of NIs under field conditions (Singh and Prasad, 1985; Sudhakara and Prasad, 1986a; Thomas and Prasad, 1987).

c. N

S AND NITROGENLOSSES AND IMMOBILIZATION

1. Urea Hydrolysis Most of the NIs, such as NP, AM, ST, ATC, KN3, CS,, and DCD, have little effect on urea hydrolysis. However, TU, ammonium, and potassium ethylxanthate and thiosulfate retard urea hydrolysis (Mahli and Nyborg, 1979; Goos, 1985; Ashworth ef al., 1980).

2. Ammonia Volatilization Since NIs retard nitrification, ammonium-N can accumulate and result in a higher soil pH (Bundy and Bremner, 1974), which is conducive to NH3 volatilization. Enhanced NH3 volatilization losses due to application of NI have been reported (Bundy and Bremner, 1974; Smith and Chalk, 1978; Prakasa Rao and Puttanna, 1987). While Bundy and Bremner (1974) reported a 28-34% N loss from volatilization of added urea N with a NI (NP, ATC, CL- 1850) and 9% without, Smith and Chalk (1978) found NH3-N losses of 86 and 92 mg kg I soil without and with NP. High NH3-N losses reported by Bundy and Bremner (1974) could ~

NITRIFICATION INHIBITORS

241

be due to high rates of N applied (400 mg N kg - I soil). Volatilization losses of NH, with or without NI can be reduced by incorporation of the fertilizer N. Clay et al. (1990) reported that NH, volatilization from bare soil was lower with urea and DCD than with untreated urea. However, when the soil surface was covered with residue, NH, volatilization was similar with or without DCD. Sudhakara and Prasad (1 986a) reported that when 120 kg N ha ' was applied 20 days after sowing rice, the NH, volatilization loss was 8.37% of the applied N from urea compared to 3.89% with neem cake-coated urea. Thus at rates of N generally applied in field crops, an increase in NH, volatilization due to NIs can be considerably reduced by the incorporation of fertilizer N and NIs in soil. Another possibility for reducing NH, volatilization is the use of dual purpose (NIIurease inhibitor) compounds such as thiophosphoryl triamide (Radel et ai., 1992). -

3. Denitrification By retarding nitrification, NIs slow down and reduce the potential for N loss by denitrification as N,O or NZ;this, however, should not be confused with the reduction of denitrification per se. For example, some workers reported that NIs (NP, DCD, NaN,, Dwell, KN3, ST, PM, ATC) directly retard denitrification (Mitsui et ul., 1964; Henninger and Bollag, 1976; McElhannon and Mills, 1981), especially when added at the rate of 50 or 100 mg kg ~I soil (Bremner and Yeomans, 1986). Such rates are too high for general applications to field crops. Bremner and Yeomans (1986) evaluated the effect of 28 NIs and found that only KN, and 2,4-diamino-6-trichloromethyl-Striazine, when added at the rate of 50 mg kg ~I soil, inhibited denitrification. The other NIs had no appreciable effect on denitrification.

4. Nitrogen Losses from Plants Plants also lose some amount of N from the foliage (Wetselaar and Farquhar, 1980; Patron et al.. 1988; Francis et al., 1993). A high loss of N was observed by Daiger e t a / . (1976) from winter wheat at different locations in western Nebraska, following different rates of N application. In general, dry matter and N content of tops and roots reached a maximum at anthesis. Thereafter, dry matter declined by about lo%, while losses of N from the tops plus roots ranged from about 20 to over 60% depending on the fertilizer N rate. Tanaka and Navasero (1964) reported a loss of 47 kg ha I in the N content of rice tops in the 3 weeks before flowering and maturity at high N rates. Patron et ai. (1988) reported a NH, loss of 60120 ng N m - > sec - I from spring wheat plants during the presenescence time period (before milk stage) and 200 to 300 ng m - ? sec ~I during final plant senescence. They found that NH, loss rates on a leaf area basis were similar for the low and high N plants despite significantly higher N concentrations in high N plants. -

2 42

RAJENDRA PRASAD AND J. F. POWER

Twice the leaf area was attained by the high N plants, resulting in similar NH, volatilization rates per plant which translates into nearly twice as high on a plant N basis for the low N plants. Farquhar et al. (1979) reported an evolution of 0.6 nmol m -,sec - I (36 g N ha I day I at LA1 5) from senescing leaves of corn. In a study at Lincoln, Nebraska, postanthesis fertilizer N losses as NH, from the aboveground biomass of corn plants ranged from 10 to 25% of the fertilizer applied (Francis et al., 1993); the apparent total N losses from the aboveground plant material ranged from 49 to 81 kg N ha-'. Francis er al. (1993) observed that postanthesis N losses from aboveground plant biomass in corn accounted for 52 to 73% of the total unaccounted for fertilizer N and suggested that failure to include such losses can lead to overestimation of N losses from soil by denitrification and leaching. Mosier er al. (1990a,b) reported a N, + N,O gas flux of 270 g N ha - I day ~I 15 days after transplanting rice where plants were included in the measuring chamber as compared to only 240 g N ha - I day - I when the plants were not included in the chamber. They concluded that young rice plants facilitated the efflux of N, and N,O from the soil to the atmosphere. Effects of NI on such losses of N from the plants have so far not been reported. ~

~

5. Immobilization Immobilization of fertilizer N by soil microorganisms is significantly enhanced in the presence of a nitrification inhibitor (Osiname et al., 1983; Juma and Paul, 1983). This has been attributed to the NIs maintaining more of the applied fertilizer N as NH,+ for a longer period of time (Prasad et al., 1983; Shyilon et al., 1984; Norman and Wells, 1989) and preferential utilization of NH,+-N by heterotrophic microorganisms (Broadbent and Tyler, 1962; Alexander, 1977). Bjarnason (1987) reported that not only is NH,' preferentially immobilized, but its remineralization is at a slower rate. Norman and Wells (1 989) found that immobilization of fertilizer N by soil microorganisms in a Crowley silt loam (Typic Albaquelf) in Arkansas was approximately the same in urea and urea DCD-amended soils during the 4-week period when the soils were not flooded (Fig. 2 ) . Immobilization appeared to level off after 2 weeks and stayed relatively constant for the remaining 2 weeks. After flooding, immobilization of fertilizer N was much greater in the urea + DCD-amended soil than in the urea-amended soil, and by the end of 8 weeks soil with urea DCD had nearly 1.5 times more NH,+ than that treated with urea only. Osiname et al. (1983) reported more N immobilization with NP than with DCD. Preferential immobilization of NH,+-N rather than NO,- -N has been suggested by a number of researchers (Wickramsingha et al., 1985; Rice and Tiedje, 1989). This would support higher immobilization of fertilizer N with NIs. Budot and Chone (1985) suggested an interesting pathway of nitrite incorpo-

+

+

243

NITRIFICATION INHIBITORS FLOODED

NONFLOODED

LSD 0.05=4.83mglkg

0

1

2

3

4

5

6

7

8

9

1011

INCUBATION TIME ( w e e k s )

Figure 2. Immobilization of fertilizer N under nonflooded and flooded conditions. Adapted from Urea; (A)urea + DCD. Norman and Wells (1989). (0)

ration into the organic N fraction via nitrite self decomposition and fixation on organic matter in a humic-rich acid forest soil (pH 4.5; organic matter 46%). Azhar et al. (1986a,b,c) also supported this pathway. NP not only reduced the loss of nitrite via chemodinitrification, but Nelson ( 1982) also discussed the incorporation of nitrite into the organic N fraction. Thus it appears that NIs increase immobilization of N by increasing the persistence of ammonium-N. Also, NIs retard nitrite accumulation in soils and thus reduce fixation of nitrites into organic matter.

III. NIs, NH,+/NO,- RATIOS, AND PLANT GROWTH Because NIs maintain a higher concentration of NH,' in the soil/solution for a longer time by retarding nitrification, these chemicals have a role in determining amounts of NH,+ and NO,- and their ratios to crop plants during different stages of crop growth. Ammonium can be more efficiently metabolized than NO, -N because it does not need to be reduced when incorporated into amino acids or other organic materials. However, NH,', or rather NH;', is toxic to all plants at certain concentrations (Magalhaes and Wilcox, 1984) and this toxicity is related to the pH of the growing media (Pill and Lambeth, 1977). Magalhaes and Huber (1989) reported that NH,' toxicity was more severe at lower (3.5) than at higher pH (5.7). There is also a difference between crops with respect to tolerance to NH,'. Prasad et al. ~

244

RAJENDRA PRASAD AND J. F. POWER

I

1

MAIZE

2

3

1

2

3

b

WEEKS AFTER FERTILIZER APPLICATION

Figure 3. Ammonium-N and nitrate-N concentration in maize and rice soils. 0,without nitranitrate-N. Adapted from Prasad ef al. (1983). pyrin; 0,with nitrapyrin; -, ammonium-N; -.-,

(1983) suggested the term "ammoniphilic plants" for species growing better with NH,'. They maintained high concentrations (40-60 mg kg I NH,+-N soil) using NP (Fig. 3) and found that while maize plants suffered in growth, rice plants did not (Table 111). Rice absorbed more N with NH4+,while maize absorbed less N in the presence of higher concentrations of NH,'. They identified rice as an ammoniphilic plant. Other species of ammoniphilic plants are known (Gigon and ~

Table 111 Plant Height and Dry Matter Accumulation in Rice and Maize Plants Affected by N-Serve (NP) Treatment" ~~~

~

Plant height (cm)

Dry matter (g per plant)

Treatment

Rice

Maize

Rice

Maize

Without N-serve With N-serve LSD ( P= 0.05)

17.1 18.2 0.66

32.9 18.6 3.47

0.24 0.26 0.023

1.45 0.90 0.13

"Adapted from Prasad e t a / . (1983).

NITRIFICATION INHIBITORS

245

Rorison, 1972; Ingestad, 1976). In view of the growing concern over nitrate pollution of groundwater, there is a need for research on high-yielding ammoniphilic cultivars of upland crop species. Leaving aside the case of very high NH,+ concentrations resulting in toxicity, Olsen (1986) cited several studies where the addition of NH,' to an all NO,system resulted in increased corn yields. Hageman ( 1 980, 1984) reviewed the effects of NH,' and NO,- nitrogen nutrition on plant growth and cited several experiments indicating that higher crop yields were obtained with a mixture of NO, and NH,' than with either source alone. Ganmore-Neumann and Kafkafi (1983), working with nutrient solutions varying in NO,- and NH,' concentrations, obtained optimal growth for strawberries with an equal ratio of NO,- to NH,'. Bock (1987) observed a 19 to 47% increase in wheat yield with basal NO,--N + urea + nitrapyrin compared to NO,--N alone. In two greenhouse studies, Camberato and Bock (1989) reported a 15- 18% increase in the grain yield of sorghum when a higher NH,' concentration was obtained using urea and NP. Under field conditions Israeli et ul. (1985) obtained a maximum yield of bananas when equal ratios of NH,' to NO,- were present in the soil extracts. Bock (1986) found that nutrient solution culture studies differed from those obtained under field conditions. Also, crop variety and stage of growth should be taken into account for optimal utilization of the NH,+/NO,- ratio. Cosgrove et al. (1985), working with snapbeans, found that the NO,- to NH,' ratio is critical for maximum yields. Teyker and Hobbs (1992) reported that with coarse-textured soils and slightly alkaline pH, an enhanced NH,' regime may be advantageous for the growth of corn. They also observed that the differences in pH regimes between the hydroponic and soil-based experiments may account for the contrasting results. In a study at Illinois (Gentry and Below, 1992), a continuous supply of mixed NO,--N and NH,+-N increased corn yield by an average of 12% compared to NO,-N alone. Shaviv er al. (1987) reported on the basis of pot culture experiments that wheat and millet (Setaria italica) exposed to NH,' only with DCD produced lower yields than those exposed to a mixture of NH,' and NO,-- with DCD. In wheat, NH,' to NO,- ratios of 50/50 and 75/25 seem to be optimal. A 25/75 NH,+/ NO,- ratio produced the highest yield at maturity. Calcium and Mg2' uptake by wheat and Mg2+uptake by millet were reduced as the proportions of NH,+ in soil were increased. In the studies of Diest (1976) and Gashaw and Mugwira (1981). maximum growth of wheat was obtained with a solution culture of 50: 50 proportion of NH,+ and NO,-. Based on data from a field study using DCD, Joseph (1992) and Joseph and Prasad ( 1993a,b) reported that the optimum concentration of NH,+-N in soil for maximum grain yield of wheat gradually decreased with the age of the crop from 54.6 to 63.6 mg kg - I soil at 15 days after sowing (DAS) to 22.7 to 26 mg k g - - ' soil at 30 DAS. In the case of NO,--N, its optimum -

2 46

RAJENDRA PRASAD AND J. I;. POWER

concentration for maximum grain yield increased with the age of the crop from 25.1 to 30 mg kg-I soil at 15 DAS to 31.6 to 34 mg kg-I soil at 45 DAS and decreased thereafter. Tsai et a/. ( 1 978) found that a greater amount of sucrose in corn (as measured by I4C)was translocated from leaves to grain under NH,+-rich conditions, resulting in higher grain yield. Warren et al. (1975) found a reduction in “stalk rot” incidence and increased yield of corn when N was kept as NH,’ for a longer period with the help of NP. As compared to NO3-, the assimilation of NH,’ in plants is not as well understood. According to Ivanko and Inguerson (1971) and Raven and Smith (1976), NH,’ is almost completely converted to organic N in roots prior to translocation. Ammonium can be assimilated either through reductive amination of aketoglutarate with the glutamine dehydrogenase enzyme (GDH) system or by incorporation into glutamate with glutamine synthetase (GS) and subsequent transfer of the amide amino group of glutamine to a-ketoglutamate with glutamate synthetase (GOGAT) (Givan, 1979; Milfin and Lea, 1976; Srivastava and Singh, 1987). Although increased activity of these enzymes does not necessarily indicate their role in assimilation, increased GDH in the presence of NH,+ has been reported in roots of pea, pumpkin, soybean, sunflower, and corn (Weisman, 1972; Magalhaes and Huber, 1989). GS activity in roots and shoots of rice is reported to be higher than in the tissues of tomato and corn; in rice it increased sharply in the presence of NH,+ (Magalhaes and Huber, 1989). The NH,+/NO,- ratios and plant growth studies lead to the following conclusions: (1) The growth of most upland crop plants is best when both NH,’ and NO3- forms of N are available for absorption; their relative amounts and ratios vary with species, cultivars and age of plant; and (2) NIs can help in maintaining NH,’ in soil in larger amounts and for longer periods of plant growth.

IV. NIs AND CROP YIELDS Experiments with NIs have been conducted with a fairly large number of crops, including rice (Oryza sativa L.), corn (Zea mays L.), wheat (Triticurn aestivum L.), grain sorghum (Sorghum bicolor L. Moench), sweet corn (Zea mays L. Rigosa), sugarcane (Saccharum oficinarum L.), bell pepper (Capsicum annum L.), potato (Solanurn tuberosum L.), tomato (Lycopersicon esculentum Mill.), cotton (Gossypium hirsutum L.), barley (Hordeum vulgare L.), oat (Avena sativa L.), sugarbeet (Beta vulgaris L.), spinach (Spinacia oleracea L.), lettuce (Latuca saliva L. var. Capitata L.), radish (Raphanus sativus L. var. radicula Pers.), cucumber (Cucumis safivus L.), cabbage [Brassica oleracea convar. Capitata (L) Alef var. Alba DC], endive (Cichorium endivia L), turnip (Brassica rapa L.), and sev-

NITRIFICATION INHIBITORS

247

era1 grasses, including Lolium prenne L., Dactylis glomerata L., and Kentucky bluegrass (Poa pmtensis L.) (Slangen and Kerkhoff, 1984; Waddington et al., 1989). This chapter is restricted to major food and fiber crops of the world: rice, corn, wheat, grain sorghum, potato, sugarcane, and cotton.

A.

RICE

The wet conditions that exist during rice production and the preference of rice for NH,+-N over NO,-N suggest that the application of NIs with NH4+-or NH,+-producing fertilizers, such as urea, would be a sound N management practice. Prasad et al. (1986) suggested the use of NP for increasing N efficiency in rice. Field experiments were conducted with NP, AM, and ST (Lakhdive and Prasad, 1970; Reddy and Prasad, 1977) and these clearly showed that on rice soils with high percolation rates, nitrification inhibitors can be usefully employed for increased rice yields and N efficiency. Nitrification inhibitors were specifically effective in reducing N losses under alternate wetting and drying conditions frequently encountered in rice fields (Rajale and Prasad, 1972). Thomas and Prasad (1987) reported that for direct-seeded rice, NP-blended urea produced 4.7 mg grain ha ' compared to 3.7 mg ha I with urea. However, under similar conditions, DCD showed no advantage (Sudhakara and Prasad, 1986b). Results from experiments conducted at different centers in Japan showed that ammonium sulfate treated with NP increased rice yield by 15-20% over untreated ammonium sulfate (Nishihara and Tsunyoshi, 1968). Similarly, in field tests carried out with AM in Japan, they showed that yields of transplanted as well as direct-seeded rice were increased by the use of 5-6 kg ha-' AM along with ammoniacal fertilizers. In the United States, Wells (1976) reported rice grain yield increases from the addition of 1.12 and 2.24 kg h a - ' of NP applied with 67 to 178 kg h a - ' of preplant-applied urea-N. In another study in Arkansas and Louisiana, no increase in yield due to NP was recorded in 1977, but in 1978 there was a positive grain yield response to NP (Touchton and Boswell, 1980). In Louisiana, Patrick et al. (1968) reported no advantage with NP for rice. Wells ef al. (1989) summarized results with DCD from Arkansas, California, Louisiana, Mississippi, and Texas. DCD delayed nitrification and tended to result in rice grain yield increases compared to urea-applied preplant without DCD in drill seeding. In water-seeded continuously flooded rice, using DCD was advantageous only if the flood was delayed for more than 14 days after urea application. At the International Rice Research Institute, application of prilled urea with 10 or 15%DCD during the final harrowing produced lowland rice yields comparable to those with split applied prilled urea without DCD (De Datta, 1986). Bains et al. (1971) reported the effectiveness of neem (A. indica Juss) seed ~

~

248

RAJENDRA PRASAD AND J. F. POWER

extract-treated urea for increasing rice yields and N efficiency. Reddy and Prasad (1975) showed that the coating of urea with neem cake controlled nitrification for a period of about 2 weeks and resulted in a significant increase in rice grain yield over prilled urea. Prasad and Prasad (1980) reported increased rice yields and N efficiency with neem cake-coated urea. These results have been confirmed by a large number of workers in India (Budhar et al., 1987, 1991; Govindaswamy and Kaliyappa, 1986; John et al., 1989; Joseph et al., 1990; Latha and Subramanian, 1986; Mishra et al., 1991; Prasad et ul., 1989; Singh et al., 1984, 1990a,b; Singh and Singh, 1991; Velu et al., 1987). NIs have therefore a definite place in rice culture, especially in conditions where N losses due to leaching and denitrification are high.

B. CORN The results of field experiments with corn in the eastern corn belt of the United States (Nelson and Huber, 1980) illustrated that 70% of the trials in Indiana showed increased yields with NIs (NP and Terrazole); the average corn yield increase from NI was 24 and 5.2% for fall-applied anhydrous ammonia and urea liquid solution fertilizers, respectively. Yield increases were also obtained in Kentucky, Michigan, and southern Illinois but not in Wisconsin and northern Illinois. Hergert and Wiese (1980), summarizing the results of experiments with NP in the western corn belt of the United States, observed that the data obtained from Minnesota, Kansas, and Nebraska indicated the largest impact of NIs on irrigated sandy soils, particularly where rainfallhrrigation provides excess water; the response of NIs on fine-textured soils was rather limited. From the results of later experiments with DCD and NP in the north central states, Malzer et al. (1989) also concluded that the greatest benefit for NI use was obtained on coarse-textured soils; their results are shown in Table IV. The data in Table IV also suggest that DCD was superior to NP when used with urea ammonium nitrate or urea. This was further confirmed in a later study conducted on installed lysimeters at the Herman Rosholt Bonanza Valley irrigation farm located in west central Minnesota (Walters and Malzer, 1990a). The soil on the experimental site was an Estherville sandy loam (Typic Hapludoll). The N1 treatment increased fertilizer use efficiency only at the 90 kg N ha I rate when the leaching load was high. It was concluded that incorporation of NI with moderate N rates coupled with conservative irrigation management should reduce the risk of yield loss and minimize nitrate movement to groundwater. Results from experiments with NIs in the southeastern United States (Touchton and Boswell, 1980; Frye et al., 1989) suggest limited benefits to corn from NIs due to relatively high soil temperatures, which permit nitrification of fall-applied ammonium-N during winter months, highly permeable coarse-textured soils, and nitrate leaching from excessive winter and spring rainfall. No yield advantage ~

249

NITRIFICATION INHIBITORS Table IV

Relative Effectiveness of Dicyandiamide (DCD) and Nitrapyrin (NP) with Several Applications and N Sources on the Corn Yield from Coarse-Textured Soils in the Midwest"."

Application Time"

No. of comparisons N sourced

No. of positive significant responses'

Average relative response (%)

DCD

NP

DCD

NP

DCD

NP -

Fall

Urea

6

-

3

-

4.9

Spr. PP

Urea

20

20

9

10

27. I

16.1

Spr. PP Spr. PP SD/split SD/split

UAN AA Urea UAN

6

6 6 15 6

4 8

2 3

4

5

28.9 20.6 5. I

2

2

1.5

11.4 8.2 4.1 I .o

12 15 6

"Adapted from Malzer et al. (1989). "Data include all N rates at or below the optimum rate fertilization within each experiment (13 experimental site years). 'Spr. PP. spring preplant: SD/split, side dress or split N application. "UAN, urea ammonium nitrate (28% N solution); AA, anhydrous ammonia. 'Significant at the 90% probability level.

with DCD was obtained in 22 comparisons in the mid-Atlantic region of the United States (Fox and Bandel, 1989). In five comparisons there was a lowering of corn yield with DCD only. The reduced yield in three of these was attributed to increased NH, volatilization losses in the presence of DCD. Townsend and McRae (1980), from Nova Scotia, Canada, also observed that except on light sandy soils, no yield advantage was gained with NP. Thus soil characteristics and the amount of precipitation received during the crop-growing season may affect the response of NI. For example, Kapusta and Varsa (1972), working on clay pan soils in Illinois known for losses due to denitrification, found a positive response in the first year which was characterized by good precipitation, but not in the next year, which was drier. Similar results were obtained in a 2-year study at New Delhi, India (Prasad and Turkhede, 1971). Benefits of the NIs in corn production are therefore limited to coarse-textured soils and in situations where excessive soil water leads to heavy N leaching.

C. GRA~NSORGHUM NP or Dwell applied with urea, anhydrous ammonia, or urea ammonium nitrate solution did not increase yield nor improve efficiency of N applied to grain sorghum during a period of 4 years (1976- 1979),even with supplementary irrigation

2 50

RAJENDRA PRASAD AND J. F. POWER

to promote leaching and/or denitrification (Westerman et al., 1981). Two tests on grain sorghum were conducted in the coastal plain of Alabama on Dothan and Norfolk sandy loam soils. In the first test (Touchton and Reeves, 1985), DCD increased grain yields when applied at the 90-kg N ha-' rate in both 1982 and 1983. Yields with 90 kg N h a - ' and DCD were equal to yields with 134 kg N ha - without DCD. In both years, conditions were favorable for N losses via leaching and denitrification. In the second test (Frye et al., 1989), no increase in yield was obtained with DCD. Mascagni and Helms (1989) also failed to obtain an increase in the yield of grain sorghum with DCD or NP on a poorly drained Sharkey sandy clay (Vertic Haplaquepts) or on a well-drained Herbert sandy loam (Mesic Ochraqualfs) soil of Arkansas. Success with NIs on sorghum has been limited.

D.

WHEAT

Extensive studies in the United States with NP and DCD showed increased yields of winter wheat due to NP in the Pacific Northwest (Washington, Idaho) (Harrison et al., 1977; Papendick and Engibous, 1980). Greater yields of wheat were obtafned with DCD in three of eight experiments in the mid-Atlantic region (Maryland and Pennsylvania) (Fox and Bandel, 1989) and in one of four experiments in the north central states (Illinois and Indiana) (Harms, 1987). In the eastern part of the Midwest (Illinois, Kentucky, Michigan, Ohio, and Wisconsin) a yield increase was on the order of 9.9 to 24% (Nelson and Huber, 1980; Shyilon et al., 1984). Nitrification inhibitors were more effective in the southern part of the Midwest due to higher rainfall and the associated nitrate leaching. In the western part of the Midwest (Nebraska, Kansas, Colorado, Minnesota), NIs were not effective in increasing wheat yields due to the virtual absence of leaching of N below the root zone (Hergert and Wiese, 1980). Little advantage with NP (Nelson et al., 1977; Boswell et al., 1976) or with DCD (Frye et al., 1989) was obtained in the southeastern states of the United States (Alabama, Virginia, Georgia, and Tennessee). Increased wheat yields with NP were obtained in Alberta, Canada (Mahli and Nyborg, 1978). Sommer and Rossig (1978) from Germany reported that injection of NH,+-N and NP gave similar yields as obtained with a split application of N. Lewis and Stefanson (1975) obtained no yield advantage with NP under field conditions in Australia. In a field experiment on a sandy loam soil at New Delhi (Singh and Prasad, 1992). wheat yield with 80 kg N ha-' + DCD was greater than that obtained with 120 kg N ha I without DCD (Fig. 4). Application of DCD beyond 15% of N as DCD reduced wheat yield. An increase of 4- 12% in grain yield of wheat due to neem cake-coated urea compared to urea was obtained in India at Kanpur (Agarwal et al., 1980), Hissar -

'I

NITRIFICATION INHIBITORS

251

5

0-0

80-0

80-5

80-10

80-15

80-20

80-25

120-0

K g N ha1-*/. D C D - N

Figure 4. Effect of DCD on wheat grain yield. Total N applied (fertilizer and DCD). Adapted from Singh and Prasad (1992).

(Bhatia et al., 1985), and Pusa (Prasad et al., 1986; Mishra et al., 1991). Success with NIs in wheat in the United States has been mixed. Nitrification inhibitors are effective in increasing wheat yields in the Pacific Northwest and the southern Midwest but not in the southeastern states and the western Midwest. The data from other parts of the world are too limited.

E. SUGARCANE In a 2-year study at New Delhi, India, Parashar ef al. (1980) found a significant increase in cane yield with neem cake-coated or mixed urea at 75 kg N ha - I and with NP applied with 150 kg N ha I . Furthermore, there was a significant residual effect on a ratoon crop and 75 kg N ha I as NP-treated urea or neem cake-coated urea produced almost the same yield as 150 kg N ha - applied as prilled urea (Sharma et al., 1981). Singh er al. (1987) also found an increased cane yield with neem cake-coated urea. Nitrification inhibitors could have a place in sugarcane culture, but more field data are needed before a definite conclusion can be drawn. ~

~

F. POTATO On sandy loam soils of Michigan, no yield advantage was obtained with NP, while the yield and number of marketable tubers increased with NP on Idaho soils (Potter etal., 1971). Broadcast application of N,as urea, with spraying of inhibitor

252

RAJENDRA PRASAD AND J. F. POWER

(NP on terazole) followed by thoroughly mixing the compounds with the soil gave some potato yield increases on Washington soils, whereas no effect was found with band (row) application (Roberts, 1979). Hendrickson et al. (1978) found a yield reduction and a decreased quality of tubers with up to 4.4 kg ha-l NP applied with ammonium sulfate and diammonium phosphate. On a Plummer fine sand (Grossarenic Paleudults) at Hastings, Florida, application of 5-6 kg ha I DCD significantly increased the tuber yield in 1983 but not in 1984 (Frye et al., 1989). Also, no increase in tuber yield was recorded due to DCD at Gainsville, Florida. On alluvial soils in Ludhiana (India), ammonium sulfate and calcium ammonium nitrate are superior to urea in the absence of NP, but urea treated with NP is comparable to ammonium sulfate and is better than calcium ammonium nitrate (Sahota and Singh, 1984). Treatment with NP increased N uptake and N recovery by potato and decreased the optimum dose by 1 1-40 kg N ha - I. Increased potato yields with neem cake-coated urea were found at Simla (Sharma etal., 1980) and Palampur (Sharma et al., 1986). ~

G.

COTTON

Reeves and Touchton (1989) in pot culture studies found that cotton was sensitive to DCD. Although significant reductions in plant growth did not occur unless DCD exceeded rates normally applied, their results suggest a need for caution when applying DCD to cotton. On a Norfolk sandy loam (Rhodic Paleudult) in Alabama there was a tendency for yields to decline with DCD, while on a Decatur silt loam in the same state and on a BeulahBosket very fine sandy loam (Typic Dystrochrepthlollic Hapludalf) in Mississippi there was no significant increase in cotton yield (Frye et al., 1989). However, in India, an increased yield of cotton due to neem cake coating of urea was reported by several workers (Seshadri and Prasad, 1979; Jain et al., 1982). Cotton seems to be sensitive to DCD and therefore this NI should not be used for cotton.

V. PHYTOTOXtCITY OF NIs Some of the results obtained in field experiments could be due to phytotoxicity of NIs, although obvious symptoms may not have appeared under field conditions at dosages used. Joseph (1992) reported that wheat benefited when DCD was applied at a 10% N level, while yield was reduced when the level of DCD-N was raised to 20%. Reeves and Touchton (1989) applied DCD at 0, 2.5,5, 10, 15, and 20 mg DCD N kg ~I soil along with urea or sodium nitrate at 50 mg N kg I soil ~

253

NITRIFICATION INHIBITORS

in a pot culture study with Norfolk sandy loam (Typic Paleudult). Six days after application of DCD at 15 or 20 mg kg ’ soil, cotton leaves developed mottled chlorosis. After 20 days, mottled chlorosis developed on leaves of all plants treated with DCD. The chlorosis intensified with DCD rates and progressed to necrosis with DCD-N rates of 20 mg kg I soil. Symptoms were similar for cotton treated with both N sources. Reductions in leaf dry weight and foliar toxicity symptoms suggested that the primary site of phytotoxicity of DCD was in leaf tissue and not in root tissue. DCD linearly increased the leaf tissue concentrations of N, P, and K and lowered concentrations of Ca” and Mg”. Lack of DCD x N source interaction suggested that reduced Ca’ ’- and Mg uptake resulted from direct effects of DCD and not from indirect effects caused by the inhibition of nitrification and an increased NH,+ uptake. It was suggested that when banded N applications are made or root growth is restricted due to compaction, phytotoxicity from DCD-N concentrations at 5 mg kg I in the root zone of cotton might diminish any potential benefits derived from increased N efficiency gained through the inhibition of nitrification. In a greenhouse study with Cherry Belle radish, Feng and Barker (1989) found that as the concentration of NP or Captan in the medium with NH,+-N increased, growth of roots and shoots in radish was restricted and leaves were stunted, showing interveinal chlorosis, marginal necrosis, and upward cupping. The roots were stunted and twisted and failed to expand properly. Ca” and Mg2‘ contents in shoots, 4 weeks after seeding, were considerably lowered when NP was applied with ammonium sulfate or urea; on the other hand, K contents were increased. Many reports (Kirkby, 1968; Wilcox ef a/., 1973) using various plants have shown that acidity of the medium and deficiencies of K + , Ca”, and Mg” are major reasons for toxic effects of NH,’. However, Goyal et a/. ( 1982) observed that even though the pH of the nutrient solution was regulated at or near neutrality, toxicity persisted in radish plants; large amounts of K and Ca2+ in the solution did not correct the toxicity. Plants grown with ammonium fertilizer and NI usually contain lower concentrations of Ca” and Mg2+(English et al., 1980; Mathers et al., 1982). This tendency is attributed to competitive absorption between NH,’ and other cations. English et al. (1980) suggested that chemical inhibition affects the permeability of plant cell membranes by altering their integrity or activity. Ca2’ and Mg” concentrations are correlated negatively to the residual NH,+ in the medium but are correlated positively to residual nitrate. Plant weight is also negatively correlated with the residual NH,’. Yield reductions and phytotoxicity from use of DCD have been reported by a number of researchers (Cowie, 1918; Maftoun and Sheibany, 1979). Symptoms of DCD phytotoxicity developed in the greenhouse within 3 to 20 days after application of DCD, depending on the crop and DCD rate (Reeves and Touchton, 1986). Symptoms expressed on corn and sorghum were chlorosis and necrosis that ~

+

~

+

+

2 54

RAJENDRA PRASAD AND J. F. POWER

began at the leaf tips and progressed down the leaf margin. Symptoms on other crops were mottled interveinal chlorosis and leaf margin chlorosis and necrosis. Based on visual symptoms, sorghum and cotton are more sensitive to DCD than corn (Reeves and Touchton, 1986). Concentrations as low as 2.5 mg DCD-N kg - I increased the stomatal conductance of water in cotton plants grown in the greenhouse (Reeves et al., 1988). This effect was noted under conditions of high transpirational demand in the afternoon. Concentrations of 5 - 10 mg DCD-N kg I increased responsiveness of stomata to decreasing soil water content over the entire range of available soil water. The effect of DCD on stomatal conductance was believed to be a direct effect of the compound and not directly due to soil water availability. When soil water is limited, DCD might increase water stress and decrease yield (Frye et al., 1989).Use of NI had a deleterious effect on the tuber grade in potato (Hendrickson er al., 1978). Although total tuber yield increased, the percentage of grade A-USDA tubers was reduced 2.4% with NP and 5. I % with DCD (Malzer et al., 1989). The studies referred to earlier indicate the following: ( I ) Some field crops such as cotton are sensitive to some NIs; and (2) phytotoxicity symptoms observed could be due to direct or indirect effects of NIs; the indirect effects being the result of higher than normal NH,+-N concentrations.

VI. HEALTH AND NITRATES NIs may possibly play a role in human and animal health by reducing the NO, content in drinking water, food, feed, and forage.

A. NITRATES AND HUMAN HEALTH The well-known problem associated with NO3- /NO,- toxicity in humans is methemoglobinemia or “blue-baby syndrome. It generally occurs when infants under the age of 4 months consume too much nitrate (Rosenfield and Huston, 1950). Microbes in the stomach reduce nitrate to nitrite. When nitrites reach the bloodstream, they convert ferrous ions in the hemoglobin to the ferric form and produce methemoglobin (MHb), which has no oxygen-carrying capacity. Very young children are susceptible because their hemoglobin has a greater affinity for nitrite than hemoglobin of older children and adults. Methemoglobinemia resulting from high nitrate concentrations in drinking water was first recognized by Comly ( I 945) at the University of Iowa. Associated symptoms are diarrhea and vomiting, and the child’s complexion becomes slate blue (Ewing and MayonWhite, 1951). In addition to drinking water, the incidence of methemoglobi”

255

NITRIFICATION INHIBITORS

nemia has occurred in young children fed unrefrigerated spinach or high nitratecontaining fruitjuices (WHO, 1978; Keating etal., 1973). In a survey of Nebraska physicians, doctors reported 15 infants with suspected nitrate-induced methemoglobinemia (Grant, 1981). In addition to water and vegetable products, infant methemoglobinemia can occur when infant foods are prepared with nitratecontaminated water (Johnson et al., 1987). This may also happen in older individuals who have genetically impaired enzyme systems for the reduction of methemoglobin. The largest outbreak was reported in Hungary (Deak, 1985) where 1353 cases occurred between 1976 and 1982. Nitrite produced from NO,- could react in the stomach with secondary amines resulting from the breakdown of meat and fish forming N-nitroso compounds, which can cause stomach cancer (Fritsch and de Saint Blanquat, 1985; Saul et al., 1981). However, it should be mentioned that nitrites which are a potential health hazard are widely used as a preservative in salted meat and sausages (Davis, 1990) where they prevent the growth of Clostridium botulinum, the organism that causes botulism (WHO, 1978). Thus the risk of stomach cancer may not be closely linked with the nitrate content in drinking water. In addition to methemoglobinemia and stomach cancer, other health disorders reported due to the large ingestion of NO, in drinking water are hypertension (Malberg et al., 1978), increased infant mortality (Super et al., 1981), central nervous system birth defects (Dorsch et al., 1984), and non-Hodgkins lymphoma (Weisenburger, 199 1); nevertheless, none of these have been conclusively proved to be due to NO,- ingestion (Spalding and Exner, 1993). Normally, in humans only about 20% of their NO,- intake comes from liquids and drinking water (Table V) (Isermann, 1983). In addition, overfertilization, heavy manuring, or irrigation with high NO3- water can also result in -

Table V Nitrate Uptake through Food and Drinks" mg nitrate person - day

Product

~

Percentage of total daily intake

0.23 5.7

0.2

Meat and meat products Cereals

1 .s

1.6

Oils and fats

-

Milk and dairy products

-

Sugar Fruits Vegetable\ (155 g day - I ) Drinks and water (2.75 liter day

~

')

"Adapted from Iserrnann (1983).

0.9 63.5 19.0

6.2

-

I .o 70.0 2 I .o

256

RAJENDRA PRASAD AND J. F. POWER

large NO,- accumulations in many vegetables, which increases the human nitrate load.

B. NITRATES AND ANIMAL HEALTH Nitrate or nitrite poisoning is also reported in animals and is again due to MHb formation in blood with consequent asphyxiation. The conversion of nitrate to nitrite is carried out by bacteria in the rumen and ruminants are therefore especially vulnerable to nitrate poisoning. Goats, especially Angora, may be more susceptible to NO,- poisoning than either sheep or cattle (Schneider et ul., 1990). Mature single-stomach animals (except horses) are more resistant to nitrate toxicosis. Other than lack of oxygen, dilation of blood vessels is another secondary effect of nitrate poisoning. Abdominal pain and diarrhea are also reported. Other effects of nitratehitrite poisoning in animals include poor growth rates, reduced milk production, increased susceptibility to infections, and even abortions late in pregnancy (Schneider et al., 1990). Nitratehitrite poisoning symptoms appear when MHb concentrations reach 20-30% of total hemoglobin, and death due to asphyxia may occur when the MHb level exceeds 75% of total hemoglobin. Blood containing MHb usually has a chocolate brown color. Feed/forage with nitrate concentrations exceeding 2.25 g kg - I NO, - -N (1 0 g kg - NO,-) have a high risk of causing acute nitrate poisoning in ruminants; about half of this concentration should not be exceeded in the diets of pregnant beef cows. Drinking water for young livestock should contain less than 35 mg liter - I NO,--N. Nitratehitrite poisoning in adult animals is likely when the N 0 3 - - N concentration in water is more than 100 mg liter-' (Schneider et ul., 1990).

c. NITRATE CONTENT OF DRINKING WATER There is growing concern regarding NO,- content in drinking water and the World Health Organization (WHO) has set a maximum limit of 100 mg NO,liter-' (22.6 mg NO,--N liter-!) and a recommended limit of 50 mg N 0 3 - - N liter I ( 1 1.3 mg N liter - I); the latter limit is also fixed by the Council of European Communities (1980). Groundwater is the source of domestic water for almost 90% of the rural population of the United States and for about 50% of the total population (Power and Schepers, 1989). In Denmark, West Germany, The Netherlands, and Great Britain the use of groundwater accounts for 99,73,70, and 30%, respectively, of the total water consumption (Strebel et ul., 1989). Groundwater forms a substantial part -

257

NITRIFICATION INHIBITORS

of the drinking water in other parts of the world also. In addition, groundwater contributes substantially toward irrigation; estimates for the United States are 75-80% of the total water used for irrigation (Power and Schepers, 1989). Maintenance of groundwater quality is thus of major concern. Nitrates in groundwater can originate from geological sources, precipitation, cultivation, animal waste, niineralization of organic N, and fertilization. Data from the U.S. Geological Survey and the Texas Department of Natural Resources over a period of 25 years showed that states where 9% or more of groundwater samples contained 10 mg N03--N liter-' (45 mg NO,- liter I ) or more were Arizona, California, Delaware, Kansas, Minnesota, Nebraska, New York, Oklahoma, Rhode Island, and Texas (Madison and Brunett, 1985). After a careful examination of the U.S. Environmental Protection Agency's National Pesticide Survey (NPS), the Monsanto Company's National Alachlor Well Water Survey, and state-wide surveys in Iowa, Kansas, Nebraska, North Carolina, Ohio, Texas, Arkansas, California, Delaware, Pennsylvania, Washington, Minnesota, and South Dakota, Spalding and Exner (1993) concluded that the highest incidence of contamination occurs in groundwater in the middle of the contiguous United States where NO, -N levels in ~ 2 0 % or more of sampled wells in Iowa, Nebraska, and Kansas exceeded 10 mg liter I; in contrast, the contamination was lower in Texas, North Carolina, and Ohio (Fig. 5). Power and Schepers (1989) observed that use of high rates of fertilizer N may be a major source of nitrates in wells in the potatoproducing area of northern Maine. The high density of septic tanks, along with application of fertilizers and manures on agricultural lands, probably contributed to high NO3- on Long Island. Intensive dairy operations with associated problems of manure disposal may be a primary source of nitrates in wells in southeast Penn~

-

~

;

Statstrca~~y Randomized :

Statewide Surveys

i

WlnerabC

I

: other

:

Surveys

Figure 5. Incidence of' NO,--N contamination in large selected surveys (number of counties surveyed is in parentheses). IA, Iowa; KS, Kansas: NE. Nebraska: NC, North Carolina: OH, Ohio; TX, Texas: AR, Arkansas: CA, California; DE. Delaware; PA. Pennsylvania; WA, Washington; MN. Minnesota; SD, South Dakota. From Spalding and Exnrr (1993).

RAJENDRA PRASAD AND J. F. POWER

258

Table VI Correlations between Groundwater Nitrate-N Concentrations and Site Characteristics in Nebraska" Correlation of groundwater NO,-N concentration with

r value

Irrigation well density Total fertilizer used N fertilizer use Irrigation well depth Water pH Livestock density Percentage land cultivated Human population Percentage land with legume

0.425* 0.283* 0.202* - 0.275* - 0.233* 0.184* - 0.068 - 0.064 - 0.042

"Adapted from Muir er al. (1973). *Significant correlations.

sylvania and northern Maryland. High NO,- concentration in Delaware and parts of North Carolina may arise from intensive poultry operations plus septic tanks. A long extended belt of high NO,- wells extends from central Minnesota and Wisconsin to west Texas. Much of this area is irrigated, often for potato, corn, and sugarbeet production. Extensive irrigated areas in Colorado, Arizona, California, and Washington also have NO,- problems in the groundwater. The NO,- found in the waters of Yellowstone Park in northwest Wyoming is probably of geological origin. From a study done in Nebraska, Muir et al. (1973) found that groundwater NO,- -N concentrations were positively correlated to total fertilizer used and irrigation well and livestock densities (Table VI). Kilmer et al. (1974) reported in North Carolina that NO, --N in groundwater under steeply sloping, moderately grazed grassed watersheds exceeded 10 mg liter - I when 1 12 kg N ha - I was applied each year. In the small pastured watersheds on well-drained residual silt loams (Typic Dystrochepts and Hapludults) in eastern Ohio, subsurface NO, - -N ranged from 3 to 5 mg liter I with applications of 56 kg N ha I year and 8.18 mg liter - I year - I with applications of 224 kg N ha - ' year - I (Owens et al., 1983). On some pastures, application of 56 kg N ha - I year - I in the first 5 years and 168 kg N ha - I year - I in the next 10 years resulted in NO,--N concentrations of 10 to 16 mg liter I (Fig. 6) in the 9th and 10th years of high N application (Owens et al., 1992). In a long-term regional study, seven creeks draining agricultural watersheds and representing agriculturally important physiographic regions of Kentucky were sampled in 1971- 1972 (Thomas and Crutchfield, 1973). These creeks were re-

-

~

259

NITRIFICATION INHIBITORS 18'

1L

-

m l o E

z 1

0

z

0

6-

2 0

I

1974

"

76

'

78

"

'

80

1

'

'

82

1

8L

'

86

'

1

88

'

19

YEAR

Figure 6. Average flow-weighted seasonal (growing and dormant) concentration of NO,--N for subsurface flow throughout the 5-year prestudy periods (1974- 1979) during which 56 kg N ha - ' was annually applied and the 10-year study period (1979- 1989) during which 168 kg N ha - I was annually applied as ammonium nitrate. Adapted from Owens rt d.( 1992).

sampled in 1989- 1990 (Thomas er al., 1991) and analyzed for nitrate concentration. The data obtained (Table VII) showed no increase in nitrate concentration in creek waters despite the fact that fertilizer N consumption in Kentucky nearly doubled during that period. Based on analysis of water samples over a 20-year period in North Carolina, Gilliam (1991) observed that drainage conditions prevailing in soil profiles affected nitrate concentrations in soil water. In the lower coastal plain region, soils Table VII Calculated Total Flux of NO, --N ( f SD) in Seven Kentucky Streams" Creek

I972

Cave Fiat McG i II s Perry ( A ) Perry ( B ) Plum Rose West Bays

17.74 2 2.97 1.93 t 1.93 2.07 ?z 1.29 3.1 1 t 3.00

1979 kg h a - '

-

4.36 t 3.22 9.70 t 7.08 3.08 t 2.16

"Adapted from Thomas cful. ( I99 I ).

15.29 t 2.33 0.67 t 0.67 2.33 t 1.12 3.74 t 2.63 12.63 t 9.42 2.54 t 1.94 4.25 -+ 3.1 1 3.94 f 0.58

2 60

RAJENDRA PRASAD AND J. F. POWER

are poorly drained and have high organic matter content and high water tables. In these soils there is sufficientorganic matter to provide an energy source for microorganisms so that denitrification occurs and reduces nitrate concentration (Gambrell et al., 1975).Trudell er al. (1986) and Gillham (1991) have confirmed anoxic conditions in shallow groundwater, which promotes denitrification. Increasing N03--N concentrations in drinking water in Europe has been a matter of great concern and by 1995, 20% of the French population will be drinking water exceeding the European Community's (EC) limit of 11.3 mg N liter - [ (Fried, 1991). Similarly, 8% of the public waterworks in Denmark and 5% of those in the former Federal Republic of Germany have groundwater that exceeds EC limits for N03--N (Fried, 1991). Handa (1987), in India, reported that numerous wells, especially those in the drier region, contain water with high nitrate contents. For example, in the state of Haryana, a well water contained as high as 296 mg NO,--N liter ~ I Handa . (1987) supports the hypothesis that the nitrate content in groundwater originates from anthropogenic activities. The effects of land use and N fertilization on NO,- concentrations in groundwater based on the experience in Western Europe are summarized in Table VIII. Arable lands, which are subjected to heavily fertilized vegetable cropping, had the highest NO,- concentration in the groundwater. Similarly, intensively grazed grassland with heavy fertil-

Table VIII Measured Site and Land Use-SpecificNitrate N Input into Groundwater(Mean Concentrationof the Annual GroundwaterRecharge)"

Soil Sand

Land use (crop rotation, N fertilizer) Arable land (cereal-sugarbeet/potatoescereal, = 120 kg N ha-' year-') Arable land (cereal-winter catch cropssugarbeet/potatoes-cereal, = 120 kg N ha-') Grassland (meadow, 250 kg N ha - year - I ) Grassland (intensively grazed pasture, 250 kg N ha-' year-I; = 2 livestock units ha-I, = 180 grazing days) Field cropping of vegetables, including special crops such as asparagus, tobacco (= 300-600kgNha-'year-') Woodland (coniferous tree stands) Woodland (alder tree stands) Arable land (cereal-sugarbeet-cereal = I50 kgN ha-' year-')

-

Loess

'Adapted from Strebel et al. (1989).

Mean nitrate concentration (mg NO,--Nliter-')

25-30

14- I6 3-7

14-20

34-70 2.5 10

7- I4

261

NITRIFICATION INHIBITORS

ization gave high concentrations of nitrates in groundwater. In England (Haigh and White, 1986; Roberts, 1987), application of 100- 1 1 1 kg N ha I frequently resulted in groundwater concentrations above the EC limit. With intensively managed grassland systems on sandy soils in the Netherlands, a strong correlation was observed between the level of N fertilization and NO,--N leaching losses to groundwater (Steenvoorden er al., 1986). In Sweden (Bergstrom, 1987), NO,fluxes from grass and lucerne lays were mostly below 5 kg NO, -N ha I year I, while that from barley receiving 120 kg N ha I as Ca(N0,)2 was 36 kg NO,- -N ha I year I. -

-

-

-

-

~

-

D. NIs AND NITRATE CONTENT INVEGETABLES About 70 to 81% of the NO,- intake in the human diet is from vegetables (White, 1975). Nitrate concentrations in vegetables may be extremely high when vegetables are grown with high levels of N and under reduced light or moisture conditions (Brown and Smith, 1966; Jackson et al., 1967). Furthermore, effects of higher N rates on NO,- concentration are more likely on fast maturing vegetables such as radish, spinach, and lettuce (Huber et al., 1977). The effects of NIs and N fertilization on NO,- concentrations in vegetables were reviewed by Slangen and Kerkhoff (1984). They concluded that a relatively high amount of NH,+-N in the growth medium achieved by the use of NIs does not assure increased yield in vegetable crops as seen in cereals, nor did it always reduce nitrate concentration in vegetables. Sommer and Mertz (1 974), in Germany, reported that an application of NP with ammonium sulfate reduced NO,- concentrations in several vegetable crops as compared to Ca(N0, ) 2 (Table IX). Moore (1973) reported a reduction of 34 and 79% in nitrate concentration in lettuce and spinach, respectively, following application of NP. With 20 ml m - I of NP, a 40% reduction ~

Table Ix

Nitrate-N Content of Some Vegetable Crops Affected by NP" NO,-N (5% of DM)

Total N (% of DM)

Crop

Without NI

With NP

Without N1

With N P

Chinese cabbage Mustard Black radish Savoy cahbage Spinach Carrots Lettuce

I .oo 0.13 0.39 0. I9 0.46 0.39 0.4I

0.13 0. 14

4.83 2.62 3.50 2.91 4.69 3.33 3.53

5.5 1 3.5 1 3.99 3.96 5.80 4.18 5.10

0.11

0.12 0.28 0.16 0.18

"Adapted from Soinrner and Mertz ( 1974).

262

RAJENDRA PRASAD AND J. F.POW,R Table X Nitrate-N Accumulation (mg kg -' dry wt) in Radish Shoots Affected by N Sources and NP" NI

Ammonium sulfate

Without NP With NP

4000a" I ooc

Urea

8000a 200b

"Adapted from Feng and Barker (1989). "Means followed by the same letter do not differ significantly ( P = 0.05).

in nitrate concentration in Chinese cabbage [ Brassica pekinensis (Lour.) Repr.] was reported by Roorda van Eysinga and Van der Meijs (1980). Feng and Barker (1989) reported that the NO,- content in radish shoots 4 weeks after seeding was 1.3I , 0.4, and 0.8% with potassium nitrate, ammonium sulfate, and urea, respectively; the values with NP (averaged over 20, 40, and 60 mg kg - I ) were I . 19, 0.01, and 0.02% (Table X). Thus, reduction in nitrate concentration in vegetables can be achieved with the help of NIs. This is one way NIs can help in preventing health hazards.

VII. NIs AND ENVIRONMENT Nitrification inhibitors can possibly play some, if not a great, role in environmental conservation. The two areas where NIs can contribute are: (1) reducing NO,- content in groundwater, and (2) reducing the evolution of N,O. Nitrification inhibitors may also help in reducing global warming but the data on this are lacking.

A. NIs AND NITRATE CONTENTINGROUNDWATER Studies on the effect of NIs on NO,- leaching are rather limited. In soil column studies, Rudert and Locascio (1979) found that NP reduced nitrate leaching losses from a Kanapha fine sand during the first 2 weeks of a 5-week leaching period. Owens (198 1) found that 42 and 53% of the applied urea N (672 kg N ha - I ) had leached from NP-treated and untreated sandy loam columns, respectively, after 144 days. When leaching conditions occurred on sandy Coastal Plain soils, the addition of NP to urea significantly reduced N losses (Chancy and Kamprath,

263

NITRIFICATION INHIBITORS

1982). From pot culture studies simulating lowland rice conditions, Prakasa Rao and Prasad (1980) reported a leaching loss of 1 1.5% applied N with prilled urea and 9.2% when prilled urea was blended with NP. Based on data with nonweighing field lysimeters, Timmons (1984) reported that NP reduced leaching losses from sandy loam soil columns and reduced annual NO, leaching losses from urea by about 7% during a 3-year period. In long-term studies on a Rayne silt loam (mesic Typic Hapludult) using monolith lysimeters at Coshcoton, Ohio, Owens (1987) found that an average N loss from the central lysimeter (no NP) during the 6-year study was 160 kg ha-' or 48% of the N applied. Lysimeters treated with NP had an average N loss of 1 17 kg ha - I or 35% of the N applied. Based on the studies made with ISN-enriched urea on field lysimeters on an Estherville sandy loam (mesic Typic Hapludoll) growing corn, Walters and Malzer ( 1990b) reported that the leaching losses of fertilizer-derived N were delayed 25 to 50 days when urea + NP were incorporated; the total N loss was, however, not affected. In addition to lysimeter percolates, Owens (1 987) also collected water samples from a spring receiving groundwater from a watershed cropped similarly to the lysimeters that received 168 kg N ha I and 1.12 kg ha - ' NP. The nitrate concentration in the groundwater was reduced when NP was used (Fig. 7). Cattle manure can substantially contribute toward the occurrence of NO,- in groundwater. NIs have been usefully employed in reduced nitrate leaching from cattle manure. From a pot culture study, Amberger and Vilsmeier (1979) showed that 15.30 kg ha - I of DCD applied with 150 m 3 cattle manure h a - ' resulted in ~

~

Conventioni I Corn

Meadow

I I

No-Ill1 Corn

I

(received ureo w i t h nitropyrtn)

,

Wheat - We

I

I

I

I I

I

I

I

..'"

....

.''

. I " ' * ' . * " . ' . * " ' . . ' I ' * * ' w s suAwssuAws SUAWSSUAWSSUAWSSUAWSSUw A s s u A w S S u A wssuAWSsuAWSSU A 1973 7L 75 76 77 70 79 80 81 82 83 1981.

01.

W

YEAR

Figure 7. Seasonal NO,--N concentration in groundwater from an adjacent watershed (19731984) cropped with no-till corn fertilized with 168 kg N h a - ' of nitrapyrin-treated urea (1977- 1982). w, winter; s, spring; su, summer; and a, autumn. Adapted from Owens (1987).

2 64

RAJENDRA PRASAD AND J. F. POWER

inhibition of nitrification over 60 days at 8"C, over 40 days at 14°C and over 20-40 days at 20°C. Gorlitz and Hecht (1980) found effective inhibition of nitrification at 20" C with 2% NP or DCD (2% of total N of the manure). For DCD the effect lasted up to 3 weeks and for NP up to 9 weeks. In field experiments, NP at 1-2% (of the total N content of the slurry) proved to be effective until March after applying cattle manure in September of the previous year. DCD was somewhat less effective. Cooper (1980) found that NP and ATC were effective inhibitors of nitrification with pig slurry, while NaNO, was ineffective.

B. OZONELAYERDEPLETION In recent years concern about air pollution has extended from the obvious effects at ground level to the depletion of the ozone layer in the stratosphere (14-32 km above the ground surface), resulting in increased penetration of ultraviolet light with a wavelength between 209 and 330 mm (UV-B). The ozone layer in the stratosphere works as a shield against ultraviolet radiation, which with prolonged exposure is associated with skin cancer in humans, particularly in fair skinned persons (NAS, 1975). Estimates (Shea, 1988) suggest a 4.6% increase in cases of skin cancer with each 1% drop in ozone. Crutzen and Enhalt ( I 977) suggest that a doubling of the atmospheric N 2 0 could cause a decrease in the ozone layer which would increase the ultraviolet radiation reaching the earth surface by 20%. The ozone hole over Antarctica at a height of 14-22 km, first identified in 1985 (Farman et al., 1985; Hofman et al., 1986), develops each southern spring and has become increasingly worse (Thompson, 1991) than when it was first detected. Recent measurements of ozone by the TOMS (Total Ozone Mapping Spectrometer) on the satellite NIMBUS-7 show much smaller ozone losses over the North Pole than over the South Pole (perhaps 10 to 60%). but the losses at middle latitudes in each hemisphere are comparable (Pyle, 1991). One consequence of the ozone hole is ozone loss beyond Antarctica during the austral spring and summer when air masses with chemically induced stratospheric ozone loss penetrate toward mid latitudes (Thompson, 1991). Stolarski et al. (1991) have shown significant total ozone loss in both northern and southern hemispheres to within 35" C of the equator. At 40" N, the stratospheric ozone has dropped by about 8% in the past decade during the late winter and early spring (Pyle, 1991). During 1972- 1975, the Climatic Impact Assessment Program (CIAP) administered by the U.S. Department of Transportation carried out extensive measurements of stratospheric nitrogen oxides and other species and concluded that an increase in stratospheric NO, would decrease the stratospheric ozone layer; the relationship suggested is shown in Fig. 8 (Grobecker et al., 1975). This proposed relationship indicates that doubling the NO, concentration would reduce ozone by

265

NITRIFICATION INHIBITORS

z

z 3

810.0

-

W z

R0 6 W

m w Q ’ 0 5 0

W

o/ /

0

c

/

I

10 0

1

100 0

J 1000 0

‘1. INCREASE IN NOx COLUMN

Figure 8. The percentage decrease of ozone as a function of the percentage increase in stratosphere nitrogen oxides, as determined by the CIAP study. The line represents the equation A(O,)/ (0,)= (1/5)A(NO~)/(NO~). The asterisk represents the ozone depletion following an explicitly assumed doubling ofNzO. All calculations are based on an injection of NO, at a 20-km altitude. Adapted from Crobecker CI a/. ( 1975).

about 20%. The NO,-ozone decomposition catalytic cycle is shown in Fig. 9, and a report by a National Academy of Sciences Panel (1 978) suggested that the NO, catalytic cycle could be responsible for up to 50-70% of the total ozone destination rate (Table XI). N 2 0 in soils is mostly produced during biological denitrification (Payne, 1981; Reddy and Patrick 1986), but could also be produced during nitrification (Bremner and Blackmer, 1978; Freney et al., 1978, 1979; Aulakh et al., 1984). Some N,O is also produced by chemodenitrification (Chalk and Smith, 1983). A global inventory of N in the biosphere shows that it is distributed in terrestrial, oceanic, and atmospheric components in the ratio 1 :70 : 1 1,8I8 (Winteringham, 1980). A number of estimates of emission of N,O and N, are available. Winteringham (1 980) estimated terrestrial and oceanic denitrification as N,O at 16-69 and 20-80 Tg (million metric tons) y e a r 1 ,respectively; the values for terrestrial and oceanic denitrification as N, were estimated at 91 -92 and 5-99 Tg year I. Tiedje ( 1988) estimated N loss due to denitrification at 105- 185 Tg year I for land and 25-250 Tg year - I for the sea. Bouwman (1990a) estimated global N,O emission at 9.7 to 12 Tg N year - I for natural ecosystems and 2.3 to 3.7 Tg N year I for cultivated lands; he also expressed the opinion that there is an annual increase of 0.25% in the concentration of N,O (Bouwman, 1990b). Seiler and Conrad (1987) estimated global N 2 0 emission from fertilized soils at ~

266

RAJENDRA PRASAD AND J. F. POWER hv

Ozone Photolysl s

Ozone formaton

hu

Ozone Photolysis

N20+0 - 2 N O

nitrous oxlde photolysis

NOx- Ozone Decomposition Catalytic Cycle

From Troposphere

To Troposphere

Y Figure 9. Main cycle of nitrogen in the stratosphere emphasizing catalytic destruction of ozone. From NAS ( 1978).

1.5 Tg N,O-N year - I . Eichner (1990) summarized N,O emission data from 104 field experiments and estimated that global release of N,O from fertilized soils to the atmosphere ranged from 0.2 to 2.1 Tg N20-N year - I. He also suggested that the fertilizer-derivedemission of N,O in the year 2000 will account for 0.1 to 1.5% of the global source and will probably not exceed 3% N20-N in the atmo-

Table XI Mechanismsfor the Decompositionof Stratospheric Ozone" Mechanism Photolysis (Chapman reactions) Transport to troposphere Hydroxyl and hydroperoxyl radicals from photolysis of water NO. catalytic cycle Chlorine from natural and man-made sources (present effect)

Percentage of total O s destruction rate

'

20 0.5

I 0' 50-70' 10-40'

OFrom NAS (1978). bThese loss processes represent an average over the ozone for-

mation region between about a 25- and 40-kmaltitude.

NITRIFICATION INHIBITORS

267

sphere. The amount of N,O emitted from a corn production system ranges from 1.3 (Mosier and Hutchinson, 1981) to 2% of applied N (Duxbury and McConnaughey, 1986). Colbourn and Dowdall (1 984) observed that denitrification losses of inorganic N range from 0 to 20% of the fertilizer applied in arable soils and from 0 to 1 % on grassland soils. NIs, which retard oxidation of ammonium to nitrate, also retard N,O emission. Bremner and Blackmer (1978) reported that application of 8 mg NP kg-I soil reduced N20-N evolved in 20 days of incubation from 148 to 10 mg kg - I soil when ammonium sulfate was the source of N; the values with urea were 122 mg kg - I soil (without NP) and 4 mg kg I soil (with NP). Smith and Chalk (1980) reported that application of 10 kg NP kg - I soil with ammonia completely prevented N 2 0 emission for 28 days; NzO emission without NP during the same period was 57 mg N kg - I soil. Some data are also available from field studies. Bremner et al. (198 1) showed that application of 0.56 kg ha - I of NP along with anhydrous ammonia ( 1 80 kg N ha I ) reduced N 2 0 emissions from 1.37 kg N ha I (without NP) to 0.55 kg N ha I (with NP) during a period of 167 days. Magalhaes er al. (1984) reported from Australia that, under fallow conditions, NP significantly reduced the anhydrous ammonia-induced loss of N,O from a calcareous soil (pH 8.5; organic C 1.3%) but not from a noncalcareous soil (pH 7.5; organic C 2%). Bronson et al. (1992), from field studies with corn on a Nunn clay loam (mesic Aridic Argiustoll) at Fort Collins, Colorado, found losses of 1.5 to 3. I kg N,O ha - I from urea alone as compared to 0.87 to 1 .O kg N02-N ha - I from urea plus NP in the 2 years of the study (Table XII). Acetylene, which inhibits nitrification in soils at partial pressures of 0.1 to 10 MPa, is often introduced into soil in field studies to measure the total denitrification by the acetylene block method (blockage of the reduction of N,O to N2) (Reyden et a/.. 1979; Rolston et a / . , 1982). Encapsulated CaCz (ECC) (Banerjee and Mosier, 1989) has been used as a slow-release source of C2H, to inhibit and reduce N 2 0 and N, emission in flooded rice paddies (Mohanty and Mosier, 1990; Bronson and Mosier, 1991; Bronson et a/., 1989). Bronson et al. (1992), from their studies in corn fields, reported a loss of 2.1 kg N02-N ha I with urea + 20 or 40 kg ECC ha I as compared to 3.2 kg N02-N ha I with urea alone in 1989; the corresponding values for 1990 were 0.33-0.38 kg ha - I with most ECC and 1.5 kg ha I with urea alone (Table XII). They also pointed out that high levels of C2H2produced in ECC-treated plots apparently blocked the N20-N2 reduction step of denitrification (Yoshinari et al., 1977), resulting in high rates of NzO emission. From a field study on a clay soil (Andaqueptic Haplaquoll) in the Philippines, John et al. (1989) reported that (N2 N2O)-ISN flux during the 19 days following an application of 29 kg N ha I as 98 atom % IsN-labeled urea never exceeded 28 g N ha I day I ; the total recovery of (N2/N,0)-N evolved from the field ~

~

~

~

-

-

~

-

+

-

-

~

268

RAJENDRA PRASAD AND J. F. P O W E R Table XI1 Cumulative Losses of N,O-N in Irrigated Corn Affected by Urea with and without Escapsulated Calcium Carbide (ECC) or Nitrapyrin during 0-97 Days after Fertilization" 1989 Treatment

Mean

Urea Urea + nitrapyrin Urea + 2OkgECCha-I Urea + 40 kg ECC ha Blank

3.3362a' 1.174bc 2.2555ab 2.2270ab 0. I 15c

1990 SDb

Mean

kg N ha-' I .65 la 0.973 0.193 0.980b 1.223 0.483bc 0.483 0.434bc 0.043 0.108~

SD

0.6 18 0.593 0.128 0.192 0.029

aAdapted from Bronson et al. (1992). 'Standard deviation. 'Means followed by the same letter are not significantly different ( P= 0.05) by Duncan's mean range test.

study was only 0.51% of the applied N, whereas total gaseous I5N loss estimated from unrecovered I5N in the I5N balance was 41% of applied N. In several other studies (Mosier et al., 1990a,b; Buresh and De Datta, 1990; Mohanty and Mosier, 1990), N gas flux measurements represented only a small fraction (1 to 10%)of the total gaseous N losses as measured using the I5N balance approach. Aulakh et al. (1992) suggested that entrapment of N gases in soil pore water and flood water in rice paddies, transmission of N gases through rice plants, and nitrogen losses through ammonia volatilization could be the cause for the low recovery of N-labeled gases. Furthermore, constancy of the N2/N20ratio is important in estimating denitrification losses from field soils where only N2 emissions into the atmosphere are measured (Reyden et al., 1979). Weier et al. (1993) found that the N,/N,O ratio was affected by the amount of available C, soil water content, and the amount of nitrates. The presence of high nitrate concentrations apparently inhibited the conversion of N,O to N2 and lowered the N2/N20ratio. They did not recommend an average N 2 / N 2 0ratio for estimation of denitrification from N 2 0 field measurements. Thus there is considerable scope and need for research on more precise direct measurements of N,O losses from agricultural fields, other lands. and water masses.

c. GLOBALWARMING The impacts of global climatic patterns of increases in greenhouse gases absorbing infrared radiation have been the focal point for many studies investigating the indirect effects of atmospheric alteration (Liverman, 1986; Smit ef al., 1988).

NITRIFICATION INHIBITORS

269

Increases in greenhouse gases are predicted to contribute to warmer and drier climates in midlatitude regions such as the midwestern United States, southern Europe, and Asia, whereas it is predicted that higher latitudes will in all likelihood be characterized by warmer but somewhat wetter climates (Smit et al., 1988). Recent models suggest that N,O also contributes to global warming (Yung et al., 1976); an increase of 0.2 to 0.3% N,O in concentration in the atmosphere would contribute about 5% to the supposed greenhouse warming (Enquette Komission, 1989). Rodhe (1990) indicated that N,O is 300 times more radiatively active than CO, and it is estimated that nitrous oxide fluxes from climates contribute to the greenhouse warming by about 4% (UNEP, 1992).

ACKNOWLEDGMENTS The senior author is grateful to the Director General, Indian Council of Agricultural Research (ICAR) and Director, Indian Agricultural Research Institute, New Delhi, for deputation to the University of Nebraska, Lincoln, which enabled the preparation of this review. The authors are grateful to Drs. James S. Schepers, Gary W. Hergert, and Merle F. Vigil for their constructive criticisms.

REFERENCES Agarwal, S. R., Shankar, H., and Agarwal, M. M. 1980. Effect of slow-release nitrogen and nitrification inhibitors on rice-wheat sequence. Indian J. Agron. 35,337-340. Alexander, M. 1977. “Introduction to Soil Microbiology,” 2nd Ed. Wiley, New York. Amberger, A,, and Guster, R. 1978. Umsatz und Wirkung Von Harnstoff-dicyandiamide-sowoie. Ammonium sulfat-dicyandiamide-productionzu weidelgras und reis. Z. P’unzenern. Bodenkd. 141, 553 -566. Amberger, A,, and Ouster, R. 1979. Zur Wirkung von rinder-gulle mit dicyandiamide-zusatz zu Wiedelgras. Z. Acker-U Pjlunzenbau ( J . Agron. Crop Sci.) 148, 198-204. Amberger, A,, and Vilsmeier, K. 1979. Versuch Zur Wirkung von cynamid, dicyandiamide, guanyharnstoff, guanidin und nitrit auf die urease aktivitat. Lnndw. Forsch. 32,409-415. Ashworth, J., Akerboom, H. M., and Cre’pu, J. M. 1980. Inhihition by xanthates of nitrification and urea hydrolysis in soil. Soil Sci. Soc. Am. J. 44, 1247- 1249. Ashworth, J., Briggs, G. G., Evans, A. A,, and Matula, J. 1977. Inhibition of nitrification by nitrapyrin, carbondisulfide and trithiocarbonate. J. Sci. Food Agric. 28,673-683. Atkinson, R. J., Mathews, W. A., Newman, P. A., and Plumb, R. A. 1989. Evidence of the mid latitude impact of Antarctica ozone depletion. Nurure 340,290-293. Aulakh, M. S., Doran, J. W., and Mosier, A. R. 1992. Soil denitrification-significance,measurement and effects of management. Ad. Soil Sci. 18, 1-57, Aulakh, M. S.. Rennie, D. A., and Paul, E. A. 1984. Acetylene and N-Serve effects upon N 2 0 emissions from NH,’ and NO1- treated soils under aerobic and anaerobic conditions. Soil Biol. Biochem. 16,351 -356. Azhar, E., Vandenabeele, J., and Verstraete, W. I986a. Nitrification and organic nitrogen formation. Plnnr Soil 94,383-399. Azhar, E., Vandenabeele, J., and Verstraete, W. 1986b. Nitrification mediated nitrogen immobilization in soils. Plan! Soil 94,401 -409. Azhar, E., Vehre, R., Proot, M., Sandra, P., and Verstraete, W. 1986~.Binding of nitrite-N on polyphenols during nitrification. Plum Soil 94,369-382.

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PRODUCTION AND BREEDING OF LENTIL F. J. Muehlbauer,'W.J. Kaiser,2S. L. Clement,zand R. J. Summerfield3 !United States Department of Agriculture Agricultural Research Service Grain Legume Genetics and Physiology Research Unit Washington State University Pullman, Washington 99164 'United States Department of Agriculture Agricultural Research Service Regional Plant Introduction Station Washington State University Pullman, Washington 99164 Department of Agriculture Plant Environment Laboratory University of Reading Shinfield Reading, Berkshire RG2 9AD, United Kingdom

I. Introduction 11. Background 111. Origin, Taxonomy, Cytology, and Plant Description A. Origin B. Taxonomy C. Cytology D. Plant Description IV.Production of Lentil A. Land Requirements B. Seed Quality and Seed Treatment C. Seedbed Preparation D. Seeding E. Lentil Cultivars V. Fertilization and Weed Control A. Fertilizers B. Weed Control VI. Principal Uses A. Food B. Fodder C. Green Manure VII. Major Constraints to Production A. Insects B. Diseases C. Environmental Stress 283 d d u m i r a in A p n o v y , Volitm~54

Copyright0 1995 by Academic Press, Inc. .All rights of reproduction in any form reserved.

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F. J. MUEHLBAUER ET AL. VIII. Hybridization Methods A. Hybridization B. Environmental Conditions C. Equipment Needed D. Emasculation of the Female Flower E. Pollination F. Other Considerations for Crossing IX.Genetic Resources A. Germ Plasm Collections B. Collection and Utilization of Wild Species X. Genetics A. Qualitatively Inherited Traits B. Quantitative Inheritance XI. Interspecific Hybridization XII. Methods Used for Lentil Breeding A. Pure Line Selection B. Bulk Population C. Pedigree Selection D. Single Seed Descent E. The Backcross Method XIII. Breeding Objectives A. Seed and Straw Yields B. Diseases C. Root Rot/Wilt Complex D. Orobanche E. Insects F. Quality G. Adaption to Mechanical Harvesting H. Other Objectives XTV Summary References

I. INTRODUCTION Since domestication in the Near East, lentil (Lens culinaris Medikus) has held a prominent place in cropping sequences in semiarid regions of the world and has provided an important source of dietary protein. Despite the great importance of the lentil crop to local populations, the crop has often been relegated to marginal areas where it is grown without the benefit of fertilization, herbicides to control weeds, pest control chemicals, or irrigation. The crop nevertheless has remained popular in those areas possibly because of tradition but more importantly because lentil may be one of only a few crops that can be grown. Research on improvement of the lentil crop has been minimal until the recent establishment of the international agricultural research centers and particularly

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the establishment of the International Center for Agricultural Research in the Dry Areas (ICARDA) in 1978 and their mandate for lentil crop improvement. Landraces of lentil and related wild species have been collected and are being maintained at ICARDA, at national research centers, and at the US.Department of Agriculture, Western Regional Plant Introduction Station at Pullman, Washington. The availability of germ plasm has been instrumental in the development of improved cultivars that have become of increasing importance in the production of the crop. Oram and Agcaoili (1994) have pointed out a significant increase of lentil production throughout the world during the 1980s because of yield increases and increases of area sown. The area sown to lentil has continued to increase in Canada, Turkey, and Australia. The lentil crop, including production practices, germ plasm constraints to production, breeding, genetics, and uses, is reviewed and discussed.

11. BACKGROUND Lentil is one of the principal food crops cultivated in the semiarid regions of the world, particularly in the Indian subcontinent and in the dry areas of the Middle East. The crop is a dietary mainstay in those areas and is mostly consumed by local populations. Of the countries that produce lentil, India is the largest producer followed by Turkey, Canada, and Syria. Ethiopia and Morocco are also major producers of lentil. Other countries of the Middle East such as Egypt, Jordan, Iraq, and Lebanon are major consumers of lentil but not major producers. World production in the areas of major use has declined in recent years as a result of a dramatic shift toward the production of cereals, with legume crops being relegated to more marginal areas with poorer soils and limited rainfall. Another major factor in the decline of lentil production in the Middle East, except for Turkey, has been the high cost of labor for harvesting the crop. In most of the countries of the Middle East, the crop is harvested almost entirely by hand (Khayrallah, 1981; Haddad and Arabiat, 1985). Turkey, on the other hand, has increased its production through partial and, in some areas, complete mechanization. Crop residues from lentil are valuable as livestock feed in many regions where grazing is limited. In dryland farming systems, the lentil crop offers farmers an alternative to cereal grains and it contributes to the nitrogen budget of infertile soil by fixing dinitrogen in symbiosis with Rhizobium. World production of lentil increased by 72% during the 1980s (Oram and Agcaoili, 1994), representing an increase of 95 1,000 tons. This dramatic increase of lentil production resulted from a 4% increase in area sown and an overall increase of 4% in yield per hectare. Production in 1990 was estimated at nearly

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2.3 million tons. Major increases in production have been recorded from Canada and Turkey. Significant shifts in lentil production have taken place throughout the last 25 years. For example, the area sown has declined sharply in Syria, Jordan, Iraq, and Ethiopia. Production in India, Pakistan, and Bangladesh has remained relatively constant, even though the crop is harvested almost entirely by hand; however, labor is readily available in those countries. Average lentil yields have varied widely from a low of 660 kg ha ' in India to over 2100 kg ha - I in Egypt (Oram and Agcaoili, 1994) where the crop is grown under irrigation. Yields have averaged over 1000 and 800 kg ha-' in the United States and Canada, respectively. Lentil production has increased to more than 600,000 tons annually in Turkey (Oram and Agcaoili, 1994) and nearly doubled between 1984 and 1990 (FAO, 1991). Turkey is now the world's largest lentil exporter. The increased production was brought about by fallow replacement in cereal production systems where sufficient rainfall is received to permit annual cropping (AGikgoz et al. 1994). Of the lentils produced in the United States, over 85% are exported; however, there is an aggressive marketing program underway which is designed to increase domestic usage. Chile and Argentina are also major exporters in the western hemisphere. Canada has recently become a significant producer of lentil, and of the nearly 230,000 tons produced annually in that country (FAO, 1991), nearly all are exported. Canada has very quickly become the world's second largest lentil exporter. Lentil has been produced on a commercial basis in the Palouse region of the United States since 1937 (Youngman, 1968). The Palouse region, located in eastern Washington and northern Idaho and characterized by loess-rolling hills with elevations of up to 900 m, is the major production area in the United States. In that region, the crop is most often grown in rotation with cereals where lentil offers a needed alternative to break cereal disease cycles, provides a crop where grassy weeds can be adequately controlled, and, through nitrogen fixation, reduces the demand for nitrogen fertilizers. -

III. ORIGIN, TAXONOMY, CYTOLOGY, AND PLANT DESCRIPTION A.

ORIGIN

Cultivated lentils originated in the Near East arc and Asia Minor (Zohary, 1972; Williams et al., 1974; Ladizinsky, 1979a, 1993; Zohary and Hopf, 1988). Lens culinaris ssp. orientalis (Boiss) Handel-Mazzeti, which closely resembles the cul-

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Figure 1. Typical habitat of wild L. orienrulis.

tivated species L. culinaris, is widely accepted as the progenitor species. L. culinaris ssp. orientalis has an extended range and can be found throughout the Near East and as far east as Afganistan. The species is found in rocky and stony habitats with very little soil (Fig. 1) and in association with other annual legumes, such as the medics, and annual grasses. The conclusion that the cultivated lentil originated in the Near East arc from L. culinaris ssp. orientalis is based on discoveries of carbonized remains of apparent cultivated lentils in the same region over which L. culinaris ssp. orientalis is distributed. Such carbonized remains have appeared in early Neolithic settlements that date back to 7000-6000 BC (Helbaek, 1959). Evidence for the center of origin and domestication of lentil has been reviewed by Ladizinsky (1979a, 1993).

B. TAXONOMY Cultivated lentil ( L . culinaris) belongs to the genus Lens which is associated with other genera of the Vicieae tribe (Kupicha, 1981). The Vicieae tribe comprises Lens, Vicia L., Pisum L., Lathyrus L., and Vavilovia A. Fed. Cicer L. had

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previously been considered as part of the Vicieae, but anatomical and morphological evidence indicates that Cicer is quite different from other members of the tribe and it is now classified in the monogeneric tribe Cicereae Alef. (Kupicha, 1975, 1977; Clarke and Kupicha, 1976). In the Vicieae, Lens is described by Davis and Plitmann (1970) as holding a position that is intermediate between Vicia and Lathyrus, but closer to Vicia sec. Ervum. Lens is distinguished from Vicia by calyx morphology, stylar characters, and pod and seed shape. The calyx tube in Lens is subequal while the calyx tube is oblique in Vicia. Other calyx traits that distinguish the two genera include the lengths of the calyx teeth relative to the corolla tube. The primary gene pool of L. culinaris comprises ssp. culinaris and its presumed wild progenitor ssp. orientalis (Ladizinsky, 1993). Three other wild Lens species are recognized in the secondary gene pool and include L. odemensis Ladizinsky, L. nigricans (M. Bieb.) Godron, and L. ervoides (Brign.) Grande. Medikus is considered the authority for cultivated lentils because the publication of L. culinaris Medikus predates that of L. esculenta Moench. (Slinkard, 1974). L. montbretii (Fischer and C. Meyer) Davis and Plitm. has been removed from the genus Lens based on the work of Ladizinsky and Sakar (1982) and placed in the genus Vicia. Morphological and karyological information indicated considerable divergence between L. montbretii and other Lens species. Their foremost observation was that L. montbretii has 2n = 12 chromosomes whereas the other Lens species have 2n = 14. Based on that information, it is clear that L. montbretii is more appropriately classified as a species of Vicia. For karyotypes of L. culinaris, L. nigricans, and L! montbretii see Ladizinsky ( 1 993). Stipule shape is a major characteristic used to distinguish the wild Lens species (Ladizinsky et al., 1988). L. culinaris ssp. orientalis has stipules that are lanceolate entire and similar to those of L. culinaris ssp. culinaris and L. ervoides. L. nigricans and L. odemensis have semihastate or dentate stipules. The stipules of L. nigricans are oriented parallel to the stem while those of L. odemensis are oriented perpendicular to the stem and is a distinguishing feature used to differentiate between the two species (Ladizinsky, 1993). Synonyms used by Barulina (1930) for the Lens species include: L. esculenta Moench for L. culinaris; L. lenticula (Schreb.) Alefed. for L. ervoides; and L. kotschyana (Boiss.) Alefed. for L. montbretii (now L! montbretii). A more complete list of synonyms can be found in Barulina (1930) and in Cubero (198 1).

C. CYTOLOGY All Lens species are diploid annuals with 2n = 14 chromosomes. They also have similar karyotypes consisting of three pairs of metacentric or submetacentric chromosomes, three pairs of acrocentric chromosomes, and one satellited pair

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of chromosomes (Sharma and Mukhopaday, 1963; Ladizinsky, 1979a; Slinkard, 1985). The karyotypes of L. culinaris and L. nigricans have been presented by Ladizinsky (1993).

D.

I”T

DESCRIPTION

Plants of L. culinaris ssp. culinaris are herbaceous annuals with slender stems and branches (Fig. 2 ) . Plant height usually ranges from 25 to 30 cm for the ma-

Figure 2. Typical plant of L. culinaris.

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jority of genotypes, but may vary from 15 to 75 cm depending on genotype and environmental conditions (Saxena and Hawtin, 198I). Plants have a slender tap root with fibrous lateral roots. Rooting patterns range from a much-branched shallow root system to intermediate types that are less branched and more deeply rooted (Nezamuddin, 1970).The tap root and lateral roots in surface layers of the soil have numerous, indeterminate nodules that vary in shape from round to elongate (Saxena and Hawtin, 1981). The herbaceous stems of lentil plants are square and ribbed and are usually thin and weak. Primary branches arise directly from the main stem and may emerge from the cotyledonary node below ground or from nodes above ground. Secondary branches arise from primary branches, but plant habit is plastic depending on available space; the number of primary and secondary branches can vary depending on the genotype, the stand density, and prevailing environmental conditions (Malhotra et al., 1974; Wilson and Teare, 1972). The leaves are relatively small compared to those of other large-seeded food legumes. They are described as pinnate or imparipinnate and comprise as many as 14 sessile, ovate or elliptic, or obovate or lanceolate leaflets that vary in length from 1 to 3 cm. Each leaf is subtended by two small stipules and it may or may not terminate in a tendril. The entire stipules are oblong lanceolate and unappendaged (Davis and Plitmann, 1970; Summerfield et al., 1982). The flowers are borne singly or in multiples on peduncles that originate from the upper nodes of the plant. Each peduncle normally bears from one to three, rarely four, flowers, although seven flowers per peduncle have been reported for plants grown in a controlled environment (Hawtin, 1977). The individual flower is complete and has a typical papilionaceous (“butterflylike”) structure (Fig. 3). Flowers are small (4 to 8 mm long), white, pale purple, or purple blue. The flower has a calyx comprised of five equally elongated sepals

Figure 3. Typical lentil flower (A) and pods and seeds (B).

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that equal or exceed the length of the corolla of the unopened flower. The corolla has a standard two wings and two lower petals that lie internal to the wings and are united at their lower margin to form the keel (reviewed by Muehlbauer et al., 1980; Summerfield et al., 1982). The stamens are diadelphous (9 + 1) with the upper vexillary stamen free. The ovary is flat and glabrous; it normally contains one or two ovules that alternate along the margin and terminate in a short curved style. The style is pilose on the inner side, it usually develops at a right angle to the ovary, and is flattened on the outer side (reviewed by Muehlbauer et al., 1980; Saxena and Hawtin, 1981; Summerfield et al., 1985). The fruits (referred to as pods) are oblong, laterally compressed, 6 to 20 mm long and 3.5 to 1 1 mm wide, and usually contain one or two, rarely three, seeds (Saxena and Hawtin, 1981). Seeds are lens shaped and weigh between 20 and 80 mg. Seed diameter ranges from 2 to 9 mm and the testa may be light green or greenish red, gray, tan, brown, or black. Purple and black mottling and speckling of seeds are also common in some cultivars and accessions (Duke, 1981; Saxena and Hawtin, 1981; Vandenberg and Slinkard, 1990). Seed size differs according to genotype, and researchers frequently follow the classification of Barulina (1930), who grouped lentils as: “macrosperma” with large seeds that range from 6 to 9 mm in diameter and “microsperma” with smaller seeds that range from 2 to 6 mm in diameter. The macrosperma types are common to the Mediterranean basin and in the Western Hemisphere, while the microsperma predominate throughout the Indian subcontinent and in parts of the Near East. Other groupings of cultivated lentils have been described and include europeae, asiaticae, intermediate, subspontaneae, aethiopicae, and pilosae. Detailed descriptions of these groups are available in Muehlbauer et al. (1985).

IV.PRODUCTION OF LENTIL A. LANDREQUIREMENTS In the United States, lentil crops are sown in the spring in rotations that include winter wheat and barley. Lentils are commonly followed by winter wheat which is planted in the fall because the land is in very good condition following the legume. In the Palouse region, soil moisture is usually fully recharged by fall and winter precipitation. Barley, which is usually spring sown, follows the winter wheat crop. Alternatively, the lentil crop is planted in alternate years with winter wheat in a 2-year rotation. Lentil crops in the Palouse have better yields when they are planted on well-

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drained soils on south- and east-facing slopes. Yields on the tops of the usually eroded hills are poor when compared to that obtained on the slopes. However, yields in the low-lying areas of fields can also be poor because of an excess of fertility and moisture. Under such conditions the lentil crop develops abundant amounts of vine and foliage at the expense of seed yield.

B. SEEDQUALITY AND SEEDTREATMENT For optimum stands and yields it is recommended that producers use certified seed with greater than 90% germination and treated with seed protectants. Foundation, registered, and certified seed of improved cultivars is generally available. Seed treatments with appropriate fungicides such as Captan ' or Apron aid in preventing damping-off and ensure good stands. Treatment of the seed with an insecticide such as Lindane is beneficial for the control of wireworms and seedcorn maggots. Molybdenum is often applied to the seed along with other seed dressings. Molybdenum is recommended for crops in the Palouse region, where the soils are known to be deficient in the element and where the crop is known to respond to small amounts when applied to the seed. In areas where there is no deficiency, the crop is not likely to respond. Lentils require inoculation with the proper strain of Rhyzobium leguminosarum for good root nodulation and dinitrogen fixation. When planting fields for the first time or when there has been a period of time without lentil, it is important for good fixation that the crop be inoculated. Seeds of K sativa have occasionally appeared in seed lots of lentil harvested throughout the region. Vicia seeds are unwanted contaminants found in many lentil landraces. These contaminants are inadvertently carried with seed that is used to plant new production areas (Erskine et al., 1994). The seeds of the Vicia contaminants have approximately the same size, shape, and color as the germ plasm that they contaminate. These Vicia rogues can be distinguished by their blunt seed edges and by the hilum area which is generally more pronounced than in lentil. In the field, Vicia rogues are conspicuous for their large blue or purple flowers, pointed pubescent leaflets, and elongated pods containing six to eight seeds. Vicia seeds are considered contaminants in lentil crops and contribute to reduced grades; however, the Vicia rogue problem can be greatly reduced or eliminated by the use of certified seed. Hard seeds of the Vicia and their germination in lentil seed fields over a period of years are the primary means of contamination of seed stocks. 'Mention of this trade name or any other trade names does not imply endorsement by the USDA-ARS to the exclusion of other products which may also be effective.

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C. SEEDBED PREPARATION Land intended for lentil is usually rough tilled in the fall either by mold board or chisel plowing. The latter is recommended in order to keep previous crop residues on the soil surface to reduce soil erosion. Fall tillage is recommended to promote water infiltration into the profile and reduce runoff (Papendick and Miller, 1977). When soils are sufficiently dry in the spring, fields are harrowed and, after one or two tillage operations, herbicides are applied and incorporated by cross tillage. Soil temperatures above 6”C are needed for good germination and seedling growth.

D. SEEDING Lentils are often planted as early as possible in the spring with the same equipment used to plant cereals. The yield advantage from early spring planting can be substantial, provided seeds are not planted when the soil is too wet (Muehlbauer and Slinkard, 1981). Seeding depths of 4 to 5 cm are optimal for germination and growth, but deeper plantings are sometimes used either to provide better access to soil moisture or to place the seeds below the zone of incorporated herbicides. Despite some success with deeper plantings, particularly when soils are dry, lentils do not generally emerge well from deep planting, especially if the soils become crusted from heavy rains. Lentil seeds can germinate in light or in darkness and in constant or fluctuating temperature regimes. However, the rates of germination, emergence, and seedling growth are markedly affected by temperature (Summerfield et al., 1982). Optimum rates of germination and growth vary with cultivar, age, and size of seeds; smaller-seeded cultivars germinate more rapidly than larger ones at temperatures between 15 and 25°C (Saint-Clair, 1972). The successive stages of canopy formation (stem elongation, leaf initiation, leaf expansion, and branching) have different optimal thermal regimes (Summerfield, 198I ). This may help explain the so-called “dormant phase” referred to by farmers in which lentil seedlings, once emerged, often grow slowly, for several days or even weeks. This indicates that successive stages of vegetative development have warmer temperature optima. Lentil yields are remarkably stable over a wide range of population densities; the plants are able to fill available space by initiating lateral branches and can readily compensate for poor emergence and thin stands. Recommended seeding rates for farmers in the Palouse region of the United States are 65 to 80 kg ha I for the most commonly grown cv. ‘Brewer.’ Elsewhere, seeding rates vary from 15 kg ha - I for the microsperma types used in northern India to 115 kg h a - ’ for ~

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the larger-seeded types used for irrigated crops in Egypt (Hawtin et al., 1980). Optimum plant density has been estimated (Muehlbauer, 1973) at 90 plants m - 2 . Smaller-seeded cultivars appear to have greater tolerance to drought; they generally mature earlier than larger-seeded cultivars and avoid drought stress. In many of the dry regions of the world, the smaller-seeded cultivars, with their earlier maturities, are prevalent, possibly because they are considered more tolerant of drought and are able to avoid extreme water stress. The development of drought-tolerant cultivars has been given priority in lentil research programs in dry areas.

E. LENTILCULTWARS Lentil cultivars with improvements for one or more traits have been developed at a number of locations throughout the world. The most prominent program in lentil breeding was established in 1978 and is located at ICARDA located in Aleppo, Syria. That program has a world mandate for the genetic improvement of the lentil crop. Major emphasis has focused on the harvesting problem in traditional production areas and the need to develop germ plasm and cultivars that can be harvested mechanically. Improved germ plasm is provided to programs throughout the world for use in breeding programs. The ICARDA program has been active in the collection and preservation of wild Lens species in the center of origin and in the collection of landraces in the traditional production areas. The lentil cultivars released prior to the 1980s were mostly selections from germ plasm collections and were not from hybridization programs (Hawtin et al., 1980). However, current national and international lentil improvement programs now provide improved resources for hybridization and selection. These programs acknowledge the importance of collecting, introducing, exchanging, and maintaining germ plasm to provide as wide a range of genetic diversity as possible for breeding programs. Improved cultivars with a larger yield potential have been a direct result of these efforts. Cultivars used by growers in the Palouse have undergone a transition from an introduced landrace (‘Chilean’) that was used to initiate commercial production in the 1930s to cultivars developed by pure line selection and, more recently, from hybridization. The cultivars in use in the United States are described as follows. ‘Chilean 78’ is a composite of pure line selections from ‘Chilean’ which were made to remove unwanted variation and Vicia rogues from the seed stock. The Chilean stock from which the selections were made was the most commonly grown type in the region until the release of Chilean 78. ‘Brewer,’ released in 1984 (Muehlbauer, 1987), has largely replaced common Chilean and Chilean 78. Brewer has uniform large seeds with yellow cotyledons

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and is 4 to 7 days earlier to mature when compared to Chilean 78. Brewer has consistently given higher yields when compared to Chilean 78. At present, it is the main cultivar grown in the Palouse region and occupies over 90% of the production area. ‘Redchief‘ is a large red cotyledon type with nonmottled seed coats (Wilson and Muehlbauer, 1991). Yields of Redchief have been consistently better than Chilean or Chilean 78. Large red cotyledon types are entirely new and it has been necessary to develop markets for the Redchief type. The demand for the large red type has slowly but consistently increased in the United States. ‘Emerald’ is a bright green-seeded cultivar with distinctive green cotyledons (Muehlbauer, 1987). Production of Emerald has been extremely limited because, similar to Redchief, market demand needs to be created for green cotyledon types. ‘Palouse’ is a yellow cotyledon cultivar with large seed size, an absence of seed coat mottling, and early maturity (Muehlbauer, 1992). It produces yields comparable to Brewer and even though it is larger seeded it has resistance to mechanical damage during threshing and processing. ‘Crimson’ is a small-seeded, red cotyledon cultivar (Muehlbauer, 199I). Crimson is well adapted to Palouse conditions and gives comparable yields to Brewer; however, the cultivar also performs well in drier areas. Crimson was derived by pure line selection from ‘Giza-9,’ a cultivar developed in Egypt. Crimson was developed for use as an export commodity for markets that prefer the small red type. ‘Tekoa’ was the first lentil cultivar to be released in the United States (Wilson et al., 1971). The cultivar has large nonmottled seeds with yellow cotyledons. The cultivar has not been widely grown in the United States because of excessive amounts of mechanical damage during harvesting and processing. However, Tekoa has been produced successfully in Chile where its apparent resistance to rust has given the cultivar a distinct advantage. ‘Spanish Brown’ or ‘Pardina’ is a small yellow cotyledon cultivar with brown and speckled seed coats. The cultivar was introduced from Spain and is now being produced extensively in the Palouse. It has produced exceptionally good yields; however, recent observations indicate susceptibility to Ascochyta blight caused by Ascochyta fabae f. sp. lentis. Cultivars developed in Canada include ‘Laird’ (some Ascochyta blight resistance with large, yellow cotyledon, nonmottled seed) (Slinkard and Bhatty, 1979), ‘Eston’ (small, pale-colored seed), ‘Rose’ (red cotyledons), and ‘Indian head’ (small seeded with black seed coats; used primarily as a green manure lentil). Cultivars developed elsewhere include ‘Precoz’ (an early maturing cultivar) from Argentina (Riva, 1975); ‘Araucana-INIA’ (rust tolerant) from Chile (Tay et al., 1981); ‘Pant L-234’ (Fusariurn resistant) (Kamboj et al., 1990), ‘Pant-209,’ and ‘Pant-406’ from India; and Giza-9 from Egypt (Hawtin et al., 1980).

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V. FERTILIZATIONAND WEED CONTROL A. FERTILIZERS Nutrient requirements of lentil crops have not been adequately determined for the major lentil production areas. However, certain applications have been worthwhile. The periodic use of molybdenum as a seed dressing in the Palouse region of the United States is essential for good nodulation and dinitrogen fixation. Applications of sulfur are important for increasing concentrations in the seeds of sulfur-containing amino acids which are nutritionally limiting in lentil seeds. Phosphorus is applied to ensure good symbiotic performance and overall plant growth. Occasionally potassium is applied. Application rates of fertilizers recommended to growers are: Molybdenum: Sodium molybdate applied as a seed dressing at 35 g ha -I. Sulfur: Applied to other crops grown in rotation with lentils at 17 to 22 kg ha - I on deficient soils. Phosphorus: If soil tests (acetate extraction method) reveal phosphorus concentrations at 4 parts per million (ppm) or less, it is recommended that 44 to 66 kg ha - I be applied. Responses to phosphorus applications are commonly evident on severely eroded soils. Potassium: On sandy or severely eroded soils, 22 kg ha - I of potassium oxide has proved beneficial for yield and may also improve the cooking qualities of seeds (Wassimi et al., 1978). Nitrogen: Well-nodulated lentil crops seldom respond to applications of inorganic N fertilizer. The “nitrogen hunger” phase, which is often experienced by grain legumes when crops are seeded early into cool, wet soil before significant symbiotic dinitrogen fixation begins, can be avoided by the application of a small starter dose of 10-25 kg ha - I inorganic nitrogen placed adjacent to, but not in contact with, the seeds (Saxena, 1981). Inoculation with an appropriate strain of R. leguminosarum is necessary when lentils are seeded into fields for the first time or after a lapse of several years. Special care should be taken when using fungicide seed dressings potentially toxic to Rhizobium.

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B. WEEDCONTROL Lentils are poor competitors and good weed control is essential for successful production. Lentil growth rates are slow during early stages of vegetative growth and weeds can quickly overgrow the crop if not adequately controlled. Hand weeding is practiced in traditional production areas, but is impractical in the extensive production systems used in the United States. It is therefore necessary that effective herbicides be used to reduce unwanted competition. Certain herbicides have been effective in controlling broadleaf and grass type weeds in lentil crops; however, only a limited number are registered for use in the United States. Wild oat can be controlled with preplant-incorporated applications of triallate (Far-go). However, triallate does not control other annual grasses. After crop emergence, sethoxydim (Poast) applications control both annual and perennial grass weeds. For broadleaf weed control, imazethapyr (Pursuit) can be applied prior to planting, followed by shallow incorporation or applied preemergence soon after planting. Metribuzin (Lexone/Sencor) can be applied preemergence, postemergence, or as a split application. Metribuzin gives good or excellent control of a wide spectrum of broadleaf weeds with few exceptions. Reemergence applications of both herbicides require adequate rainfall in order to distribute the herbicide into the zone where weed seeds germinate. Under conditions of excessive rainfall and on soils with minimal organic matter, metribuzin may leach deeper into the profile and cause crop injury. Injury is the most severe on the tops of eroded hills where soils have minimal organic matter and where lentils may have been seeded too shallow. Dry conditions reduce the effectiveness of soil-active herbicides and weed control may be poor.

VI. PRINCIPAL USES A. FOOD A major food use of lentil is as dhal, decorticated and split lentils, which is a principle ingredient in soups and other dishes prepared on the Indian subcontinent. Besides their use in soups, whole lentil seeds are often ground into flour and added to cereal flour in the preparation of breads and other baked products. Lentils are also used in dishes containing rice and cereal grains. When combined with cereal grains, lentil provides a nutritionally well-balanced diet for consumers. The relatively large concentrations of lycine compensate for the minimal concentrations

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in the cereal grains while the cereal grain compensates for the minimal concentrations of sulfur-containing amino acids in lentil.

B. FODDER Feed for livestock is an important use for the residues from the lentil crop. In the traditional production areas of the Middle East, West Asia, North Africa, Ethiopia, and the subcontinent of India, residues from threshing of the lentil crop are essential for livestock feeding. In some years the residues have commanded prices equal to or greater than that of the grain.

C. GREENMANURE A green manure lentil cultivar (Indian head) is used to a limited extent in Canada. The cultivar is capable of producing an abundance of foliar material which can be incorporated and improve soil nutritional status.

Vn. MAJOR CONSTRAINTS TO PRODUCTION A. INSECTS Lentils are attacked by a variety of insects during crop development and in storage (van Emden et al., 1988). The principle soilborne insects, which attack seeds and developing seedlings soon after planting, include seedcorn maggots [ Delia platura (Meigen)], wireworms (Limonius spp. and Ctenicera spp.), and cutworms (Agrotis spp.). The larvae of leaf weevils (Sitona spp.) feed below ground on the root nodules which inhibits nitrogen fixation. A variety of insects feed on the leaves, stems, and flowers: thrips (Frankliniella spp.), aphids [Aphis craccivora (Koch) and Acyrthosiphon pisum (Harris)], leaf weevils [Sitona lineatus (L.)], lepidopterous larvae (Helicoverpa and Spodoptera spp.], and grasshoppers. The most important insect pests of the pods and seeds include lygus bugs (Lygus spp.), bruchid beetles (Bruchus spp. and Callosobruchus spp.), and lepidopteran pod borers [(Helicoverpaarmigera (Hub.), Cydia nigricana (F.), and Etiella zinckenella (Treitschke)]. Bruchid beetles are also major postharvest pests, except in the United States. Economically important pests of lentil in the Palouse region are the pea (A. pisum) and cowpea aphids (A. craccivora), lygus bugs, the western yellow-striped armyworm [Spodoptera praejica (Grote)], and, to some extent, seedcorn maggots

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and wireworms. Lentil fields in the Palouse can be devastated by aphid-vectored pathogenic viruses (see subsequent section) and aphid-induced feeding damage when pea aphid densities intermittently reach outbreak levels. The cowpea aphid (black and smaller than the light green pea aphid) damages lentil through direct feeding: its role in vectoring viruses is poorly understood. Several factors seem to favor pea aphid outbreaks: (1) fall buildup of aphids on alfalfa and other perennial host plants: (2) mild fall and early winter temperatures favoring abundant egg laying by aphids, and thus a large overwintering population; (3) mild winter temperatures: and (4) spring conditions conducive to early movement of aphids from overwintering hosts to lentils (Homan et al., 1991). Aphids have many natural enemies, including lady bird beetles, parasitic wasps, lacewings, and syrphid flies, but chemical control may be necessary if these insects do not combine to keep aphids at subeconomic levels. Insecticide treatment for pea aphid control is considered when an economic threshold of 30 to 40 aphids are collected per 180” sweep of a 38-cm-diameter insect net, with few natural enemies present, and aphid numbers do not decline over a 2-day period (Homan et al., 1991). Lygus bug feeding on the immature reproductive structures of lentil causes seed and pod abortion as well as a serious seed quality problem known as “chalky spot” in crops grown in northern Idaho and eastern Washington in some years (Summerfield et al., 1982). Lygus bugs feed with piercing-sucking mouthparts and inject toxic saliva into the immature seed. This action results in the formation of a depression around the feeding area and a chalky blemish. Adult lygus bug activity can be monitored during bloom and podding by making 25 of 180”sweeps in at least five randomly selected places in a field. Chemical control is warranted when 7 to 10 adult lygus bugs are collected per 25 sweeps (O’Keeffe et al., 1991). The western yellow-striped armyworm is usually a late season pest. When heavy infestation develops, larvae can defoliate plants and consume pods.

B. DISEASES Some of the more serious disease problems of lentil include the following. 1. The Root Rotmilt Complex

Probably the most important disease problems of lentils worldwide are root rots and wilts caused by P-ythium, Rhizoctonia, Sclerotinia, and Fusarium species (Kaiser, 1987). Research is underway toward selection for resistance to the various components of the root rot/wilt complex. Reports on the inheritance of resistance to Fusarium wilt have been made in germ plasm from India (Kamboj eral., 1990). Two other important diseases of lentil in many countries, especially in wetter areas or during years with heavy rainfall, are rust and Ascochyta blight.

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2. Rust Caused by Uromyces viciae-fabae Pers., rust is a serious problem in areas where mild temperatures and humid conditions favor development of the disease. Some sources of resistance have been identified and progress toward developing resistant cultivars is being made (Khare, 1981). Fortunately, rust of lentils has not yet 'appeared in the Palouse region of the United States.

3. Ascochyta Blight Blight caused by Ascochyta fabae Speg. f.sp. lentis Gossen et al., a seedborne disease, causes severe damage in many cool, wet regions (Fig. 4). Work in several countries has identified good sources of resistance and these lines are being incorporated into breeding programs. Ascochyta blight is becoming a major problem in the United States and it continues to be an economic problem in the lentilproducing areas of Canada. Breeding programs have been initiated to introduce into other lentil cultivars the resistance shown by the cultivar Laird (A. E. Slinkard, personal communication). Thiabendazole seed treatment can reduce the incidence of seedborne A. fabae f. sp. lentis, but the compound is not registered for use on lentil in the United States (Kaiser, 1987).

4. Seedborne Fungi In the Palouse, reduced seed quality can result from infection of seeds by different pathogenic fungi, some of which are also pathogens of chickpea and pea (Kaiser, 1992). The incidence of fungi associated with commercial lentil seeds in the Palouse varies greatly from year to year and is influenced by weather conditions, particularly rainfall. The seedborne pathogens most frequently isolated from discolored Palouse grown lentil seeds are Botrytis cinerea, Phoma medicaginis var. pinodella (= Ascochyta pinodella), and two Fusarium species ( E acuminaturn and E avenaceum). The amount of rainfall during July, when the crop is approaching maturity or is about to be harvested, appears to affect the incidence, prevalence, and severity of seedborne pathogenic fungi. If excessive rainfall occurs during harvest or when plants are drying in windrows, lentils that remain on or near the moist soil surface may have discoloration of their seeds resulting from these conditions that favor colonization and infection of the pods and seeds by several pathogenic and saprophytic fungi.

5. Viruses Viruses are a major lentil disease problem in the Palouse. The viruses that infect peas also infect lentil: alfalfa mosaic, bean (pea) leaf roll (BLRV), bean yellow

PRODUCTION AND BREEDING OF LENTIL

Figure 4.

Ascochyta blight of leaves (A), pods (B), and seeds (C) of lentil.

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mosaic, pea enation mosaic (PEMV), and pea streak. These viruses are transmitted by pea aphids, generally from infected alfalfa and clover plants. Control of the aphid vectors can reduce infection, but the economic thresholds are not known and insecticides are often ineffective in preventing the spread of stylet-borne viruses. In the Palouse region, PEMV and BLRV are the most important virus diseases of lentil, but the crop is also a host of pea seedborne mosaic virus (PSbMV). Indeed, PSbMV may cause stunting and malformation of leaves, stems, flowers, and fruits. There may also be a reduction in yield and the production of smaller, misshapen seeds. Fortunately, PSbMV of lentil has not been found under field conditions in the Palouse. Sources of resistance to PSbMV have been identified and are being used in the development of lentil cultivars (Kaiser, 1987). Sources of tolerance to PEMV have been identified (F. J. Muehlbauer, personal observations) and are currently being incorporated into improved cultivars.

c. ENVIRONMENTAL STRESS Drought is considered to be the major environmental stress that limits lentil yields. Lentil crops are often grown in marginal areas where limited rainfall and deficient soil moisture are encountered. The relegation of lentil to marginal lands is a consequence of the increased area sown to more renumerative crops (e.g., wheat). It is not surprising then that average lentil yields have declined in those regions. In general, drought and heat stress are commonly encountered by lentil in the Middle East region and are experienced during the reproductive period (Erskine, 1985a). Drought-tolerant cultivars are required to stabilize lentil production and also to extend lentil cultivation into those areas that receive less rain. As with other crops, success in screening for drought tolerance is not yet possible because of the lack of efficient screening techniques and knowledge of what to screen for. Late planting has been used to simulate drought and heat stress; however, meaningful comparisons between late and normally planted lentil might be difficult because of the difference in plant growth duration. A line source irrigation system was used to create a moisture gradient in an area with otherwise insufficient rainfall (Karaki, 1986). Under the line source system, there was considerable variation among 10 lentil genotypes in response to moisture stress which gives encouragement for further work. Another approach to drought resistance screening is to select for drought avoidance through early maturity. This could be a suitable approach when the major moisture stress occurs toward the end of the growing season. However, in seasons that receive adequate rainfall, early genotypes may not have the ability to respond to above-average moisture if and when available. Agents that simulate moisture stress have been tested for use in drought resistance screening. Haddad et al. (1987) screened 40 lentil genotypes by growing

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1-week-old seedlings in different concentrations of polyethylene glycol (PEG) solutions that created different degrees of moisture stress. These genotypes were grouped into strongly resistant, moderately resistant, and susceptible types. Landraces are potential sources of drought tolerance because they have demonstrated their ability to survive under extremely stressful environments. The wild species such as L. culinaris ssp. orientalis might also have considerable drought tolerance. These potential sources of drought tolerance need to be considered by breeders. Hot weather is thought to cause aborted seeds, empty pods, and reduced yields in Brazil (Manara and Manara, 1983). Elsewhere, cold tolerance and salt tolerance, if found, could be used by breeders to extend the range of adaptation of the crop. Jana and Slinkard (1979) found differences in salt tolerance in the cultivated lentil, but the level of tolerance was too small to be of any breeding value.

VIII. HYBRIDIZATION METHODS A. HYBRIDIZATION Lentil flowers are cleistogamous and naturally self-pollinated, with extremely infrequent natural cross pollination (estimated to be less than 0.8%)which is presumed to be caused by small insects such as thrips (Wilson and Law, 1972). Hybridization of lentil flowers with hand emasculation and pollination is difficult because they are small and delicate and must be handled with care. However, the selection of flowers in the correct development stage followed by careful emasculation and pollination can lead to a high percentage of successful cross pollinations. To obtain successful hybridizations it is essential that the reproductive system crossing techniques and environmental conditions be fully understood.

B. ENVIRONMENTAL CONDITIONS Environmental factors play a major role in the degree of success in lentil hybridization. Flowering and seed set, for example, are improved by a photoperiod (16 hr or longer) and good irradiance. Flowers usually open before 10.00 hr on cloudless days, but when the sky is overcast, they may not open until 17.00 hr. The corolla petals fade within 3 days, and pods are visible 3 or 4 days later (Meimandi-Nejad, 1977; Muehlbauer et al., 1985; Summerfield et al., 1985). Lentils are considered to be either long-day or day-neutral plants (Shukla, 1955; Moursi and Ab El-Gawad, 1963; Saint-Clair, 1972; Summerfield et al., 1985). Under greenhouse and growth chamber conditions, day temperatures of about 25" C and night temperatures of about 18"C are reasonable combinations for good

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plant growth and seed set. Seed set can also be improved by maintaining relative humidity at about 50% (Wilson, 1972).

C. EQUIPMENT NEEDED The equipment used to hybridize lentil consists of magnifying glasses (usually 7 or 10 X ), sharply pointed forceps, small tags, and 95% alcohol. Persons with keen eyesight can hybridize lentils without the aid of magnification. The forceps used for emasculation and pollination are immersed in the alcohol between crosses to prevent contamination by unwanted pollen. Tags are used to record the parents, the date of the cross, and sometimes the initials of the person who made the cross.

D. EMASCULATION OF THE FEMALE FLOWER Emasculation is necessary to prevent self-pollination.Flowers in which the corolla has reached a length equivalent to about 75% of that of the sepals (Fig. 5A) are generally at the proper stage for emasculation. At this stage of development,

Figure 5. Cross-pollination in lentil.

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the anthers are intact and the stigma is receptive. When the lengths of the corolla and sepals are equal, the flowers generally will have pollinated. The flower bud selected for emasculation is held between the thumb and forefinger so that the suture of the keel is accessible. The peduncle holding the flower is weak and tender and must be handled with care. The sharply pointed forceps are used to remove the sepals closest to the keel. The wings and the standard are folded back and held between the thumb and forefinger. The keel is then split longitudinally to expose the staminal column and stigma. While carefully holding the flower, the stamens are removed by grasping the filaments with the forceps and breaking them free from the staminal column to complete the emasculation (Fig. 5B).

E. POLLINATION Pollination should be performed immediately after emasculation for best results (Wilson, 1972; Malhotra et al., 1978; Muehlbauer et al., 1980; Muehlbauer and Slinkard, 1981). Flowers in which the corollas have elongated to a length about equal with that of the sepals are selected as sources of pollen (Fig. 5C). The flowers at this stage of development are open or slightly so and have anthers that have very recently dehisced. Viable pollen suitable for transfer is identified by its bright orange-yellow color and is contained in flowers in which the keel and wing petals are turgid. Pollen retains its viability for several days if flowers are collected immediately after anthesis, placed in petri dishes, and stored in the dark at 4- 10”C. The flower chosen as a source of pollen is held between the thumb and forefinger and the standard and wings are folded back and held. At this point, two methods of gaining access to the pollen can be used and are equally successful. One approach is to grasp the keel with the forceps and remove it from the flower to expose the pollen laden stigma. This pollen laden stigma can then be used as a “brush” to apply pollen to the emasculated flower to complete the crossing procedure (Fig. 5D). Another method is to puncture one side of the keel with a single prong of the forceps and peel the keel away from the stigma. This approach often leaves more pollen on the stigma compared to the former approach. Pollination of the emasculated flower is performed as described earlier. Sufficient pollen should be applied to the emasculated flower so that it is readily visible on the stigma; this may require several male flowers. The petals of the pollinated female flower are then carefully returned to their original position to protect the stigma. Pollinated flowers seldom need protection from foreign pollen. After pollination has been completed, a tag is fixed to the internode directly below the pollinated flower to identify the cross. An alternative method of identifying crosses is to use color-coded thread tied to the peduncle of the flower or to the internode directly below.

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Successful pollinations and seed setting vary according to individual skill and prevailing environmental conditions. Wilson (1972) reported up to 82% successful manual pollinations in a greenhouse environment. Success in that study was achieved with adequate irradiance, about 50% relative humidity and temperatures between 15 and 25°C. Pollination success greater than 80% was obtained by Slinkard in controlled environment chambers with dayhight temperatures of 21"/15"C (Muehlbauer and Slinkard, 1981). The percentage of successful pollinations decreases under field conditions, especially during hot weather with dry air. Malhotra et al. (1978), for example, reported 27 to 43% success under field conditions in India. Mera and Erskine (1982) found that enclosing plants in perforated polyethylene bags improved the success rate compared to unbagged controls. They also discovered that pollinations of the first flowers on a plant had a 44.8% success rate compared to only 17.3% when later-formed flowers were used.

F.

OTHER CONSIDERATIONS FOR CROSSING

A reliable genetic marker for the identification of F, hybrid seed is red cotyledon color, a trait controlled by a single dominant gene. In using this marker, F, hybrid seeds from crosses between yellow cotyledon female parents and red cotyledon male parents can be identified by their red cotyledons. Seeds with yellow cotyledons can be discarded as selfs. The gs gene for stem coloration is also a useful marker to identify F, hybrids where the male parent has Gs for purple stems and the female parent has gs for green stems. Because of the differences in seed size between lentil accessions, it is advisable to use large-seeded parental lines as the female parents. This reduces losses from shattering since the pod of the large-seeded parent can accommodate the hybrid seed while that of the small-seeded parent often cannot. Erskine (personal communication) uses the reverse because the smaller-seeded parent will often yield two hybrid seeds when used as the female. However, timely harvest is necessary to prevent losses due to shattering. Pod and seed development can be observed within 3 days of pollination. When cross-pollination is successful, ovary development is rapid and the swelling seeds in the pod are obvious. But, when crosspollination is unsuccessful, the ovary often enlarges but aborts later even when the developing pod may have reached as much as 50% of its potential size. Harvesting and separating the seeds from the pods are always done by hand, as soon as the pods become yellow-brown, so as to avoid seed losses from pod dehiscence. Harvested pods should be allowed to dry completely in envelopes or bags before removing the seeds. Hybrid seeds can usually be kept at room temperature provided they are to be sown in a reasonable period of time. For long-

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term storage, lentil breeding material may be stored at about 10"C with 30% relative humidity or in a freezer at - 20" C.

IX. GENETIC RESOURCES A. GERMPLASMCOLLECTIONS Germ plasm resources for lentil improvement programs are maintained at a number of places, including the USDA-ARS Regional Plant Introduction Station located at Pullman, Washington (Table I). Large collections are also maintained by ICARDA and by the Indian Agricultural Research Institute in New Delhi, India. Smaller collections are maintained by programs in several countries. [For more information on germ plasm collection, curators, and addresses, see Bettancourt et al. (1 989).] These germ plasm collections comprise the landraces and, in the case of the larger collections, the related wild species (Muehlbauer, 1992). The ICARDA collection is by far the largest and comprises over 6000 accessions, including wild species from 53 countries. The USDA collection numbers Table I Lentil Germ Plasm Collections Institute/location 1.

2. 3. 4. 5.

6.

7.

8.

9.

Ethiopian Genebank/Addis Ababa, Ethiopia ICARDA/Aleppo, Syria National Seed Storage Laboratory/ Fort Collins, CO Pakistan Agricultural Research Council/Islamabad,Pakistan Regional Plant Introduction Station/ Pullman, WA Vavilov Institute of Plant Industry/ St. Petersburg, Russia ZG Kulturpflanyenforschung/Gatersleben, Germany Institute of Crop Germplasm ResourceslBeijing, Peoples Republic of China Indian Agricultural Research Institute/ New Delhi, India

Number of accessions 413 6000 702 144

1973 2470 160 336

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over 3500 accessions and includes more that 150 accessions of related wild Lens species. The collection maintained at the Indian Agricultural Research Institute in New Delhi exceeds 3000 accessions. Samples for breeding and other research purposes are available on request. An evaluation of 4500 accessions from ICARDA’s collection suggested the presence of considerable variation for important agronomic traits such as grain yield, straw yield, 100 seed weight, seed number per pod, time to 50% flowering, time to maturity, plant height, height of lowest pod, and pod number per peduncle (Erskine, 1985b). Thus, significant variation for important traits is available for breeding purposes.

B.

COLLECTION AND UTILIZATION OF WILD SPECIES

All of the recognized wild Lens species have been collected and are being maintained in germ plasm repositories, including L. culinaris ssp. orientalis, L. ervoides, L. nigricans, and L. odemensis (Ladizinsky, 1979c; Muehlbauer, 1981). The wild material has been the basis of a series of genetic and cytogenetic studies (Ladizinsky, 1979a,b). Recent efforts have concentrated on collection and preservation to ensure that the wild relatives are available for future breeding. Even though collections have been made in southern Europe, the Middle East, and Asia Minor, wild species are still relatively underrepresented in the germ plasm collections (Muehlbauer, 1981; Erskine, 1985b). It is believed that the wild species will contribute to the improvement of pest resistance, seed quality, and tolerance to environmental stress.

X. GENETICS

A. QUALITATIVELY INHERITED T ST R A I Lentil genetic information has progressed from relatively few characterized genes in 1981 (Muehlbauer and Slinkard, 1981) to the characterization of additional genes, isozyme loci, restriction fragment length polymorphisms (RFLPs), and random amplified polymorphic DNAs (RAPDs) (Zamir and Ladizinsky, 1984; Havey and Muehlbauer, 1989; Simon et al., 1993). A genetic linkage map for these loci now contains nearly 100 loci (Simon et al., 1993). The map (Fig. 6) has shown considerable conservation with the linkage map for pea (Weeden et al., 1994). The described genes of lentil are listed in Table 11.

1

2

4

3

5

i i

EMH14b

Gsyn-c

Pgm-c Fk CMHS8

CMH65a

PM H l 1l c CMHS2a

Rp122 Aat-mb

Aat-m Me-2

Adh

CMH4I

Dashed lines indicate regions showingsimilar linkage in pea. Regions have been situated to correspond to placement on the pea map. with the following exceptions. A: Linkage region found on chromosume5 or pea B: Linkage region found on chromosome 3 of pea

Est-1 Gal-2

EMH14a

[=Ten centimorgans \indicates

Figure 6.

suspected arrangement based on the pea map

The lentil genetic linkage map.

Table I1 List of Gene Symbols in Lentils Symbol/ allele

Fn Gh

Gs I 0 Ggc TRC

P Pi

Sbv SCP V W YC

GlP GrP

Tnl Chl

Character

Reference

Number of flowers per inflorescence Plant growth habit Epicotyl color Cotyledon color Cotyledon color (synonymous with Yc) Gray seed coat ground color Tan seed coat ground color Flower color Pod indehiscence Resistance to pea seedborne mosaic virus Seed coat spotting Flower color

Gill and Malhotra (1980) Ladizinsky (1979b) Ladizinsky (1979b) Slinkard (1978b) Singh (1978) Vandenberg and Slinkard (1990) Vandenberg and Slinkard (1990) La1 and Srivastava (1975) Ladizinsky (1979b) Haddad et al. ( 1978) Ladizinsky (1979b) La1 and Srivastava (1975); Wilson and Hudson (1978) Wilson and Hudson (1978) Slinkard (1978b) Vandenberg and Slinkard (1989) Vandenberg and Slinkard (1989) Vandenberg and Slinkard (1989) Vandenberg and Slinkard (1989)

Flower color Cotyledon color Glabrous pod Green pod color Tendril-less leaf Chlorina chlorophyll mutant

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1. Cotyledon Color Tschermak (1928) determined that the red cotyledon color of lentils was dominant to yellow. This finding was later confirmed by Wilson et al. (1970). Hybrid seed from reciprocal crosses indicated that the red cotyledon color was completely dominant to yellow. The F,, which segregated into 3 red to 1 yellow, confirmed that the red cotyledon color was controlled by a single dominant gene. Wilson (1972) also noted that red cotyledon seeds resulting from crosses between yellow cotyledon maternal parents and red cotyledon paternal parents were readily distinguishable from selfed maternal seeds with yellow cotyledons and that the cotyledon color trait would be a valuable genetic marker in hybridization programs to confirm that resulting seeds were indeed hybrids. Both Singh (1978) and Slinkard (1978b) confirmed the mode of inheritance of cotyledon color. Singh (1978) referred to the red cotyledons as orange and proposed the gene symbols 0 for orange and o for yellow cotyledons. He also noted that the intensity of the orange cotyledon color did not vary in reciprocal crosses and so concluded that there was no cytoplasmic effect on gene expression. In addition to the inheritance of red and yellow cotyledons, Slinkard (1978b) reported on the inheritance of green cotyledons. Crosses between red cotyledon parents and green cotyledon parents produced F, progenies that contained red, yellow, and green cotyledon types. Those progenies indicated that red was dominant to both yellow and green, and that yellow was dominant to green. A study by Muehlbauer and Short (manuscript in preparation) confirms the findings that red cotyledon color is dominant to both yellow and green, and that yellow is dominant to green. However, there was no evidence for the presence of a recessive color inhibitor as suggested by Slinkard (1978b). The data clearly show that dominant red cotyledon color is epistatic to both yellow and green. The gene symbols proposed are: Yc = red cotyledon regardless of the presence of I or i. In the absence of dominant Yc, yellow and green cotyledon types are expressed such that y c yc I- = yellow cotyledons and yc yc i i = green cotyledons.

2. Flower Color Flower color in lentil is reportedly controlled by two genes. La1 and Srivastava (1975) obtained F, ratios of 3 violet: 1 white from crosses of violet- and pinkflowered parents. However, when pink- and white-flowered parents were crossed, a F, ratio of 9 violet: 3 white: 3 pink : 1 rose was obtained which indicated the presence of two independent genes with complete dominance and no epistasis. The gene symbols proposed were: V for violet flower with color expressed only in the presence of P; p produces pink flowers in the presence of v; and the double recessive vv p p produces rose-colored flowers. Thus, the homozygous genotypes and phenotypes are W PP (violet), W p p (white), vv PP (pink), and v v p p (rose).

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Wilson and Hudson (1978) studied the progenies of crosses between white- and violet-flowered parents and obtained a F, ratio of 9 violet: 6 intermediate : 1 white, suggesting additive effects of the genes involved. They noted that the intermediate class consisted of two types that were difficult to distinguish from each other: one was darker and had pale violet standards and white wings and keels while the other type was lighter with a pale violet standard and white wings and keel. The white flowers often had pale pink or pale violet veins deep within the throat of the standard. They proposed V as the symbol for violet and ww for white. The homozygous genotypes and phenotypes were suggested as follows: WW W (violet), W w w and vv WW (intermediate),and vv ww (white). Gill and Malhotra (1980) obtained a 3 violet: 1 white flower color ratio in F, progenies which was the same as found by La1 and Srivastava (1975). They also studied the linkage between genes for flower color, presumably V, and the gene for the number of flowers per inflorescence ( F n ) and found no association between the two genes. The differing results between the studies of La1 and Srivastava (1975) and those of Wilson and Hudson (1978) suggest that additional work is needed on the genetics of flower color in lentils. The complexity of flower color inheritance in other grain legumes suggests that a similar complex system may exist in lentils. The work done by Ladizinsky (1979b), using interspecific crosses of L. culinaris ssp. culinaris with the closely related L. culinaris ssp. orientalis, indicates that genes in addition to the Vgene may be involved in the inheritance of flower color. In those interspecific crosses, he found what appeared to be an additional gene, a conclusion he based on segregation for bluish flower color which he presumed was different from the two genes previously reported (La1 and Srivastava, 1975).

3. Seed Coat Color Seed coat coloration is controlled by several genes, and pleiotropic action is likely for epicotyl and flower color because of what appears to be concurrent variability for the traits. Pleiotropic action of genes for coloration is expected and has numerous parallels in other genera, including Cicer and Pisum. A single gene for seed coat spotting was proposed by Ladizinsky (1979b), who found a 3 : 1 ratio of F, plants with spotted and nonspotted seedcoats and proposed the symbol Scp for the gene controlling this trait. Nonspotted individuals would have the scp scp genotype. Five alleles of the Scp gene have been reported (Vandenberg and Slinkard, 1990). The background color of lentil seed coats is reportedly (Vandenberg and Slinkard, 1990)controlled by two genes. Dominant Ggc determines gray ground color while the dominant Tgc gene produces tan ground color. When both dominant genes are present (Ggc Tgc), brown seed coat color is produced. The double recessive for these genes (ggc fgc)has a green seed coat color.

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Vaillancourt et ul. (1986) reported a recessive gene tun for zero tannin in the seed coat. The trait appears to be useful in the development of lentil cultivars in which the seeds do not darken with aging. Vandenburg and Slinkard (1987) reported a recessive gene xun for a xantha mutant. The xun gene is epistatic to and independent of the two loci conditioning cotyledon color. Thus, cotyledons of seeds homozygous for xantha are yellow regardless of the genotype for cotyledon color. A chlorina chlorophyll mutant was reported to be controlled by a single recessive gene designated as chf (Vandenberg and Slinkard, 1990) and produces pale yellow foliage.

4. Epicotyl Color Epicotyl color is simply inherited with purple epicotyl dominant to green epicotyl (Ladizinsky, 1979b). The trait is useful as a genetic marker to identify F, hybrids. The gene symbol Gs has been proposed with purple epicotyl having the Gs GS genotype and green epicotyl having gs gs. The intensity of the pigmentation is suspected as being influenced by environmental conditions (e.g., temperature, irradiance, and soil fertility). These environmental effects can sometimes make classification of plants difficult.

5. GrowthHabit Erect, intermediate, and prostrate growth habits were found in ratios that indicated a single gene with incomplete dominance (Ladizinsky, 1979b). Based on that study, the prostrate type was designated as gh gh and the erect type as Gh Gh. This gene has significance for lentil breeders who are attempting to develop upright and lodging-resistant cultivars. A single recessive gene designated as tnl by Vandenberg and Slinkard (1990) reportedly controls the presence or absence of tendrils on the ends of the leaves. This trait also has significance for lentil breeding in that good tendril activity is important for canopy formation and lodging resistance by the plants with tendrils being able to intertwine and provide mutual support.

6. Flower Number In crosses of parents with two-flowered racemes with parents with threeflowered racemes, Gill and Malhotra ( 1 980) obtained a F2ratio of 3 two-flowered : 1 three-flowered plants. They proposed that the gene symbol Fn be used for the trait, with Fn Fn being two-flowered andfnfn being the three-flowered genotype.

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7. Pod Indehiscence Genes which are expressed in the pod include Glp and Grp (Vandenberg and Slinkard, 1990). The dominant allele of Glp produces pod pubescence while the homozygous recessive allele (glp) produces glabrous pods. The dominant Grp gene produces red pods while the homozygous recessive grp allele produces green pods. Pod indehiscence is the major distinction between wild and cultivated species of Lens and is considered to be the trait most critical to domestication of the species. In crosses of the putative progenitor, L. culinaris ssp. orientalis, with L. culinaris ssp. culinaris, Ladizinsky (1979b) found that pod indehiscencewas controlled by a single recessive gene and assigned the symbol pi. Therefore, the indehiscent-cultivated types were designated as pi pi and the wild progenitor, L. culinaris ssp. orientalis, as Pi Pi. Variability for shattering susceptibility within cultivated germ plasm, even in the presence of recessive pi, indicates the presence of genes that modify the indehiscent pod trait. Breeders of the crop need to improve shatter resistance, which has proved troublesome in certain circumstances.

8. Virus Resistance Resistance to PSbMV was found in lentil germ plasm (Haddad et al., 1978). In crosses of two resistant accessions (PI 368648 and PI 212610) with known susceptible cultivars, they obtained a F2 ratio of 3 susceptible: 1 resistant, indicating that a single recessive gene conferred resistance. The recessive gene for PSbMV resistance was designated as sbv with dominant Sbv conferring susceptibility. Similarly, Aydin el al. (1987) found resistance to pea enation mosaic virus in lentil germ plasm. Resistance to PEMV does not exhibit an immune response to the virus; however, symptoms on inoculated plants were extremely mild, indicating a very useful degree of tolerance. Short (1994) has shown that resistance to PEMV is conferred by a single recessive gene.

9. Isozymes Polymorphisms at isozyme loci have become an important tool for studying genetic variation and for lentil improvement. Using isozymic variation in conjunction with morphological genes, genetic linkage maps have been established that should prove to be useful tools in the future. Linkage of enzymic and morphological genes in lentil was first reported by Zamir and Ladizinsky (1984). The inheritance of eight isozyme loci was determined and two linkage groups involving five loci were established, one of which

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included epicotyl color (Gs).Linkage between Gs and an isozyme of glutamateoxaloacetate-transaminase (Got-2) (14 map units apart) and between Gs and an isozyme of Malic enzyme ( M e - I ) (25 map units apart) was shown. Linkage between Got-3 and an isozyme of alcohol dehydrogenase (Adh-I) (21 map units apart) was also shown. Segregation for epicotyl color in the hybrids confirmed that epicotyl color was controlled by a single gene, previously designated as Gs with purple dominant to green. Codominance was evident for the alleles at the loci involved in the control of the isozymes. Additional linkage groups and preliminary gene maps of lentil were subsequently reported (Muehlbauer et al., 1989; Havey and Muehlbauer, 1989). Allozymic polymorphisms for 18 loci and the genes for four morphological traits were analyzed by Muehlbauer et al. (1989) to produce a linkage map for lentil consisting of six linkage groups. Havey and Muehlbauer (1 989) constructed a more detailed map for lentil by including RFLPs. This latter map contained 34 loci arranged in nine linkage groups. Simon and Muehlbauer (1992) reported a lentil linkage map (Fig. 6) containing over 100 loci that include morphological and isozymic markers, RFLPs and RAPDs.

B. QUANTITATIVE INHERITANCE Estimates of genetic parameters for quantitative traits are very useful since they provide information on the inheritance of such traits and help to identify appropriate breeding methods. Such parameters include the subdivision of genetic variance into proportions resulting from additive, dominance, and epistatic effects of genes. Moreover, parameters like heritability, expected genetic advance in response to selection, and the degree of association between traits are also important for the design of more effective breeding and selection programs.

1. Genetic Variance In lentil, a self-pollinated species, genetic variance is expected to be primarily additive; however, the nonadditive proportion of genetic variance could also be important. Singh and Jain (197 1) observed heterosis in F, hybrids for the respective numbers of branches per plant, pods per plant, pod clusters per plant, and also for seed yield, indicating nonadditive genetic variance for these traits. Similarly, Sagar and Chandra (1980) found nonadditive genetic effects that influenced seed yields in the F,’s and F,’s of nine lentil crosses. On the other hand, Goyal et al. (1976, 1977) reported greater variances due to general combining ability effects (GCA) than to specific combining ability effects (SCA) for seed yield, number of seeds per pod, seed size, and number of primary branches, indicating the impor-

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tance of additive genetic effects for those traits. Their SCA variances were also significant. GCA and SCA were equally important for the respective number of pods per plant and of secondary branches. Surprisingly, there was no heterosis for plant height. Haddad et al. (1982) estimated genotypic variances for several traits in three lentil crosses and partitioned them into components due to additive, dominance, and additive X additive variances. Additive genetic variance was a major component of total variance in one of the crosses for all traits examined except for plant height and mean seed dry weight. Dominance variance estimates were unexpectedly large in two of the crosses. Where parental means were similar, additive genetic variance estimates were small and, in many cases, negative. Where the parents differed appreciably, additive genetic variance was the major component of total variance for days to flowering and days to maturity, height to the lowest pod, and growth habit. Significant dominance variance estimates were obtained for plant height in all three crosses and for mean seed dry weight in two crosses. Sakar (1983) estimated the genetic effects for several lentil traits by using two lentil cultivars that differed for plant height, maturity, and seed size. Using the F, , FZ,B ,, and B2populations, he found that additive genetic effects were significant for days to first flower and mean seed dry weight. Additive and dominance genetic effects were large and equally important for both plant height and basal pod height with small additive X additive epistatic components. Nonadditive genetic effects were important for flowering duration (the number of days from first flower to the last flower), days from first flower to maturity, days from sowing to maturity, and seed yield per plant. In his study, Sakar (1983) estimated that three genes were responsible for the variation in plant height in the cross of ‘Laird’ (Slinkard and Bhatty, 1979) with ‘Precoz’ (Riva, 1975). Four genes were estimated to be responsible for variations in basal pod height, and at least two genes were responsible for variations in mean seed dry weight. As expected, the same genes were considered to influence plant height and basal pod height (i.e., they influenced node number per stem, internode length, or both). Current information on the components of genetic variance in lentil suggests that additive genetic effects are the major component of the genetic variance for most traits and that a considerable nonadditive component can be anticipated in early generations.

2. Heritability Estimates Most of the heritability estimates (Table 111) reported thus far for lentil are based on single experiments conducted at one location and in only 1 year. Therefore, the

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Table 111 Heritability Estimates for Important Traits in Lentils Range of heritability estimates Trait Yield and yield components Seed yield per plant Weight of 100 seeds No. of seeds per pod No. of pods per plant Plant traits Plant height Height of lowest pod Plant type (growth habit) No. of secondary branches per plant Reproductive traits Days from sowing to first flowering Days from sowing to maturity Quality factors Cooking time Protein concentration (%)

Broad sense '

Narrow sense

0.42-0.80 0.59-0.98 0.74' 0.45-0.80

0.07' 0.52-0.68

0.16-0.83 0.17-0.67 0.05 -0.44

0.0-0.38 0.36'

0.55'

0.47-0.78 0.54-0.96

0.53c 0.42'

0.82'

0.71''

'Sources for these broad-sense heritability estimates are from Erskine et al. (1985), Haddad (1979). Muehlbauer (1974). Sakar (1983), and Sindhu and Misra ( 1982). bSources for narrow-sense heritability estimates are from Haddad (1979) and Sakar (1983). 'Only one estimate was considered.

estimates of genetic variance on which they are based are likely biased by the inclusion of environmental variance components. Nevertheless, the estimates provide an indication of the relative ease of making progress through breeding. The estimates for seed yield and its components are moderately large which indicate that good progress can be expected from effective selection. The small narrowsense estimate of 0.07 (Sakar, 1983) indicates some difficulty in selection for yield in the hybrids he used. The heritability of several plant traits was quite variable and sometimes small, indicating that selection progress for traits such as plant height and height of the lowest pod will depend heavily on the variation available in the source material and on the effects of climate and weather. Reproductive traits, such as days from sowing to flowering and to maturity, had moderately large heritabilities, indicating that these traits would respond to selection. Quality factors such as seed size and seed thickness seemed to be strongly heritable.

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3. Association among Traits Larger and more stable yields are primary goals in most lentil-breeding programs. However, selection for productive genotypes can be difficult because of the large number of genes involved and the relatively small heritabilities. Correlations between traits in lentil need to be given very careful consideration and interpretation by plant breeders because the degree of association between traits might well differ according to the type of correlation measured. Correlations between seed yield and the numbers of pods per plant, seeds per pod, and secondary branches per plant; plant height; and straw yield have been positive and significant (Muehlbauer, 1974; Haddad, 1979; Chauhan and Sinha, 1982; Kumar et al., 1983; Sanvar et al., 1982; Tyagi and Sharma, 1985). Seed yield was negatively correlated with mean seed dry weight and with times to flowering and maturity in a range of locations (Muehlbauer, 1974; Haddad, 1979; Sakar, 1983; Tyagi and Sharma, 1985). Path coefficient analysis applied by Chauhan and Sinha (1982) indicated that the number of pods per plant and the number of secondary branches per plant were the most important characters contributing to large seed yield in lentil. However, Singh ( 1977) and Kumar et al. ( 1983) found that pod number per plant and plant height have the greatest direct effects on seed yield. On the other hand, Tikka et al. (1977) found that pod length had the largest direct effect on seed yield, followed by plant height, days to flower, and number of secondary branches per plant.

XI. INTERSPECIFIC HYBRIDIZATION Based on the crossability studies of Ladizinsky et al. (1984), L. orientalis ssp. and L. odemensis share a common gene pool with the cultivated lentil; interspecific hybrids between the cultivated lentil and L. culinaris ssp. orientalis and L. odemensis are easily obtained. Slight chromosomal rearrangements may cause partial sterility, but there are still ample opportunities for gene flow and for the utilization of these wild forms for breeding purposes. L. nigricans should not be overlooked as a source of needed germ plasm for breeding cultivated lentils. The lack of success in hybridizations with the cultigen is a significant barrier, but these difficulties in hybridization can be overcome by embryo rescue (Cohen et al., 1984) as was possible, for example, in the cross of the cultigen with L. ervoides that yielded partially fertile F, hybrids. Thus, all the wild Lens species can be considered as belonging to a common gene pool with the cultigen.

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Xn. METHODS USED FOR LENTIL BREEDING Lentils are of ancient origin and were probably one of the first plant species to be domesticated (Zohary, 1972; Ladizinsky, 1979~; Muehlbauer et al., 1985). Despite this antiquity, lentil breeding is a recent endeavor when compared with breeding efforts devoted to cereals or some of the other food legumes. Landraces still occupy most of the cultivated land sown to lentil in the major producing countries (Erskine, 1985a). Moreover, most of the cultivars released to date have been derived from selection within heterogenous laridraces and are not the result of hybridization (Muehlbauer, 1992). Nevertheless, it is expected that new and improved cultivars will become available in many lentil-producing areas due to the increased efforts in lentil breeding in both national and international programs. The methods of breeding lentil are similar to those utilized in breeding other self-pollinated crops and include pure line selection or hybridization followed by the bulk method, the pedigree method, the single seed descent, or some modification of these procedures.

A. PURE L m SELECTION Mass selection and pure line selection within introduced germ plasm accessions or local landraces have been the principle methods of lentil improvement in developing countries. Also, as the lentil crop has been introduced to relatively new production areas (United States and Canada), the initial cultivars used were developed from selection within introduced landraces. Genetic variability in the germ plasm collections worldwide has not been fully exploited to improve important agronomic traits. Mass and pure line selections have provided improved cultivars in a number of cases. Examples include ‘Tekoa,’ a pure line selection from the PI 25 1784 (Wilson et al., 1971); ‘Chilean 78,’ a cultivar developed by mass selection for large seed size and desirable color (Muehlbauer and Slinkard, 1981); and Crimson, a small red cultivar developed by pure line selection from Giza-9 introduced to the United States from Egypt. ‘Laird,’ a pure line selection derived from PI 343028, was developed and licensed in Canada (Slinkard, 1978a). Laird was selected for its high yield and large seed size. In India, the cultivar Pant-L-406 was selected from P-495, an Indian genetic stock, and has improved yield potential and resistance to rust and wilt (Pandya et a/., 1980). ‘Araucana-INIA,’ a cultivar released in Chile, was developed by mass selection from N-1284, an accession from the Chilean Germplasm Collection. The cultivar is tall, has a good tolerance to rust, and yields well (Tay et al., 1981).

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B. BULKPOPULATION Bulk population breeding has been the preferred method for lentil because of its ease of application and because of the problems involved with alternative methods. The method is simple, requires minimal record keeping, and is not labor intensive. Simplicity makes it attractive for programs that are designed to develop cultivars adapted over a wide geographical area because subsamples of populations can be widely tested. The advantage of wide testing includes the possibility of natural selection favoring genotypes adapted to particular local environments. However, caution is needed when using the system to ensure the survival of desirable genotypes through successive generations of bulking. It is expected that seed size differences between parents and the larger numbers of seeds produced on small-seeded genotypes compared with those on large-seeded genotypes may cause rapid shifts in a bulk population toward a preponderance of small-seeded types. To increase the proportion of desired phenotypes, populations can be subjected to mass selection either on the basis of seed size or color or on plant traits such as, for example, flowering time, plant height, branching characters,or disease resistance. Selection in early generations, such as in the F, to F4,might be effective in eliminating many undesirable genotypes. Selected plants can then be grown in bulk for several generations, followed by reselection after the populations have reached homozygosity. Because of the problems of genetic shifts during generation advance, modifications of the bulk population method have been devised. These include mass pedigree, modified bulk, single seed descent, and other schemes designed to control genetic shifts or to channel the shifts in the desired direction. ICARDA uses a bulk pedigree method in which crosses are advanced in bulk to the F4, after which the pedigree method is used. The generations advance by bulking, which allows an early evaluation and selection of bulks on which to concentrate efforts. Selection of plants in the F4is based only on highly heritable plant characters, and thereafter the progenies are managed by the pedigree method. Visual selection of F4 plants according to available selection criteria should lead to greatly improved types.

C.

PEDIGREE

SELECTION

The pedigree method of breeding is not the choice of most lentil breeders for managing lentil-breedingmaterial. However, if the method is used, between 5 and 15 F, plants are sufficient to provide from 200 to 2000 F2 seeds. To allow for successful selection, lentil plants need to be widely spaced so that individuals can be observed for possible selection. The plastic branching habit of lentil plants is a

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disadvantage of this method because when widely spaced for observation, their performance may be entirely different from that in more densely sown stands as used in commercial production. Also, for pedigree selection to succeed, readily identifiable traits need to be available, which is not the case in lentil. However, selection of the F, plants for traits with large heritability estimates (such as flowering date, relative maturity, and seed size) is likely to be successful. Characteristics that are considered desirable in lentil, such as upright growth habit, greater branch number, earlier flowering, and suitable maturity dates, should be readily distinguishable among F4families. The F5 provides the first opportunity to observe selections in comparison to standard check cultivars. Selected F, lines are usually sown in multirow plots that have within- and between-row spacings similar to those used in farmer’s fields. Preliminary yield trials may also be conducted. With this approach, line characteristics can be observed in solid sowings and yield potential can be gauged. Lines that meet the selection criteria of plant type, relative earliness, degree of branching, seed size and color, and yield are then retained and entered into advanced yield trials in succeeding generations. Slinkard, in Canada, has proposed a modification of the bulk method in which individual F, plants are selected and evaluated for yield in the F, and later generations. The method, designated as the “F,-derived family method,” places early emphasis on yield potential with the expectation that genes for yield can be actively selected for in early generations (Muehlbauer and Slinkard, 1985).

D. SINGLESEEDDESCENT Single seed descent, in contrast to bulk population breeding, is not affected by the method of plant culture since it does not depend on the numbers of seeds produced by the genotypes involved. Therefore, this method is suitable for rapid generation advance in greenhouses and growth chambers and, since only a small population is needed, less time and space for advancing generations are required. Haddad and Muehlbauer (1981) found that more genetic variability was maintained in the single seed descent method when compared to the bulk method and that natural selection was operated in the bulk method against less competitive, short-statured lentil genotypes. The single seed descent-derived populations had 10, 9, and 13% more erect lines in three hybrids when compared with the same hybrids advanced by the bulk method.

E. THEBACKCROSS METHOD The genes for resistance to pea seedborne mosaic virus and similar simply inherited genes in lentil are well suited to transfer to acceptable cultivars by means

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of the backcross method. Good sources of resistance are available and resistant plants are easily identified in segregating populations. Nevertheless, the backcross method has not been widely used in lentil improvement programs.

XIII. BREEDING OBJECTIVES Lentil-breeding programs throughout the world have similar objectives with larger and more stable seed yield being the most important. Adaptation to stress environments,especially to drought, and resistance to diseases and insects are also major breeding objectives. Priorities and breeding goals usually differ between regions depending on specific problems and special considerations related to farmers’ needs and consumer demands. In the developing countries, for example, one of the major breeding goals is the development of genotypes suitable for mechanical harvesting. Moreover, improving straw yield is also important because of the value placed on lentil straw as animal feed and as residues for the control of soil erosion. On the other hand, increased seed yield, improved disease resistance, and improved seed quality are principal breeding goals in the major exporting countries. Current objectives for lentil breeding in the major producing areas are as follows.

A. SEEDAND

STRAW YIELDS

Increased seed and straw yields with acceptable quality are the principal objectives in lentil breeding, but strategies for improvement differ. In North America, Muehlbauer (1992) and Slinkard (1985) have emphasized the importance of environmental adaptation and disease resistance. In the Middle East, where erratic and limited rainfall prevails in the lentil-producingareas, genotypes better adapted to drying soil and hot weather are desired. Erskine (1985a) suggested that improved yield could be achieved through the exploitation of genotype X environment interactions to identify genotypes for specific local environments instead of relying on fewer genotypes that are more widely adapted. Selection for improved yield and wider adaptation can be practiced within landraces; however, little progress in yield can be anticipated when compared to adapted landraces. The introgression of microsperma with macrosperma types holds promise for crop improvement because the two types evolved from and became important in different ecological regions and, therefore, are likely to possess different genes and adaptive complexes. Summerfield (1981) pointed out that no single factor has been or is likely to be identified that explains relative adaptation to environments and that well-adapted genotypes would probably be endowed

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with several individually unspectacular traits, the best combinations of which are difficult to predict. Larger straw and seed yields are often emphasized in those developing countries where straw is important for feed. The correlation between seed and straw yields is strong and positive so simultaneous selection for both traits should be possible.

B. DISEASES The diseases of lentil are, in general, relatively less damaging than those of most other food legume crops. However, there are some important and potentially devastating diseases that include the wilthoot rot complex in the Indian subcontinent, rust in India and South America, and Ascochyta (Ascochytafabae f. sp. lentis) blight and viruses in North America. Lentil genotypes resistant to various races of Fusarium oxysporum f. sp. lentis have been identified and can be used in breeding programs (Kannaiyan et al., 1978; Khare, 1980). Screening for resistance under field conditions can best be accomplished in wilt-sick plots (W. Erskine, personal communication). Lentil rust ( U f a b a e ) is an important disease in India, Morocco, Pakistan, Ethiopia, Argentina, and Chile. Infection of susceptiblecultivars has caused up to 70% yield losses in Chile and total field losses were observed in Morocco (Sakr, 198913). Sources of resistance have been identified in cultivars such as ‘Tekoa,’ ‘Laird,’and ‘Arancana-INIA,’which are now in use in South America, as is ‘PantL-406’ in India and ‘Precoz’ in Morocco (Pandya et al., 1980; Tay er al., 1981; Muehlbauer and Slinkard, 1985; Sakr, 1989a).Several other lines resistant to rust in India and Morocco have been used in breeding programs (Agrawal er al., 1976; Khare et al., 1979). “Hot spots” for rust, such as Debre Zeit in Ethiopia and Pantnagar in India, were suggested (Erskine, 1985a) to be useful locations for establishing screening nurseries for rust resistance. Chemical control of rust was very effective using Dithane-M45 as a foliar spray (Singh er al., 1985; Sakr, 1989b). Lentil Ascochyta blight attacks the leaves, stems, and pods and is an important disease in parts of western Canada where frequent rains occur between flowering and harvest. According to Gossen (1983, the fungus can be found in Argentina, Brazil, Syria, Greece, Chile, and Pakistan. When seeds from 30 countries were screened for Ascochyta infection, the fungus was isolated from seeds of 16 countries, including Australia, Canada, Ethiopia, Hungary, India, Italy, Morocco, Russia, Spain, Turkey, and Yugoslavia (Kaiser and Hannan, 1986). In some cases, infection of seeds is so severe that lentils are unmarketable (Kaiser, 1981; Gossen and Morral, 1983). Resistance sources have been identified in North America; Laird, ILL 5588, and ILL 5684 have good resistance to the prevailing race(s) and

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are used as resistant parents in crosses. Resistance in Laird is controlled by a single recessive gene, ral,, while that of ILL 5588 and ILL 5684 is due to two dominant genes, Ral, and Ral,. ILL 5588 also carries the ral, gene (Tay, 1989). Natural infection, which can be obtained in cooler, moister parts of Saskatchewan, is used for making selections. The mode of inheritance of resistance is not yet known. Several viruses are reported to infect lentils (Kaiser and Eskandari, 1970; Kaiser, 1972; Haddad et al., 1978), and PSbMV is potentially serious because it can be seedborne and is transmissible by aphids. Screening of the U.S. Department of Agriculture collection indicated that PI lines 212610, 151786, 297745, and 368648 were immune to the virus. Jermyn (1980), in New Zealand, confirmed PI 2 12610 as resistant to aphids, pea seedborne mosaic virus, and other viruses. Even though PSbMV has not been detected in farmers’ lentil fields in the United States, Muehlbauer has begun to develop breeding materials immune to the virus. Incorporation of multiple disease resistance into breeding material and acceptable cultivars is possible with the use of resistant germ plasm already identified. Pea enation mosiac virus, a natural pathogen of lentil, became a serious problem in lentil production in the United States during the late 1980s and, as a result of screening germplasm, PI 472547 and 472609 were identified as tolerant (Aydin et al., 1987).

C. ROOT R O T ~ I L T COMPLEX Root rot/wilt caused by E oxysporum f. sp. lentis, Rhizoctoma solani, and Sclerotium rolfsii is an important disease complex of lentil in India where several resistant lines have been identified (Pandey et al., 1988). Resistance to E oxysporum in India was controlled by two dominant genes with duplicate interactions in one line (L234) and complementary effects in two other lines (IL446 and LP286). The genes in L234 were not allelic to those found in either ILL446 or LP286 (Kamboj et al., 1990). Root rot caused by Thielaviopsis basicola was first noticed in 1984 under field conditions in eastern Washington and northern Idaho by Bowden el al. (1985). Sources of partial resistance were identified in lentil-breeding lines.

D. OROBANCHE Lentil is susceptible to several species of Orobanche, including 0. crenata and 0. ramosa (Basler, 1981). 0. crenata is the most important species, especially in Mediterranean countries (Erskine, 1985a). Control of Orobanche is difficult because of the large number of wind-blown seeds which can be produced each year and which may remain dormant in soil for several years thereafter.

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Research at ICARDA has shown that lentil accessions can have different susceptibilities to the various Orobanche species. This finding suggests that selection for resistance or tolerance should be possible (Basler and Haddad, 1979). Sauerborn et al. (1987) have developed a rapid test to screen lentil for resistance to Orobanche under laboratory conditions. After 35 days of incubation of lentil seeds at 20-25" C in clay-filled petri dishes, Orobanche attachments to lentil roots can be counted directly. Several lentil genotypes from India have shown satisfactory tolerance to 0. crenata at ICARDA, but were poorly adapted to the cooler weather of the Mediterranean region (Erskine, 1985a). Therefore, this resistance source and others which might come to be identified in the future should be recombined with locally adapted material.

E. INSECTS Little progress has been made in the identification of insect-resistant lentil germ plasm (Clement et al., 1994). It is expected, however, that breeding for insect resistance will become more important if insect problems increase with the spread of newly developed cultivars.

F. QUALITY Cultivars proposed for release must have quality that is acceptable to farmers and consumers. Lentil quality is either related to obvious seed characters such as seed size, testa, and cotyledon color or to the nutritional quality of seeds such as their protein and methionine concentrations. Breeders in the Americas are concerned about the development of large-seeded (macrosperma) lentils with yellow cotyledons and light-green seedcoats (Muehlbauer and Slinkard, 1985) because of export market demand. However, small-seeded red cotyledon lentils are often desired elsewhere. Evaluation of germ plasm for nutritional quality and seed decortication has begun at ICARDA. Variability for each of these characters is available in the germ plasm collection (Solh and Erskine, 1981). Erskine et al. (1985) studied 24 small-seeded lentils and found a small range in protein concentration of between 25.5 and 28.9% and a negative correlation of protein concentration with seed yield ( r = - 0.94). However, increased seed yield could be found without a significant decrease in protein concentration. In the same study, they found that cooking time is more related to seed size and less to environment, with a positive genetic correlation ( r = 0.92) between the two traits.

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G. ADAFTATION TO MECHANICAL HARVESTING Development of germ plasm that can be mechanically harvested is a principal goal for many breeders in national programs within the Middle East, Southwest Asia, and at ICARDA. Several traits are considered to be important for the success of mechanical harvesting and include increased plant height, pods borne well cabove the soil surface, erect growth habit, improved standing ability, reduced pod dehiscence, and reduced pod drop. A clearance of about 15 cm between the soil surface and the lowest pod is considered necessary for successful mechanical cutting or pulling of lentil plants (Khayrallah, 1981). This leads to the view that mechanical harvesting of lentil would be facilitated by the introduction of tall cultivars with the lowermost pods borne well above the soil surface (Solh and Erskine, 1984). Considerable genetic variability for plant height and lowest pod height was found in the ICARDA collection with ranges from 10 to 45 cm and from 6 to 30 cm for the two traits, respectively (Solh and Erskine, 1981). It was also found that the two traits are positively correlated (Haddad, 1979; Sakar, 1983) which indicates that selection for both traits is possible. However, tall plants had a tendency to lodge (Haddad, 1979) and the traits were highly influenced by the environment (Saxena and Hawtin, 1981). Relative pod indehiscence has been identified in lentil, and selection was feasible for this trait simply by delaying harvest and allowing breeding materials to be exposed to conditions conducive to seed and pod shatter followed by selection of the most indehiscent plants. However, significant variability for pod retention, which accounts for as much as twice the loss caused by pod dehiscence, does not seem to be available (Erskine, 198%). Nonlodging lentil cultivars could be a very important development toward the success of mechanical harvesting in stony areas and also to reduce losses in those areas where lentil is mechanically harvested. Erskine (1 985a) suggested that stem thickness, stem lignification, and greatei tendril production may be important contributions to lodging resistance in lentil. Tall erect lentil types considered important for successful mechanized harvest of lentil may have reduced yield potential. In the experience of the authors, erect genotypes with acute branch angles tend to be relatively poor yielders and do not compete well with weeds. Their poor competitive ability is the result of a reduced ability of strongly erect genotypes to fill available space with a spreading branch habit. By not covering the soil surface as rapidly as more spreading types, there can be losses of limited soil moisture. Also, the slower rates of canopy closure in upright types tend to provide an advantage to weeds, which then deplete water even more. Genotypes that rapidly cover the soil surface and develop a full canopy should allow for successful mechanical harvest of acceptable seed and straw yields.

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It seems that variability for several traits contributing to successful mechanical harvesting is available in lentil germ plasm. However, for traditional farmers in the developing countries, mechanization of lentil harvest is a multidimensional problem that can only be solved by careful attention to the cultivars in use and by the local management practices employed. Proper equipment that provides a smooth seedbed, planting with seed drills to ensure good crop establishment, and harvesting equipment that is designed to collect the maximum amount of biomass are all necessary for successful mechanical harvesting of lentil.

H. OTHEROBJECTIVES There are several other objectives which are either important for certain areas or have been recently identified by breeders and which might be given more attention in the future: photothermal insensitivity, resistance to MCPB herbicide, reduced tannin concentration in testa, cytoplasmic male sterility, development of lentil as an annual green manure crop, improving the seasonal fixation of nitrogen, and understanding the empty pod syndrome (Slinkard, 1980; Vaillancourt and Slinkard, 1983; Erskine, 1985a; Muehlbauer and Slinkard, 1985).

xrv.

SUMMARY

The accelerated progress made in recent years toward a better understanding of the genetics of lentil and the relation among wild forms should be the basis for substantial future gains by breeding. Several topics that are in obvious need of attention by breeders include: larger and more stable seed and biological yields; resistance to diseases and insects; and better tolerance to heat and drought. The germ plasm pool for lentils has been expanded by the availability of the wild species and by the research that has shown that all the related forms of Lens share a common gene pool. This common gene pool has not yet been exploited to any significant extent for lentil crop improvement, but several programs are actively utilizing the wild species for genetical studies and progenies are being evaluated for important traits. The limitations on lentil yields brought about in some regions by the crop being grown on progressively poorer land because of the competition imposed by more remunerative crops is a barrier that may be impossible to overcome. Breeding programs have not focused on improving nitrogen fixation by lentil crops. Estimates of fixation are small and indicate only nominal contributions to the nitrogen status of the soil. However, the ability of lentils to fix some nitrogen

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in marginal areas, albeit small amounts, may represent important contributions to farming systems. Breeding efforts have resulted in the development and release of cultivars which have distinct advantages over previously grown landraces. These efforts have been based on the ready availability of germ plasm and on accumulating genetic information. Expanding efforts on genetics and genetic markers hold particular promise for the eventual development of marker-based selection for genes that are difficult to identify and manipulate. Excellent communication among lentil researchers has developed and is fostered by the annual newsletter. The Lentil Experimental News Service (LENS), published by ICARDA, is available to all interested researchers on request.

ACKNOWLEDGMENT Research on lentil at the University of Reading is generously supported by a grant to Rodney J. Summerfield from the Overseas Development Administration of the UK Foreign and Commonwealth Office-Plant Sciences Programme Adaptive Project R5496cb.

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USE OF APOMIXIS IN CULTWAR DEVELOPMENT Wayne W. Hanna U.S. Department of Agriculture Agricultural Research Service Coastal Plain Experiment Station Tifton, Georgia 3 I793

I. Introduction II. The Gene(s) Controlling Apomixis A. Sources B. Expression C. Genetics 111. Breeding A. Advantages B. Identifying Apomictic Plants C. Breeding Methods D. Genetic Vulnerability W. Impact on Seed Industry V. International Impact VI. Evaluation References

I. INTRODUCTION Plants produce seed by both sexual and asexual (apomictic) methods. In sexual reproduction, an embryo is formed from the union of chromosomally reduced female (egg) and male (sperm) gametes produced during meiosis. Sexual reproduction results in genetic recombination during both microsporogenesis and megasporogenesis and allows crossing of compatible (ploidy, species, etc.) plants to produce new gene combinations. In apomictic reproduction, an embryo is formed directly from a chromosomally unreduced magaspore mother cell or from a somatic cell of the nucellus or ovule. Apomixis makes vegetative reproduction or cloning through the seed

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possible. It fixes hybrid vigor by allowing a plant to clone itself indefinitely through seed. The various types of apomictic mechanisms are briefly described in Section 1II.B. Apomixis has received increased research emphasis in the past 20 years due to discoveries of partially apomictic (facultative) plants in cultivated species (Arthur et al., 1993; Hanna et al., 1970; Hanna and Powell, 1973; Schertz, 1992), discoveries of sexual plants in apomictic species (Bashaw, 1962; Hanna er al., 1973; Smith, 1972; Voigt, 1971), new information on genetic control (summarized by Bashaw, 1980b; Nogler, 1984; Asker and Jerling, 1992), and a broader awareness of the impact that apomixis could have on cultivar development. Research in the United States on apomixis in agronomic grain and forage crops as well as citrus has been summarized (Elgin and Miksche, 1992). Research on apomixis in rice (Oryza sativa L.) has also been summarized (Wilson, 1993; Rutger, 1992). Gustafsson (1946) has published a comprehensive report on apomixis. More recently, the apomictic mechanisms, genetics of apomixis, methods of identifying and confirming apomixis, and the potential of apomixis in crop improvement have been discussed in books and reviews (Asker and Jerling, 1992; Bashaw, 1980b; Bashaw and Hanna, 1990; Hanna, 1991; Nogler, 1984). Collections of papers (translated from Russian to English) dealing with various aspects of apomixis in a number of plant species have been published by Khokhlov (1976) and Petrov (1984). Apomixis could have a major impact on seed-propagated food, forage, and fiber production around the world. It would especially be beneficial in the major annual grain crops such as wheat (Triticum aestivum L.), rice, and soybean (Glycine m a Merr.), where hybrid vigor is present but systems for commercially producing hybrids may not be available and/or economical. In crops such as maize (Zea mays L.), sorghum (Sorghum bicolor L. Moench), and pearl millet [Penniseturn glaucum (L.) R. Br.], commercial hybrid production systems are available but apomixis could have a major impact by simplifying hybrid seed production (Section 1II.A) and by making hybrids readily available and/or affordable in developing countries (Section V). Apomixis also would allow breeders to easily capture hybrid vigor in vegetable, minor grain and forage, and turf species.

II. THE GENE(S) CONTROLLINGAPOMIXIS Genetic studies on the apomictic mechanisms have been difficult to conduct and many times are inconclusive because apomixis may not allow needed crosses and backcrosses to be made and segregating progenies to be observed. Understanding the inheritance of apomixis becomes more complex when plants reproduce by

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facultative apomixis (both sexual and apomictic reproduction can occur in a plant concurrently at various frequencies). Despite difficulties encountered in studying the genetics of apomixis, progress is being made in understanding the genetic control of apomixis in various crops. Genetics of apomixis has been summarized for a number of crops (Asker and Jerling, 1992; Nogler, 1984). It is generally concluded that apomixis is controlled by qualitative inheritance. The simple genetic control of apomixis improves the potential for manipulating this reproductive mechanism and transferring it to other species.

A.

SOURCES

The best source of the gene(s) controlling apomixis would be within the species targeted for improvement. Unfortunately, genes controlling apomixis have not been discovered in most of our major cultivated species. However, genes controlling apomixis can probably be found in the wild species of the genus or related genera of most major cultivated crops in the world (Hanna and Bashaw, 1987). Before genes controlling apomixis can be used, they have to be identified. Cytological, genetic, and morphological approaches to identifying and confirming apomixis have been previously discussed (Bashaw, 1980b; Hanna and Bashaw, 1987). Apomictic reproduction is mainly found in polyploid species. However, it appears that polyploidy is not necessary for the expression of apomictic reproduction. Nogler (1984) cited examples of apomixis in diploid species. Obligate apomixis was reported in a polyhaploid of an interspecific Pennisetum hybrid backcross derivative with between one and two sets of chromosomes from the apomictic species, indicating that apomixis is possible in nonpolyploid plants (Dujardin and Hanna, 1986). Use of mutagens to produce mutant genes that cause plants to reproduce apomictically may be another source. Plants that reproduce by facultative apomixis have been induced in P. gluucum (Hanna and Powell, 1973; Arthur et al., 1993) and S. bicolor (Hanna et al., 1970). Apomixis is expressed at a variable but generally low level in these mutants. Apomictic reproduction in facultative types should be at a high enough level to provide the uniformity needed and to preserve the vigor of a particular genotype for its intended use. Developments in molecular biological techniques should make it possible to clone a desirable apomixis gene(s) and transfer it into any crop. Although the previous statement sounds simple, much research is needed to locate molecular markers, clone the gene(s), insert the gene(s) into a recipient species, and get the gene(s) to express itself phenotypically. Progress is being made in developing molecular markers (Ozias-Akins et d., 1993; Miles et d.,1994).

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B. EXPRESSION Phenotypic expression of a gene(s) controlling apomixis is important in both the originating species and in plants of the recipient species, genera, or family if the gene is transferred. First, it should be noted that technically there are few obligate apomicts because offtypes can usually be found if large enough populations are observed in most apomictic species. For breeding and practical purposes, this discussion assumes that plants producing more than 99% maternal types are obligate. Obligate apomixis is preferable to facultative apomixis when sexual counterparts are available in a species for making hybrids and when the desirable genotype to be propagated is apomictic. Various levels of apomictic reproduction can be found and selected in facultative species such as Kentucky bluegrass (Poa pratensis L.) (Bashaw and Funk, 1987). Levels of apomixis can be increased by crossing selected facultative apomictic types (Pepin and Funk, 1971). Apomictic reproduction is an advantage in species like Kentucky bluegrass with high and diverse ploidy levels because it helps to bypass sterility problems associated with unstable chromosome numbers. The amount of facultative reproduction that can be tolerated in a cultivar depends on its use. More offtype variation may be tolerated in a forage cultivar than in a grain crop where uniformity in height and maturity is critical for mechanical harvesting. Gene expression becomes even more critical when genes controlling apomixis are transferred from different species, genera, and/or families. Within an originating species, apomixis may be obligate. However, in a different genetic background (genotype or species), apomixis may be expressed differently. Sexual pearl millet X obligate apomictic P. setacum (Forsk.) Chiov. hybrids are obligate apomicts but are highly male and female sterile (Hanna, 1979). High male sterility and facultative apomixis were observed in sexual maize X Tripsacum dactyloides (L.) L. (Savidan et al., 1993) and in sexual wheat X Elymus rectisetus (Nees in Lehm.) (Carman and Wang, 1992) crosses. Crosses between sexual pearl millet and obligate apomictic P. orientale result in partially male sterile facultative apomictic hybrids and derivatives (Dujardin and Hanna, 1987). Male fertile obligate apomictic hybrids and derivatives were produced from crosses between sexual pearl millet X obligate apomictic P. squamulatum Fresen crosses (Hanna et al., 1992). Fortunately, several Pennisetum species reproduce by obligate apomixis, and a gene(s) in a wild species that expressed obligate apomixis through the BC, generation has been found (Dujardin and Hanna, 1989a). The highest level of maternal types due to apomixis observed in the BC, generation was 90% (Hanna et al., 1993). The reason for the reduction in apomictic reproduction from BC, to BC, is unknown. It might be possible to recover obligate apomictic BC, plants if larger populations of apomictic plants are screened.

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Can the gene(s) conditioning obligate apomixis in the BC, generation of the Pennisetum crossing project confer obligate apomixis in crops such as wheat, rice, maize, sorghum, and soybean if it is isolated and inserted in the genomes of these crops by molecular techniques? This question waits to be answered but it is reasonable to assume that the gene(s) would induce obligate apomixis in these and other species. Environmental conditions are able to influence apomictic expression in some species (Asker and Jerling, 1992; Hussey e f al., 1991). Burton (1992) observed no morphological variation in progeny established from seed of obligate apomictic Paspalum notatum Flugge plants grown at varying elevations and subjected to water and nutrient stresses. Gounaris ef al. (1991) reported that application of inorganic salts to the growing media changed the frequencies of sexual and apomictic embryo sacs in Cenchrus ciliaris L. (buffelgrass) plants but the study was not documented with progeny tests. Artificial control of method of reproduction, apomictic to sexual or sexual to apomictic (especially the latter), would be a breakthrough for using apomixis in cultivar development.

C. GENETICS A number of reports discuss present knowledge of the genetics of apomixis in various crops (Asker and Jerling, 1992; Bashaw and Funk, 1987; Nogler, 1984). Fortunately, apomixis appears to be qualitatively controlled by a single or, at most, a few genes. Both recessive and dominant gene actions have been reported in the same and different species. The potential for using apomixis in cultivar development and cloning the gene(s) for use in other species and genera is enhanced by its qualitative inheritance. Obligate and facultative apomixis in the same species and varying degrees of facultative apomixis in the same species indicate that apomixis may also be affected by modifying genes and/or genetic background.

III. BREEDING Apomixis is only in the early stages of making contributions to cultivar development. Efforts in the past have been concentrated on identifying and studying the various apomictic mechanisms and on the genetics of apomixis. More recently, efforts have moved toward transferring the gene(s) controlling apomixis from wild to cultivated species (Hanna et al., 1992; Ozias-Akins et al., 1992). The discovery of a sexual plant in what was considered obligate apomictic buffelgrass provided an opportunity to manipulate apomixis in a breeding program

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(Bashaw, 1962). Bashaw (1980a) used the sexual plant as a female parent in a breeding program with obligate apomicts to develop the improved cultivars: ‘Nueces’ and ‘Llano.’ Selection of highly apomictic clones in facultative apomictic Kentucky bluegrass has been the basis for improved cultivars in this species (Bashaw and Funk, 1987). Apomixis is used in Citrus to produce uniform virusfree rootstock (Hearn et al., 1992). It is yet to be determined if apomixis can be used to produce true-breeding hybrids at the diploid level. This is an important consideration since many of our major food crops are diploid and apomixis is usually found at a polyploid level. It may be necessary to use apomixis at the tetraploid or a higher ploidy level. Although poor seed set is usually associated with autotetraploids, significant improvements in seed set have been made in sorghum (Doggett, 1964) and pearl millet (Dujardin and Hanna, 1989b). Tetraploid pearl millet (2n = 4x = 28) has been used in the apomixis gene transfer program in Pennisetum (Hanna et al., 1992).Pearl millet plants (BC,) with 2n = 29 chromosomesare obligate apomicts (Dujardin and Hanna, 1989a).

A. ADVANTAGES Some advantages of apomixis in a breeding program have been previously discussed (Hanna and Bashaw, 1987). In a commercial hybridization program where male sterility is used, apomixis eliminates the need to develop and maintain Alines or cytoplasmic-nuclear male sterile systems, B-lines or male fertile maintainers of the A-lines, and R-lines or restorer lines for male fertility restoration of the A-lines. The A-, B-, and R-lines require time and testing for their development and space as well as isolation to maintain them. The development of A-lines rapidly narrows both the nuclear and cytoplasmic gene pools that can be used to develop stable male sterility systems. Likewise, a search for R-lines to completely restore the A-lines again narrows the gene pool. Gene pool vulnerability is further discussed in Section 1II.D. The only requirement for producing apomictic hybrids is that a cross-compatiblefemale with some degree of sexual reproduction is available for crossing with an apomictic pollinator. In apomictic species, the availability of a sexual female is the most limiting factor. When a gene@)for apomixis is introduced in a sexual species, all germ plasm within a species has potential as a parent of a new hybrid. The genotype of every apomict is fixed in the F, generation and every apomictic genotype from a cross has the potential of being a cultivar. Gene combinations and vigor are not lost as in each segregating generation of sexual F, hybrids. Recessive genes controlling apomixis may be used to fix the genotypes of transgressive segregates in certain crosses (further discussed in Section 1II.C). Planting true-breeding seeds from apomictic reproduction would have many

USE OF AF'OMIXIS

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advantages over tuber propagation in crops such as potato, Solanum tuberosum L. Seeds would reduce the propagation and spread of diseases and viruses which are readily transmitted through the tubers (Asker and Jerling, 1992; Hermsen, 1980). In addition, seed propagation by apomixis would greatly reduce the storage, shipping, and planting costs and volume compared to tuber propagation. Apomixis would allow breeders to precisely engineer plants. It would allow one to develop genotypes with characteristics such as quality, responses to management, and maturity that are highly reproducible from field to field and year to year. At the same time, a number of apomictic genotypes could be mixed together in various combinations to enhance genetic diversity to accomplish a specific goal. Apomixis would change how commercial cultivars are produced and marketed. Some may consider this a disadvantage but the author prefers to view it as an opportunity for the future. In summary, apomixis provides a unique opportunity to develop and maintain superior gene combinations in cultivars and it would simplify hybrid seed production.

B. IDENTIFYING APOMICTIC PLANTS It is necessary to determine the reproductive behavior of selected plants in a breeding program involving apomixis. This can be done by progeny testing openpollinated seed from selected plants. Morphologically variable progeny from a plant would indicate sexual origin. The frequency of uniform or maternal progeny from a plant would indicate the level of apomictic reproduction. At least 20 to 25 progenies are needed to obtain a reliable estimate of a plant's reproductive behavior, especially if it reproduces by facultative apomixis. Fewer progeny may be needed to identify sexual plants, but the same number of progenies should be grown for all selected plants since the reproductive behavior may not be known before they are progeny tested. Cytological observations are more rapid than progeny testing for identifying the method of reproduction. New ovule-clearing techniques (Young et al., 1979) allow one to classify the reproductive behavior of a plant within 2 or 3 days after collecting the ovaries. In Pennisetum, Paspalum, and Panicum, it is possible to collect a few florets at the beginning of anthesis and to classify the reproductive behavior of the plant before it completes anthesis. Apospory and adventitious embryony are the apomictic mechanisms easiest to identify at anthesis. Apospory can be identified by the presence of multiple embryo sacs, lack of antipodal development, and/or shape and orientation of embryo sacs in the ovule. In adventitious embryony, the embryo develops as a bud-like structure through mitotic division of somatic cells of the ovule, integuments, or ovary wall. No embryo sac is formed in which these embryos develop. However, a sexual embryo sac may de-

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velop in the same ovule and is essential for endosperm development. Diplospory is more difficult to identify and requires cytological observations at earlier ovule development than the two previous mechanisms. Lack of meiosis or a linear tetrad of megaspores is the best evidence for diplospory. Fertilization of the polar nuclei by a sperm, pseudagamy, is necessary for endosperm development in apospory and diplospory. Bashaw (1980b) provides a more detailed discussion on the apomictic mechanisms. It has been reported that the lack of fluorescing callose in the walls of dyads, tetrads, and megaspore mother cells is also an indication for diplospory (Carman and Wang, 1992). Molecular markers linked to the gene(s) controlling apomixis can be used to identify apomictic plants in the seedling stage (Hanna et al., 1993; Ozias-Akins et al., 1993). This eliminates the need to grow the plants in the field unless they are needed in future hybridization studies. However, this molecular approach cannot be used to distinguish between obligate and facultative apomixis at this time.

C. BREEDINGMETHODS In a breeding program, it must be remembered that obligate apomictic plants can only be used as male parents in crosses. Microsporogenesis does function in apomictics with resultant genetically recombined and chromosomally reduced male gametes. An apomictic plant must have some pollen fertility if it is to be used in a breeding program. The ideal apomictic mechanism in a breeding program would be one that is controlled by a dominant gene(s), is environmentally stable, and reproduces only by obligate apomixis, especially when sexual counterparts are available for crossing with the apomicts. Breeding procedures for utilizing apomixis have been previously described for forage and turf grasses. Taliaferro and Bashaw (1966) and Bashaw and Funk (1987) outlined a procedure for buffelgrass, based on the genetic control of apomixis in this species. Burson et al. (1984) described schemes for breeding obligate and facultative apomictic species. Nakajima (1990) and Savidan (1981) discussed the use of apomixis in breeding guinea grass (Panicum maximum Jacq.). Burton and Forbes (1960) showed that by doubling the chromosome number of diploid sexual ‘Pensacola’ Bahia grass (Paspalum notatum Flugge), it could be crossed with tetraploid obligate apomictic ‘common’ Bahia grass to release the genetic variability of this apomictic species. Others have pollinated obligate apomicts to produce B,,, hybrids resulting from the fertilization of an unreduced apomictic egg by a chromosomally reduced sperm from the pollen (Bashaw et al., 1992; Bashaw and Funk, 1987). Production of B,,, hybrids allows one to develop hybrids in apomictic species where no sexual plants are available for use as female parents. New gene combinations are developed by adding one or more whole genomes of the species or an alien species.

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In order to simplify the following discussion and figures, it will be assumed that apomixis is controlled by a single gene in a diploid plant. The results from various crosses would be modified and made more complex by modifier genes, genetic background, ploidy, and if more than one gene controls apomictic reproduction.

1. Dominant Gene Apomixis controlled by a dominant gene would be the easiest to use in a breeding program because all apomicts would be heterozygous for the method of reproduction. Therefore, sexual X apomictic crosses result in both sexual and apomictic F, progenies (Fig. 1). One would theoretically expect one-half of the F, plants to be sexual and one-half of the plants to be apomictic if apornixis is controlled by a single dominant gene. Sexual F, plants can be discarded or used in crosses with other apomictic plants to produce new apomictic hybrids and sexual plants with new gene combinations. Using improved sexual plants in crosses with improved apomictic plants from other crosses in each generation increases the likelihood of developing superior apomictic hybrids in succeeding generations. F, apomicts with desirable agronomic traits produced from the cross in Fig. 1 can be selected and immediately placed in replicated tests to evaluate desired traits. Progeny tests for genotype stability are not necessary if the plants are obligate or at least highly apomictic. Superior genotypes can be released as cultivars. 2. Recessive Gene All sexual plants are heterozygous for a recessive gene controlling apomixis in crosses between sexual plants homozygous for method of reproduction and apomictic plants in which apomixis is controlled by a recessive gene (Fig. 2). Compared to the cross in Fig. 1, this cross requires selfing the F,, a loss of vigor in

SEXUAL

7

OBLIGATE APOMICT (Dominant gene)

SEXUALS

APOMICTS

1. Use s e l e c t e d p l a n t s i n

1. S e l e c t best phenotypes

crosses w i t h o t h e r apomicts 2 . Discard unsel ected p l a n t s

2. P l a n t i n r e p l i c a t e d t e s t s !Test APOMICTIC CULTIVAR RELEASE

Figure 1. The breeding procedure when obligate apomixis is controlled by a dominant gene(s).

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W. W. HANNA4 SEXUAL

OBLIGATE APOMICTIC

(Recessive gene) Fl

Self

+-F*

SEXUAL

APOMICTIC

(Follow procedure as with sexual F,s in Fig. 1 i f plants a r e superior - otherwise discard)

(Follow procedure as with apomictic F, selections in Fig. 1)

Figure 2. The breeding procedure when obligate apomixis is controlled by a recessive gene(s) and when sexual plants are homozygous for the method of reproduction.

progenies due to selfing results, and only one-fourth of the F2 progenies are apomictic. However, it is possible to select superior apomictic transgressive segregates in the F, generation that are superior to the F,. Selection and testing of apomictic plants and release of apomictic cultivars would be similar to the procedure followed for the cross in Fig. 1. Crosses between two sexual plants, both heterozygous for method of reproduction, results in F, plants that (1) breed true for sexuality, (2) are sexual but heterozygous for apomixis, and (3) breed true for apomixis (Fig. 3). This procedure can capture heterosis in apomictic plants in a similar way to the apomictic plants produced in the cross in Fig. 1. Only 25% of the F, plants can be apomictic in this cross whereas 50% of the plants in the cross in Fig. 1 can be apomictic. Sexual F, plants heterozygous for the gene controlling apomixis could be handled similarly to the F, sexual plants in Fig. 2. True-breeding sexual plants should be handled similarly to the sexual F, plants in Fig. 2. SEXUAL (H) (Recessive gene)

i

SEXUAL (H) (Recessive gene)

t i ' -

SEXUAL

SEXUAL (H)

(Follow procedure as (Follow procedure as with with sexual F, p l a n t s F, sexual plants in Fig. 2 in Fig. 2 ) . o r use in crosses with other sexual (H) or apomictic crosses).

APOMICTIC

(Follow procedure as with apomictic F, selections in Fig. 1).

Figure 3. The breeding procedure when obligate apomixis is controlled by a recessive gene and when both parents are heterozygous (H)for the method of reproduction.

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USE OF APOMIXIS SEXUAL (H)

SEXUAL

i

1"i

SEXUAL

SEXUAL (H)

(Follow the same procedure as for the F, sexual and sexual (H) plants in Fig. 3). Figure 4. The breeding procedure when obligate apomixis is controlled by a recessive gene and when one of the parents of a cross is heterozygous (H)for the gene controlling apomixis. A reciprocal cross would produce the same results.

A cross between a plant homozygous for sexuality and a sexual plant heterozygous for the method of reproduction is probably the most inefficient cross to make for producing superior apomictic plants (Fig. 4). About 50% of the progeny of this cross should be sexual and heterozygous for the gene controlling apomixis as in the Fig. 3 cross, but no apomictic F, progeny are produced. A sexual female plant heterozygous for the gene controlling apomixis pollinated with an obligate apomict is the most efficient way to develop superior apomictic cultivars when apomixis is controlled by a recessive gene (Fig. 5 ) . The outcome of the cross and the selection and testing of apomictic plants are similar to that for the cross in Fig. 1. Sexual plants from the cross in Fig. 5 are heterozygous for genes controlling apomixis whereas sexual plants in Fig. 1 are homozygous for sexuality because no other genotype is possible for sexuality when the gene controlling apomixis is dominant. Population breeding methods may also be applied to improving apomictic species. Five cycles of recurrent restricted phenotypic selection (RRPS) increased yields of diploid Pensacola Bahia grass, Puspalurn notutum, but failed to produce

SEXUAL (H)

i

OBLIGATE APOMICT

(Recessive gene)

7

SEXUAL (H)

(Follow same procedure as for F, sexual (H) selection in Fig. 3)

APOMICTS

(Follow same procedures as for apomictic selections in Fig. 1)

Figure 5. The breeding procedure when obligate apomixis is controlled by a recessive gene and when the sexual parent is heterozygous (H)for the method of reproduction.

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high-yielding obligate apomictic plants in a population of tetraploid Bahia grass with the recessive gene for apomixis (Burton and Forbes, 1960; Burton, 1992). Apomictic plants homozygous for the recessive gene controlling apomixis occurred at a low frequency, less than the 1 in 36 expected in tetraploid material, and failed to yield as well as the sexual plants in the population. Burton (1992) developed another P. notutum population of apomictic and sexual plants with apomixis controlled by a dominant gene. After three cycles of RRPS, the best apomictic plants yielded more dry matter than Argentine Bahia grass.

3. Facultative Apomixis Facultative apomixis is useful in species where obligate sexual plants are not available for crossing with apomictic pollinators and where the frequency of apomixis can be increased by crossing diverse facultative types (Bashaw and Funk, 1987). It can be a disadvantage because it can complicate and make the breeding process unpredictable. The same procedures could be used for breeding facultative apomicts as for obligate apomicts, except that more progeny testing would be required to establish the stability and frequency of apomixis of various apomictic genotypes.

4. Interspecific Hybrids Interspecific hybridization between a sexual and apomictic species can be used to release the genetic variability in apomictic species. Lutts et ul. (1991) crossed induced tetraploids of sexual Bruchiuriu ruziziensis with apomictic B. decumbens which released the genetic variation of the apomictic species. Burson (1989) identified sexual progenitors of obligate apomictic pentaploid Puspalum dilututum Poir for use as a female parent to release the genetic variability of the apomictic species. Hanna and Dujardin (1990) developed apomictic interspecific hybrids with over 20 different chromosome and/or genome combinations from crosses among five Pennisetum species. A number of the interspecific hybrids produced high yields of high quality forage (Hanna et al., 1989).

5. Chemical Control Chemical control of the apomictic mechanisms by turning them “on” or “off’ at will would have a major impact on using them to produce hybrids. Presently, little information is available on this subject. The ability to turn apomixis “off’ would make sexual plants in apomictic species available and allow one to release at will the genetic variability of any genotype that reproduces by apomixis. If apomixis could be turned “on” at will with a chemical, a sexual hybrid could be

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made temporarily apomictic in a commercial production field to increase seed. The hybrid would be sexual in the farmer’s field if not treated with the chemical. Chemical control of the apomictic mechanisms presents a challenge to biochemists and genetic engineers in the future.

D. GENETIC VULNERABILITY A major concern for using apomixis in cultivar development is that a few superior cultivars would occupy most of the area planted to a particular crop. A report published by the National Academy of Science (1972) showed that the area commercially planted to most of the major sexually reproducing agronomic and horticultural crops in the United States is already represented by a limited number of cultivars for each crop. The impact of the corn blight in 1970 due to susceptibility of the major male sterility-inducing cytoplasm used to produce hybrid maize was discussed in the same report. Sorghum uses the milo cytoplasm in most of its commercial hybrid production (Bosques-Vega et al., 1989; Schertz and Pring, 1982). Use of apomixis in cultivar development could actually enhance genetic diversity. Each apomictic plant from a sexual X apomictic cross is potentially a unique cultivar regardless of the heterozygosity or homozygosity of its parents. Apomixis would allow breeders to build and fix unique genotypes that would not be possible or at least very difficult with sexual reproduction. Vulnerability due to cytoplasm would virtually be eliminated because a specific cytoplasmic-nuclear male sterility-inducing cytoplasm would not be needed to commercially increase a hybrid. There could be as many different cytoplasms as there are commercial hybrids if apomixis is used in cultivar development.

IV. IMPACT ON SEED INDUSTRY Apomixis would no doubt have an impact on the way commercial cultivars are produced and increased. production practices would be radically changed and at the same time greatly simplified. The need to maintain and increase parental lines (except for breeding) and the need to be concerned about isolation to prevent outcrossing would be eliminated. The major concern in seed production would be to prevent mechanical mixtures. Outcrossing would only be a problem when a cultivar reproduced by some degree of facultative apomixis. Offtypes in facultative apomictic cultivars would need to be rogued. The land needed to produce hybrid seed would be significantly reduced.

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Will farmers save their own seed instead of purchasing new seed each year? Some will probably save their own seed since obligate apomictic hybrids will breed true. In the author’s opinion, most farmers will continue to purchase seed because they recognize the advantages of planting high quality, treated and sized seeds. Apomixis would lower seed production costs for industry. It will probably be more economical for farmers to purchase seed each year than to purchase and operate the equipment needed to process their own seed. Another concern is control of rights to germ plasm. Rights to specific apomictic cultivars can be controlled through patents since apomictic cultivars are vegetatively propagated through seed. Cultivars would need to be documented by morphological, biochemical, and molecular methods and descriptors. Documentation methods would need to be refined and precise because of a proliferation of cultivars in the market, some with only small genetic differences.

V. INTERNATIONALIMPACT All farmers can benefit from apomictic hybrids because apomixis maximizes the opportunity to develop and make available superior genotypes to be grown on the farm. However, the greatest impact of apomictic hybrids would be in lesser developed countries where the largest portion of the world’s population is located, hybrids may not be widely grown, and farmers are accustomed to saving their seeds from year to year. Hybrids usually result in an increase in production, with the amount depending on genotypes and crop. In countries where yields are low and food supplies are limited, any increase in production due to hybrid vigor is welcomed. Ouendeba et al. (1992) obtained an 81% increase in grain yield for pearl millet in a population cross between landraces from Sudan and Nigeria. If the vigor of that landrace hybrid could be fixed, it would revolutionize pearl millet grain production in West Africa and at the same time maintain the adaptability and diversity of local germ plasm. Up to 73% heterosis has been reported for rice hybrids (Virmini et al., 1982). Using apomixis to fix hybrid vigor in rice would have a major impact on food production around the world. The widespread use of a few apomictic cultivars should be a concern but probably not a reality. The ability to rapidly create new stable cultivars using apomixis would greatly reduce problems due to pest epidemics. If an apomixis gene(s) was readily available to be used in cultivar development, there would be a proliferation of new cultivars with different heights, maturities, qualities, adaptations, etc. Various combinations of these apomictic cultivars could be mixed in numerous combinations to provide reliable production in diverse environments to meet the needs of the farmer.

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VI. EVALUATION There is no genetically controlled character that could have a greater impact on food, forage, and fiber production around the world than apomixis. Apomixis is being used to develop cultivars in forage and turf grasses and in Citrus rootstock. We have made significant progress in transferring a gene(s) controlling obligate apomixis from a wild species to cultivated pearl millet. Apomictic cultivars in pearl millet should be possible when the problem related to retaining seed set on apomictic backcross-derived plants is solved (Dujardin and Hanna, 1989a). Wild apomictic species have been crossed with maize and wheat, but high sterility and facultative apomictic behavior have been encountered. Male fertility is needed in these species crosses to transfer apomixis to the cultivated species and to use it in cultivar development.Facultative apomixis has been reported in sorghum and rice but no obligate apomixis has been reported in the cultivated or wild species. It appears that molecular methods may be needed to transfer genes controlling apomixis to our major grain crops such as maize, wheat, rice, sorghum, and soybean and many other important food, forage, and fiber crops if apomictic cultivars are to be developed in these species. This will require isolation of a stable gene(s) (preferably dominant) controlling obligate apomixis, insertion of the gene(s) into the genome of a target species, expression of obligate apomixis in the target species, and replication of the gene(s) controlling apomixis in the genome of the target species. One can readily see that many questions need to be answered and many obstacles overcome regarding the wide use of apomixis in cultivar development. It will not be easy to isolate and transfer the gene controlling obligate apomixis and use it to produce apomictic cultivars in our major world crops, but it should be possible, especially with the major advances being made in molecular biology. It is worth the effort because of its potential impact around the world.

REFERENCES Arthur, L., Ozias-Akins, P., and Hanna, W. W. 1993. Female sterile mutant in pearl millet: Evidence for initiation of apospory. J. Hered. 84, 112- 115. Asker, S. E., and Jerling, L. 1992. “Apomixis in Plants.” CRC Press, Boca Raton, FL. Bashaw, E. C. 1962. Apomixis and sexuality in buffelgrass. Crop Sci. 2,412-415. Bashaw, E. C. 1980a. Registration of Nueces and Llano buffelgrass. Crop Sci. 20, 1 12. Bashaw, E. C. 1980b. Apomixis and its application in crop improvement. In “Hybridization of Crop Plants” (W. R. Fehr and H. H. Hadley, eds.), pp. 45-68. Amer. SOC.Agron., Madison, WI. Bashaw, E. C., and Funk, C. R. 1987. Breeding apomictic grasses. In “Principles of Cultivar Development: Crop Species” (W. R. Fehr, ed.), Vol. 2, pp. 40-82. MacMillan Co., New York. Bashaw, E. C., and Iianna, W. W. 1990. Apomictic reproduction. In “Reproductive Versatility in the Grasses” (G. P.Chapman, ed.), pp. 100- 130. Cambridge Univ. Press, England. Bashaw, E. C., Hussey, M. A,, and Hignight, K. W. 1992. Hybridization (N + N and 2n + N)

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of facultative apomictic species in the Penniseturn agamic complex. Ini. J. Plant Sci. 153, 466-470. Bosques-Vega, A., Sotamayor, A., Torres-Cardona, S., Perrecly, H. R., and Schertz, K. F. 1989. Maintainer and restorer reactions with A,, A, and A, cytoplasms of lines from the sorghum conversion program. Publ. MP-1676. Texas Agric. Exp. Station, College Station. Burson, B. L. 1989. Phylogenetics of apomictic Paspalurn dilataium. In “Proc. XVI Int. Grassl. Congr.,” pp. 479-485. Nice, France. Burson, B. L., Voigt, P. W., and Bashaw, E. C. 1984. Approaches to breeding apomictic grasses. In “Proc. 40th Southern Pasture and Forage Crop Improvement Conf.,” pp. 14-17. Baton Rouge, LA. Burton, G. W. 1992. Manipulating apomixis in Paspalurn. In “Proc. Apomixis Workshop,” pp. 16- 19. Atlanta, GA. Burton, G. W. 1982. Effect of environment on apomixis in bahiagrass, Paspalum notafum. Crop Sci. 22,109-111. Burton, G. W., and Forbes, I., Jr. 1960. The genetics and manipulation of obligate apomixis in common bahiagrass (Paspalurn noiafum Flugge). In “Proc. 8th Int. Grassland Congr., Univ. Reading, England,” pp. 66-71, Alden Press, Great Britain. Carman, J. G., and Wang, R. R.-C. 1992. Apomixis in the Triticeae. In “Proc. Apomixis Workshop,” pp. 26-29. Atlanta, GA. Doggett, H. 1964. Fertility improvement in autotetraploid Sorghum. I. Cultivated autotetraploids. Heredity 19,403-419. Dujardin, M., and Hanna. W. 1986. An apomictic polyhaploid obtained from a pearl millet X Penniseturn squamularum apomictic interspecific hybrid. Theor. Appl. Genet. 72,33-36. Dujardin, M., and Hanna, W. 1987. Inducing male fertility in crosses between pearl millet and Penniseium orieniale Rich. Crop Sci. 27,65-68. Dujardin, M., and Hanna, W. W. 1989a. Developing apomictic pearl millet: Characterization of a BC,. J. Genet. f lanr Breed. 43, 145- 15 I. Dujardin, M., and Hanna, W. W. 1989b. Fertility improvement in tetraploid pearl millet. Euphytica 42, 285-289. Elgin, J. H., Jr., and Miksche, J. P. (ed.) 1992. Proc. of the Apomixis Workshop, U.S. Department of Agriculture, Agricultural Research Service, ARS- 104. Gounaris, E. K., Sherwood, R. T., Gounaris. I., Hamilton, R. H., and Gustine, D. L. 1991. Inorganic salts modify embryo sac development in sexual and aprosporus Cenchrus ciliaris. Sex. Plant Reprod. 4, 188-192. Gustafsson, A. 1946. Apomixis in higher plants. I. The mechanism of apomixis. Lunds Univ. Arsskr. Avd. 2,42, 1-66. Hanna, W. W. 1979. Interspecific hybrids between pearl millet and fountaingrass. J. Hered. 70, 425-427. Hanna, W. W. 1991. Apomixis in crop plants: Cytogenetic basis and role in plant breeding. In “Chromosome Engineering in Plants: Genetics, Breeding, Evaluation” (P. K. Gupta and T. Tsuchiya, eds.), Part A, pp. 229-242. Elsevier, New York. Hanna, W.W., and Bashaw, E. C. 1987. Apomixis: Its identification and use in plant breeding. Crop Sci. 27, 1136-1 139. Hanna, W.W.,and Dujardin, M. 1990. Role of apomixis in building and maintaining genome combinations. In “Proc. Second Int. Symp. on Chromosome Engineering in Plants.” pp. 112- 117. Univ. Missouri, Columbia. Hanna, W. W., Dujardin, M., and Monson, W. G. 1989. Using diverse species to improve quality and yield in the fenniseium genus. In “Proc. XVI Int. Grassl. Congr.,” pp. 403-404. Nice, France. Hanna, W. W., Dujardin, M., Ozias-Akins, P., and Arthur, L. 1992. Transfer of apomixis in fenniserum. In “Proc. Apomixis Workshop.” pp. 30-33. Atlanta, GA.

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Hanna. W., Dujardin, M., Ozias-Akins, P.. Lubbers, E., and Arthur, L. 1993. Reproduction, cytology, and fertility of pearl millet X Pennisetum squatnulrrtum BC, plants. J . Hered. 84,213-216. Hanna, W. W., and Powell, J. B. 1973. Stubby head, an induced facultative apomict in pearl millet. Crop Sri. 13,726-728. Hanna, W. W., Powell, J. B., Millot, J. C., and Burton, G. W. 1973. Cytology of obligate sexual plants in fanicum maximum Jacq. and their use in controlled hybrids. Crop Sci. 13,695-697. Hanna, W. W., Schertz, K. F., and Bashaw, E. C. 1970. Apospory in Sorghum bicolor (L.) Moench. Science 170,338-339. Hearn, C. J.. Barrett, H. C., and Niedz, R. P. 1992. Apomixis in citrus. In “Proc. Apomixis Workshop,” pp. 49-52. Atlanta, GA. Hermsen, J. G. 1980. Breeding for apomixis in potato: Pursuing a utopian scheme. Euphytico 29, 595 -607. Hussey, M. A., Bashaw, E. C., Hignight, K. W., and Dahmer, M. L. 1991. Influence of photoperiod on the frequency of sexual embryo sacs in facultatively apomictic buffelgrass. Euphyrica 54, 141-145. Khokhlov, S. S. (ed.) 1976. “Apomixis and Breeding.” [Translated from Russian by Amerind Publishing Co. Pvt. Ltd., New Delhi, for USDA-ARS] Lutts, S., Ndikumana, J., and Louant, B. P. 1991. Fertility of Brachiaricr ruziziensis in interspecific crosses with Brachinria decumbens and Brrrrhictricr brizantha: Meiotic behavior, pollen viability and seed set. Euphyrica 57,267-274. Miles, J. W., Pedraza, F., Palacios, N., and Tohme, J. 1994. Molecular marker for the apomixis gene in Brachiarifr. In “Plant Genome 11,” p. 51. San Diego, CA. [Abstract] Nakajima, K. 1990. Apomixis and its application to plant breeding. In “Proc. Gamma Field Symposia No. 29,Ohmija-machi,” pp. 71 -92. Ibaraki-ken, Japan. National Academy of Science. 1972. Genetic vulnerability of major crops. NAS Printing and Publishing Office, Washington, D.C. Nogler, G. A. 1984. Gametophytic apomixis. In “Embryology of Angiosperms” (B. M. Johri, ed.). Springer-Verlag, New York. Ouendeba, B., Ejeta, G., Nyquist, W., Hanna, W., and Kumar, A. 1992. Heterosis and combining ability among African pearl millet landraces. Crop Sci. 33,735-739. Ozias-Akins, P., Lubbers, E. L., and Hanna, W. W. 1992. Molecular research on apomixis in fenniseturn. In “Proc. Apomixis Workshop,” pp, 34-35. Atlanta, GA. Ozias-Akins, P., Lubbers, E. L., Hanna, W. W., and MacNay, J. W. 1993. Transmission of the apomictic mode of reproduction in Pennisetum: Coinheritance of the trait and molecular markers. Theor. Appl. Genet. 85,632-638. Pepin, G. W., and Funk, C. R. 1971. Intraspecific hybridization as a method of breeding, Kentucky Bluegrass (fooprrrtensis L.) for turf. Crop Sci. 11,445-448. Petrov, D. F. (ed.) 1984. “Apomixis and Its Role in Evolution and Breeding.” [Translated from Russian by Amerind Publishing Co., Pvt. Ltd., New Delhi for USDA and National Science Foundation] Rutger, J. N. 1992. Searching for apomixis in rice. In “Proc. Apomixis Workshop,” pp. 36-39. Atlanta, GA. Savidan, Y. I98 I . Genetics and utilization of apomixis for the improvement of guineagrass (fcinicuriz tnnxitnutn Jacq.). In “Proc. XIV Int. Grassl. Congr.,” pp. 182- 184. Westview Press, Boulder, CO. Savidan. Y.. LeBlanc, O., and Berthaud, J. 1993. Progress in the transfer of apomixis in maize. In “Agronomy Abstracts,” p. 101. Madison, WI. Schertz, K. F. 1992. Apomixis i n sorghum. In “Proc. Apomixis Workshop,” pp. 40-42. Atlanta, GA. Schertz, K. F., and Pring, D. R. 1982. Cytoplasmic sterility systems in sorghum. In “Proc. Interal. Symp. Sorghum’’ (L. R. House, L. K. Mughogho, and J. M. Peacock, eds.), pp. 373-383.

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A

Smith, R. L. 1972. Sexual reproduction in Punicum maximum Jacq. Crop Sci. 12,624-627. Taliaferro, C. M., and Bashaw, E. C. 1966. Inheritance and control of apomixis in breeding buffelgrass, Pennisetum ciliare. Crop Sci. 6,473-476. Virrnini, S. S., Aquino, R. C., and Khush, G. S. 1982. Heterosis breeding in rice. Theor. Appl. Genet. 63,373-380. Voigt, P. W. 1971. Discovery of sexuality in Erugrostis curvulu (Schrad) Nees. Crop Sci. 11,424-425. Wilson, K.J. (ed.) 1993. In “Proc. Int. Workshop on Apomixis in Rice, Changsha, Peoples Republic of China.” The Rockefeller Foundation, New York. Young, B. A,, Sherwood, R. T., and Bashaw, E. C. 1979. Cleared-pistil and thick-sectioning techniques for detecting aposporus apomixis in grasses. Can. J. Bor. 57, 1668- 1672.

Index A Acetolactate synthatase inhibitors, crop resistance, 84-87 Acetyl-CoA carboxylase inhibitors, crop resistance, 87-89 Agriculture definition, 4 impact on subsurface microbial ecology, 1-57 background definitions, 3-4 dynamic bounding sphere metaphor, 2 function, 22-35 metabolic status, 30-32 nutrient cycling, 32-35 responsiveness to change, 22-30 habitat structure, 5-22 geology, 5-8 hydrology, 5-8 organisms, 8-22 actinomycetes, 19-20 fungi, 19-20 protozoa, 21 -22 management practices, 36-46 crop, 39-43 livestock, 45-46 pest, 43-45 soil, 39 water, 37-38 measurement, 47-56 geochemical changes, 52-54 integrated effects testing, 54-56 physical changes, 49-52 subsurface versus surface habitats, 46-47 Aluminum phytotoxicity, 120- 122 wheat tolerance binding in cell wall, 129- 130 exclusion at the plasmalemma, 131- 132 genetic basis, 137- 143 manganese tolerance relationship, 145- 146 organic acid accumulation, 127- 129 root mucilage production, 130- 131 tolerant protein synthesis, 132- 134

351

Ametryne, crop resistance, 80-84 Ammonia nitrate ratio effect on plant growth, 243-246 volatilization, nitrification inhibitor induced, 240- 24 I Apomixis, 333-347 breeding, 337-345 advantages, 338-339 genetic vulnerability, 345 methods, 340-345 chemical control, 344-345 dominant gene, 341 facultative apomixis, 344 interspecific hybrids, 344 recessive gene, 341 -344 plant identification, 339-340 controlling genes, 334-337 expression, 336-337 genetics, 337 sources, 335-336 impact on seed industry, 345-346 international impact, 346 Arylox yphenoxypropionates, crop resistance, 87-89 Ascochyta blight, in lentil, 300 Asulam, crop resistance, 99 Atrazine, crop resistance, 80-84

B Bacteria, see Microbes Bipyridiliums, crop resistance, 98-99 Blight, ascochyta, in lentil, 300 Breeding, see Plant breeding Bromoxynil, crop resistance, 92-93

C Chromate, microbial reduction, 210-215 bioremediation of contaminated soils, 214-215 mechanisms, 212-214 microorganisms, 210-2 12 Contamination, see Environmental contamination

352

INDEX

Corn, nitrification inhibitors effect on yield,

248-249

Fungi seedborne disease, in lentil, 300 subsurface habitat structure, 19-20

Cotton, nitrification inhibitors effect on yield, 252 Crops, see speciJiccrop Cyanamide, crop resistance, 96 G Cyanazine, crop resistance, 80-84 Cyclohexanediones, crop resistance, 87-89 Genetics, see Plant breeding Geology, subsurface microbial habitat structure,

D

Dalapon, crop resistance, 96-97 Denitritication, rate reduction by nitrification inhibition, 241 2,4-Dichlorophenoxyaceticacid, crop resistance,

93-94 Dicyandiamide nitrification inhibition, 234-243 nitrogen loss and immobilization, 240-243 relative effectiveness, 236-238 soil factors affecting effectiveness, 239-

240 phytotoxicity, 252-254 Dihydropteroate synthase inhibitors, crop resistance, 99 Diquat, crop resistance, 98-99 Dynamic bounding sphere metaphor, impact of agricultural practices on subsurface microbial ecology, 2

E Ecology, see Microbes, subsurface ecology Environmental contamination bioremediation by microbial reduction chromate, 214-215 organic compounds, 195- 197 selenium, 208-2 10 uranium, 204-205 nitrates, 256-264 ozone layer depletion, 264-269

F Fertilizers impact on subsurface microbial habitats,

39-43,52-54 lentil requirements, 296 nitrogen immobilization by microorganisms,

242-243 nitrogen use, 234-235

5-8 Global warming, 268-269 Glufosinate, crop resistance, 94-96 Glyphosate, crop resistance, 89-91 Greenhouse gases, 268-269 Groundwater, see also Water management definition, 3-4 nitrate contamination, 256-264 nutrient cycling, 32-35 subsurface microbial habitat structure, 5-8

H Herbicides, crop resistance, 69- 101 chemical families, 80- 100 acetolactate synthatase inhibitors, 84-87 acetyl-CoA carboxylase inhibitors, 87-89 bipyridiliums, 98-99 bromoxynil, 92-93 cyanamide, 96 dalapon, 96-97 dihydropteroate synthase inhibitors, 99 glufosinate, 94-96 glyphosate, 89-91 mitotic inhibitors, 99- 100 phenoxycarboxylic acids, 93-94 phosphinothricin, 94-96 phytoene desaturase inhibitors, 97-98 protoporphyrinogen-oxidaseinhibitors, 98 triazines, 80-84 mechanisms, 71 -77 exclusion, 72-76 site of action alteration, 76-77 site of action overproduction, 77 variant selection, 77-80 biotechnological techniques, 78-80 genetic sources, 78 traditional plant-breeding techniques, 78 Human health, nitrate effects, 254-256 Hydrogen, oxidation by iron and manganesereducing microorganisms, 176- 181 Hydrology, see Groundwater: Water management

INDEX I Imidazolinones, crop resistance, 84-87 Iron, microbial reduction, 176-201 activity monitoring, 186- 187 effects on plant growth, 201 effects on soil properties, 199-200 electron flow in anoxic soils, 192-195 electron transport, 183- 184 interaction with other microbially catalyzed redox processes, 190- 192 isolation, 184- 186 mechanisms, 187- 190 microorganisms, 176- 183 hydrogen oxidation, 176- 181 magnetotactic bacteria, 182- 183 organic matter oxidation, 176- 18 I sulfur-oxidizing reducers, 181- 182 organic contaminant degradation, 195- 197 oxide formation, 197- 199 Irrigation, impact on subsurface microbial habitats, 37-38.49-52

L Lentil, 283-327 background, 284-291 Cytology, 288-289 origin, 286-287 plant description, 289-291 taxonomy, 287-288 breeding methods, 3 18-32 I backcross, 320-321 bulk populations, 319 pedigree selection, 3 19-320 pure line selection, 3 18 single seed descent, 320 breeding objectives, 32 1 -326 cultivar quality, 324 diseases, 322-323 insects, 324 mechanical harvesting adaptation, 325-326 orobanche, 323-324 root rot/wilt complex, 323 seed yields, 32 1-322 straw yields, 321-322 fertilization. 296 genetics, 307-3 17 germ plasm collection, 307-308 inherited traits, 308-3 17

353

association among traits, 317 cotyledon color, 3 10 epicotyl color, 3 12 flower color, 3 10-3 1 1 flower number, 312 genetic variance, 314-315 growth habit, 312 heritability estimates, 3 15-3 I6 isozymes, 313-314 pod indehiscence, 3 13 seed coat color, 3 11-3 12 virus resistance, 3 13 interspecific hybridization, 3 17 wild species, 308 hybridization methods, 303-307 environmental conditions, 303-304 equipment, 304 female flower emasculation, 304-305 pollination, 305-306 production, 291 -295 cultivars, 294-295 land requirements, 291 -292 seedbed preparation, 293 seeding, 293-294 seed quality, 292 seed treatment, 292 production constraints, 298-303 diseases, 299-302 environmental stress, 302-303 insects, 298-299 uses, 297-298 weed control, 297 Livestock management, impact on subsurface microbial habitats, 45-46

M Magnetotactic bacteria, iron reduction, 182-183 Malate, aluminum chelation in tolerant wheat, 128-129 Manganese microbial reduction, 176-201 activity monitoring, 186- 187 effects on plant growth, 201 effects on soil properties, 199-200 electron flow in anoxic soils, 192-195 electron transport, 183- 184 environmental reduction mechanisms, 187- I90

3 54

INDEX

Manganese, (continued) interaction with other microbially catalyzed redox processes, 190- 192 isolation, 184- 186 microorganisms, 176- 183 hydrogen oxidation, 176- 181 organic matter oxidation, 176- 181 sulfur-oxidizing reducers, 181- 182 organic contaminant degradation, 195- 197 oxide formation, 197- 199 phytotoxicity, 120- 122, 134-135 wheat tolerance aluminum tolerance relationship, 145- 146 distribution, 134- 135 genetic basis, 143- 145 mechanisms, 135-136 uptake in roots, 134- 135 Methane, production inhibition by iron oxides, 191 Microbes chemical reduction, 175-217 chromate, 2 10-2 15 bioremediation of contaminated soils, 214-215 mechanisms, 2 12-214 microorganisms, 210-212 iron, 176-201 activity monitoring, 186-187 effect on plant growth, 201 effect on soil properties, 199-200 electron flow in anoxic soils, 192-195 electron transport, 183- 184 environmental reduction mechanisms, 187- 190 interaction with other microbially catalyzed redox processes, 190- 192 isolation, 184- 186 microorganisms, 176- I83 hydrogen oxidation, 176- I8 1 organic matter oxidation, 176- 181 reduction by magnetotactic bacteria, 182- 183 sulfur-oxidizing reducers, 181- 182 organic contaminant degradation, 195197 oxide formation, 197- 199 manganese, 176-201 activity monitoring, 186- 187 effect on plant growth, 201 effect on soil properties, 199-200

electron flow in anoxic soils, 192-195 electron transport, 183-184 environmental reduction mechanisms, 187-190 interaction with other microbially catalyzed redox processes, 190- 192 isolation, 184- 186 microorganisms, 176- 183 hydrogen oxidation, 176- 181 organic matter oxidation, 176- 181 sulfur-oxidizing reducers, 18 1 - 182 organic contaminant degradation, 195197 oxide formation, 197- 199 selenium, 205-2 10 bioremediation of contaminated soils, 208- 2 10 enzymatic mechanisms, 207-208 microorganisms, 205-207 uranium, 202-205 bioremediation of contaminated soils and water, 204-205 enzymatic mechanisms, 203 enzymatic versus nonenzymatic reduction, 203-204 microorganisms, 202-203 immobilization of nitrogen fertilizer, 242243 metabolic status in subsurface habitats, 3032 responsiveness to environmental change, 22-30 subsurface ecology, 1-57 agricultural impact, 35-57 management practices, 36-46 crop, 39-43 livestock, 45-46 pest, 43-45 soil, 39 water, 37-38 measurement, 47-56 geochemical changes, 52-54 integrated effects testing, 54-56 physical changes, 49-52 subsurface versus surface habitats, 46-47 background definitions, 3-4 dynamic bounding sphere metaphor, 2 function, 22-35 metabolic status, 30-32

INDEX nutrient cycling, 32-35 responsiveness to change, 22-30 habitat structure, 5-22 geology, 5-8 hydrology, 5-8 organisms, 8-22 actinomycetes, 19- 20 fungi, 19-20 protozoa, 21 -22 Mitotic inhibitors, crop resistance, 99- 100

N Nitrapyrin nitrification inhibition, 234-243 nitrogen loss and immobilization, 240-243 relative effectiveness, 236-238 soil factors affecting effectiveness, 239240 phytotoxicity, 252-254 Nitrates ammonium ratio, effect on plant growth, 243 - 246 environmental effects, 262-269 global warming, 268-269 groundwater content, 262-264 ozone depletion, 264-268 health effects animal, 256 in drinking water, 256-261 human, 254-256 in vegetables, 261 -262 iron reduction inhibition, 190 Nitrification inhibitors, 233-269 ammoniumhitrate ratios, 243 -246 effect on crop yields, 246-252 corn, 248-249 cotton, 252 potato, 251 -252 rice, 247-248 sorghum, 249-250 sugarcane, 25 1 wheat, 249-250 environmental effects, 262-269 global warming, 268-269 groundwater content, 262-264 ozone depletion, 264-268 and nitrates animal health, 256 in drinking water, 256-261

355

human health, 254-256 in vegetables, 261 -262 nitrogen loss, 240-242 ammonia volatilization, 240-241 denitrification, 241 immobilization by microorganisms, 242243 from plants, 241 -242 urea hydrolysis, 240 phytotoxicity, 252-254 relative effectiveness, 236-238 soil factors affecting effectiveness, 239-240 organic matter, 238-239 pH, 240 soil water, 240 temperature, 239-240 Nitrogen annual fertilizer use, 234-235 immobilization by microorganisms, 242-243 IOSS, 240-242 Nitrous oxide, ozone depletion, 264-269 Nutrient cycling, in subsurface microbial habitats, 32-35

0 Organic matter effect on nitrification inhibitor effectiveness, 238 - 239 oxidation by iron and manganese-reducing microorganisms, 176- I8 1 Orobanche, in lentil, 323-324 Ozone, depletion, 264-269

P Paraquat, crop resistance, 98-99 Pesticides, see also speciJic chemical compound impact on subsurface microbial habitats, 54-56 Pest management impact on subsurface microbial habitats, 43-45 in lentil, 298-299,324 pH, effect on nitrification inhibitor effectiveness, 240 Phenoxycarboxylic acids, crop resistance, 93 -94 Phosphinothricin, crop resistance, 94-96 Phytoene desaturase inhibitors, crop resistance, 97-98

3 56 Phytotoxicity acid soils, 120-122, 134-135 nitrification inhibitors, 252-254 Plant breeding acid tolerance in wheat, 146- 161 approaches, 159- 161 cultivar development, 161 genetic pool variation, 149- 151 justification, 147- 149 screening strategies, 151- 159 apomixis, 333-347 advantages, 338-339 controlling genes, 334-337 expression, 336-337 genetics, 337 sources, 335-336 genetic vulnerability, 345 impact on seed industry, 345-346 international impact, 346 methods, 340-345 chemical control, 344-345 dominant gene, 341 facultative apomixis, 344 interspecific hybrids, 344 recessive gene, 341 -344 plant identification, 339-340 for herbicide resistance, 78 lentils genetics, 307-3 17 germ plasm collection, 307-308 inherited traits, 308-317 association among traits, 3 17 cotyledon color, 3 10 epicotyl color, 3 I2 flower color, 3 10-3 I 1 flower number, 3 12 genetic variance, 314-315 growth habit, 3 12 heritability estimates, 315-3 16 isozymes, 313-314 pod indehiscence, 313 seed coat color, 3 I 1-3 12 virus resistance, 3 13 wild species, 308 hybridization, 303-307 environmental conditions, 303-304 equipment, 304 female flower emasculation, 304-305 pollination, 305-306 methods, 3 18-32 1

INDEX backcross, 320-321 bulk population, 319 pedigree selection, 3 19-320 pure line selection, 3 18 single seed descent, 320 objectives, 32 1-326 cultivar quality, 324 diseases, 322-323 insects, 324 mechanical harvesting adaptation, 325326 orobanche. 323-324 root rot/wilt complex, 323 seed yields, 321 -322 straw yields, 32 1-322 screening strategies field evaluation, 157- 159 nutrient solution culture, 152- 156 soil bioassays, 156- 157 tissueculture, 151-152 Plant growth annual nitrogen fertilizer use, 234-235 effects of microbial reduction, 201 Plasmalemma, aluminum exclusion in tolerant wheat, 131-132 Pollution, see Environmental contamination Potato, nitrification inhibitors effect on yield, 25 1-252 Prometryn, crop resistance, 80-84 Protoporphyrinogen-oxidase inhibitors, crop resistance, 98 Protozoa, subsurface habitat structure, 8-22

R Reduction, see Microbes, chemical reduction Reproduction, see Plant breeding Rice, nitrification inhibitors effect on yield, 247-248 Root mucilage, production in aluminum tolerant plants, 130- 131 Root rot/wilt complex, in lentil, 299-302, 323 Rust, in lentil, 300

S Seed industry, see Plant breeding Selenium, microbial reduction, 205-2 10 bioremediation of contaminated soils, 208210

INDEX enzymatic mechanisms, 207-208 microorganisms, 205-207 Simazine, crop resistance, 80-84 Soil acidity causal elements, 117- 120 global severity, 122- 124 phytotoxicity, 120- 122 acid tolerance in wheat, 124-164 aluminum tolerance binding in cell wall, 129- 130 exclusion at the plasmalemma, 13I - 132 genetic basis, 137- 143 manganese tolerance relationship, 145-. 146 organic acid accumulation, 127- 129 root mucilage production, 130- 131 tolerant protein synthesis, 132- 134 uptake in roots, 124- 127 breeding, 146- 161 approaches, 159- I6 I cultivar development, 161 genetic pool variation, 149- 15 1 justification, 147- 149 screening strategies, 15 1- 159 manganese tolerance aluminum tolerance relationship, 145146 distribution, 134- 135 genetic basis, 143- 145 mechanisms, 135- 136 uptake in roots, 134- 135 sustainable production, 161- 162 contamination, bioremediation by microbial reduction chromate, 2 14-2 I5 organic compounds, 195- 197 selenium, 208-210 uranium, 204-205 effects of microbial reduction, 199-200 electron flow to iron and manganese in anoxic sediments, 192- 195 horizons definition, 3 subsurface microbial habitat structure, 5-9 management, impact on subsurface microbial habitats, 39 nitrification inhibitor effectiveness factors, 239 - 240 organic matter, 238-239

357

pH, 240 soil water, 240 temperature, 239-240 screening for plant breeding, 156- 157 subsurface microbial ecology, see Microbes, subsurface ecology Sorghum, nitrification inhibitors effect on yield, 249-250 Sugarcane, nitrification inhibitors effect on yield, 25 1 Sulfonylureas, crop resistance, 84-87 Sulfur, oxidation by iron and manganesereducing microorganisms, 181- 182

T Triazolopyrimidine sulfonanilides, crop resistance. 84-87

U Uranium, microbial reduction, 202-205 bioremediation of contaminated soils and water, 204-205 enzymatic mechanisms, 203 enzymatic versus nonenzymatic reduction, 203-204 microorganisms, 202 - 203 Urea, hydrolysis retardation by nitrification inhibitors, 240

V Vadose zone, microbiology, I9 Viruses, in lentil, 300-302

W Water management, see also Groundwater impact on subsurface microbial habitats, 37-38 Weed control impact on subsurface microbial habitats, 43-44 in lentil, 297 Wheat acid soil tolerant. 124- 164 aluminum tolerance binding in cell wall, 129- 130 exclusion at the plasmalemma, 13I - 132

358 Wheat, (conrinued) genetic basis, 137- 143 manganese tolerance relationship, 145146 organic acid accumulation, 127- 129 root mucilage production, 130- 131 tolerant protein synthesis, 132- 134 uptake in roots, 124- 127 breeding, 146-161 approaches, 159-161 cultivar development, 161 genetic pool variation, 149- 151

INDEX justification, 147- 149 screening strategies, 151 - 159 manganese tolerance aluminum tolerance relationship, 145I46 distribution, 134- 135 genetic basis, 143-145 mechanisms, 135-136 uptake in roots, 134- 135 sustainable production, 161 - 162 nitrification inhibitors effect on yield, 249250