Phytobacteriology: (Cabi)

Phytobacteriology principles and practice

Explanation for Figures on cover page Top left: Top centre: Top right: Lower left: Lower centre: Lower right:

Symptoms of bacterial brown rot, caused by Ralstonia solanacearum in potato. Fluorescent cells of Erwinia chrysanthemi in immuno-fluorescence microscopy. Potato tuber showing symptoms of soft rot caused by Erwinia carotovora subsp. carotovora. Water-soaked spots on pods of pea (Pisum sativum), caused by Pseudomonas syringae pv. pisi. Pure culture of Pseudomonas savastanoi pv. fraxini, 3-day-old culture on nutrient agar. Galls on root collar of Gladiolus corms, caused by Rhodococcus fascians.

Explanation for Figures on title page Left:

Right:

Electron-microscopic (EM) photo of dividing cells of Xanthomonas hyacinthi, causal agent of yellow disease in hyacinth (Hyacinthus orientalis). Shadow cast technique. Cells 0.9 µm in length. Photo: Laboratory for Bulb Research (LBO), Lisse, The Netherlands. EM photo of a single cell of Xanthomonas hyacinthi, showing the one polar flagellum, typical for the genus Xanthomonas. Shadow cast technique. Cell 1.4 µm in length. Photo: Laboratory for Bulb Research (LBO), Lisse, The Netherlands.

Fig. 1 Top:

Principal bacterial morphologies (straight rod, curved rod or vibrio, coccus, spirillum and curved with tapered end) of fresh water bacteria, using a long-forgotten Congo Red negative stain. Bottom: Large fresh water Spirillum species showing flagellar bundles in the same stain as mentioned above.

Phytobacteriology principles and practice

Dr. J. D. Janse Head of Department Bacteriology Plant Protection Service Wageningen The Netherlands

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© J.D. Janse 2005. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. All queries to be referred to the publisher. A catalogue record for this book is available from the British Library, London, UK. Library of Congress Cataloging-in-Publication Data Janse, J.D. (Jacob Dirk), 1953Phytobacteriology : principles and practice / J.D. Janse p. cm. Includes bibliographical references and index. ISBN-13: 978-1-84593-025-7 (alk. paper) ISBN-10: 1-84593-025-8 (alk. paper) 1. Bacterial diseases of plants. 2. Phytopathogenic bacteria. I. Title. SB734.J36 2006 632'.32—dc22 2005015226 ISBN-13: 978-1-84593-025-7 ISBN-10: 1-84593-025-8

Printed and bound in Singapore from copy supplied by the author by MRM Graphics and Kyodo.

v

Contents

Page

Preface Chapter I 1. 2. 3. 4. 5. 6. 7. 8.

1

-

Introduction to bacteriology and bacteria

Notes on the history of bacteriology Place of bacteria in the living world Morphology of bacteria Physiology and growth of bacteria Metabolism of bacteria Molecular biology and genetics of bacteria Genetic exchange between bacteria Taxonomy of bacteria

Chapter II -

Phytobacteriology and diagnosis of bacterial diseases of plants

1. Notes on the history of phytobacteriology 2. Phytopathogenic bacteria 3. Diagnosis of bacterial plant diseases a) Assessment of symptoms b) Isolation c) Pure culture d) Detection and identification - Conventional detection methods - Conventional identification methods - Newer detection methods - Newer identification methods e) Pathogenicity test f) Reisolation g) Reidentification h) Diagnosis report

Chapter III -

Disease and symptoms caused by plant pathogenic bacteria

1. The pathogenic bacterium 2. The host plant 3. Molecular basis for interaction between a pathogenic bacterium and a (non-) host: pathogenicity, virulence, HR reaction and resistance 4. Phases in pathogenesis 5. Symptoms a) Leaf spots b) Excrescences and galls c) Tumours d) Vascular disease and wilting e) Necroses and cankers f) Rotting g) Bacteria embedded in slime h) Symptoms of fastidious, (non-)culturable bacteria, including Xylella fastidiosa, phytoplasmas and spiroplasmas

Chapter IV 1. 2. 3. 4.

Epidemiology

Environmental effects and disease development Survival Dissemination and transmission of the pathogen and epidemiological cycles Geographical distribution of some bacterial pathogens

3 3 7 15 15 23 25 27 27 35 35 35 39 39 43 43 43 43 47 57 65 77 79 79 79 85 85 91 95 99 105 105 105 109 113 113 115 115 117 119 119 121 127 140

vi

Chapter V -

Damage and losses caused by bacterial plant diseases

143 143 143

Prevention and control of bacterial pathogens and diseases

149 149

1. Damage 2. Losses

Chapter VI -

1. Principles of control of plant pathogenic bacteria and/or the diseases they cause 2. Prevention of introduction and dispersal after interception of bacterial plant pathogens by quarantine measures and legislation 3. Control aimed at eradication 4. Prevention and control at farm or nursery level: the integrated approach 5. The role of education and hygiene 6. The role of healthy basic material and indexing/testing in control strategies 7. Breeding for resistance 8. Biological control 9. Chemical control 10. Sanitation and disinfection

Chapter VII -

Examples of bacterial diseases of cultivated and wild plants

1. Bulbaceous plants 2. Plants with bulbous roots (corms) 3. Gramineous plants 4. Palm trees 5. Orchids 6. Arable and cash crops 7. Fruit and nut trees, fruits 8. Ornamental plants 9. Stone fruits 10. Vegetables 11. Bacterial pathogens that attack many host plants

Annexes Annex 1 Annex 2 Annex 3 Annex 4 Annex 5ab Annex 6ab-

Newer classification of bacteria List of plant pathogenic bacteria and their main hosts List of plant pathogenic bacteria that appear on quarantine lists List of some (important) phytoplasmas Diversity of Ralstonia solanacearum and schemes for detection of potato ring rot and brown rot EU schemes for detection and identification of Ralstonia solanacearum in potato

149 153 155 155 161 163 167 169 173 175 175 181 183 195 197 201 209 231 241 247 269 283 283 285 295 298 299 301

Suggested reading and literature cited

303

List of host plants mentioned in Chapter VII

333

Index

341

Photographic credits Photographs and figures by the author, except where indicated with the illustration. PD = photograph of Plant Protection Service, Wageningen, The Netherlands. Special thanks to Dr. M. Scortichini for valuable contribution of illustrations, as indicated.

Acknowledgements I would like to thank all those students of my courses in plant bacteriology and microscopy at the International Agricultural Centre, Wageningen, The Netherlands, International Centre for Advanced Mediterranean Agronomic Studies, Bari, Italy and the School of Applied Environmental Sciences, University of Natal, Pietermaritzburg, SA that inspired me to write the following text. Furthermore I thank my wife and children for giving me time and having patience, my colleagues of the Division of Diagnostics and Department Bacteriology, Plant Protection Service, Wageningen and the General Management of the Plant Protection Service for constant support, Dr. N. Klijn, Division of Diagnostics, Plant Protection Service, Dr. J. Elphinstone, Central Science Laboratory, York and Dr. J. van Vaerenbergh, Institute for Crop Protection (CLO), Merelbeke, Belgium for critical reading of the manuscript and the staff of CABI for excellent co-operation and realization of the book.

Preface

1 The Camellia flower while about falling, stuck fast in the branches Shoha

Preface The objective of this introduction to phytobacteriology is to focus attention on and to discuss several aspects of this fascinating field of plant pathology. Chapter I briefly outlines the history and science of bacteriology and gives an overview of the diversity and versatility of the very small but immensely complex bacteria. Chapter II explains characterization, identification and naming of bacteria, which is not an easy task, with many pitfalls present. It will be shown that a diagnosis of a bacterial disease (i.e. linking a specific bacterium to symptoms observed in the field) may be even more difficult, requiring a conscientious attitude. An incorrect diagnosis may expose growers and countries to the adverse effects of new, imported bacterial diseases. It may also imply the erroneous destruction of large quantities of plants or erroneously blame the grower or exporting countries as responsible for the introduction of new alien pests. How bacteria cause disease and how plants react is described in Chapter III. Comprehension of the basis of symptom formation will enhance optimal application of preventive and control measures. In Chapter IV epidemiology of bacterial diseases is discussed, viz. the way in which bacteria can be dispersed and how diseases develop in space and time. The economic importance of bacterial diseases and strategies for control and reduction of crop losses are outlined in Chapters V and VI. In many cases bacterial diseases cannot be controlled chemically, leading to sometimes devastating epidemics, in important (food) crops like potato, tomato, cassava (manioc), rice, cotton and fruit trees. Finally some fifty examples of well- and lesser-known plant pathogenic bacteria and the diseases they cause are presented in Chapter VII.

Jaap D. Janse

Fig. 2 Bacteria on the root surface of Alnus glutinosa (some showing long flagellar tufts or adherence structures, presumably of polysaccharidal nature). Bar represents 1 µm.

2

Chapter I

Bacteria were first seen by Antoni van Leeuwenhoek in 1683

Archive Handke

Fig. 3 Left: Bacteria in dental plaque as they were observed for the first time by Antoni van Leeuwenhoek in 1683 (from his letter to the British Royal Society of 17 September 1683). A = rod-shaped bacterium; B = Selenomonas sputigena, an actively motile oral bacterium; E = Micrococcus species; F = Lepthotrichia buccalis; G = probably a spirochete. Right: Bacteria in dental plaque in phase-contrast microscopy, showing similar forms to those observed by van Leeuwenhoek.

Fig. 4 Left: Robert Koch at work in his laboratory, where he discovered that bacteria can cause disease (experiments with Bacillus anthracis, causing anthrax in sheep). Right: One of Koch’s microscopes as still present in the Robert Koch museum in Berlin.

Introduction to bacteria and bacteriology

3

CHAPTER I - INTRODUCTION TO BACTERIOLOGY AND BACTERIA1) 1. Notes on the history of bacteriology For many ages bacterial fermentation capacity has been widely used by mankind for preservation and production of foods such as butter, cheese and yoghurt. Preservation methods to avoid their action such as drying, salting and dehydration (by high sugar concentration) have been widely known as well. The first man to see bacteria was the Dutch merchant and microscopist Antoni van Leeuwenhoek (1632-1723). He observed them in suspensions of white material he obtained from his teeth (dental plaque). His drawings in a letter to the British Royal Society show the basic morphological forms, viz. rods, curved rods, cocci and spirillae (Fig. 3). Thereafter it took almost 150 years before naming of bacteria was started by C.G. Ehrenberg (1795-1876) in Germany, who introduced names like Bacillus and Spirillum, still used today. The idea that microbes, including bacteria, arose spontaneously was proven to be wrong by the famous and thorough scientific experiments of the Frenchman Louis Pasteur (1822-1895). He proved that fermentation and spoilage occurred only under the influence of micro-organisms (bacteria, yeasts) and that these organisms and their action could be stopped by heat-killing (sterilization) their vegetative cells or spores. It was the German country doctor Robert Koch (1843-1910, Fig. 4) who showed for the first time in 1876 that bacteria can cause disease. He proved that the rod-shaped particles he found in blood of sheep (Fig. 5) that died from anthrax, were infectious, living bacteria. He did so by cultivating the anthrax bacteria (Bacillus anthracis) in pure culture outside the body of the sheep (in the sterile liquid of the eyeballs of a cow) and re-inoculated these cultures into mice. The mice died from anthrax and showed the same particles (bacteria) in their blood as the sheep. These principles of isolation from diseased tissue, production of a pure culture and host test to prove pathogenicity are called Koch’s postulates (Fig. 6 and 7) and are still very important in bacteriology. Koch also described bacterial endospores for the first time, introduced agar media for bacterial growth and discovered the causal organisms of e.g. tuberculosis, Mycobacterium tuberculosis and cholera Vibrio cholerae. The Russian Sergei Winogradsk (1856-1953) discovered and described in 1889 the autotrophic bacteria that play an important role in iron and sulphur cycles on earth and later described free-living nitrogenfixing bacteria such as Azotobacter and Nitrobacter. After the discovery in 1928 of penicillin (produced by the fungus Penicillium notatum) by the Englishman Alexander Fleming (18811955), the American Selman A. Waksman (1888-1973) discovered in 1944 the antibiotic streptomycin (produced by the Actinomycete Streptomyces griseus). Oswald T. Avery (18771953) and co-workers demonstrated in 1944 that DNA is the genetic material of bacteria. Classification using (sequences of) ribosomal RNA and detection of the Archaea started in the late seventies and early eighties of the last century. The first complete sequence of a bacterial genome (Haemophilus influenzae) was published by Fleischer et al. in 1995 and the first one of a plant pathogen (Xylella fastidiosa) by Simpson et al. in 2000.

Development of Koch’s postulates based on his studies with Bacillus anthracis Fig. 5 Rod-shaped cells of Bacillus anthracis in a blood smear of a sheep, in Gram stain. L = leucocyte; BA = B. anthracis cell.

1)

For more information see Bulloch (1935); Sneath and Sokal (1973); Starr (1981); Lederberg et al. (2000); Madigan et al. (2000); Boone et al. (2001); Woese et al. (1990).

4

Chapter I

KOCH’S POSTULATES 1. The suspected pathogenic organism (here: the bacterium) must

always be present in lesions of the diseased tissues of an organism in question and absent in healthy organisms (here: plants). 2. The suspected organism must be isolated from the diseased tissues

and grown in pure culture. 3. When the pure culture of the organism is inoculated into a healthy

host (here: plant) in the laboratory it must produce a similar disease in this host. 4. The same organism must be found and reisolated from the

experimentally inoculated host (here: plant) in which disease developed.

Period between 1876 and 1915 Epidemics of cholera were controlled by a system also developed by Robert Koch and causal bacteria of diseases such as tuberculosis, leprosy, pneumonia, plague, typhus, paratyphus, botulism, tetanus, bacterial meningitis and Weil’s disease were discovered. Fig. 6 Top: Koch’s postulates. Lower left: Hand-drawn and coloured microscopic view of comma-shaped cholera bacteria (Vibrio cholerae) in slimy intestinal content of a patient. Lower right: Similar to left, but now in a section of the intestinal wall tissue. From: Kolle and Hetsch (1916).

Introduction to bacteria and bacteriology

5

Healthy plant

Diseased plant Positive: suspected bacterial cells

Negative: no bacterial cells

+ve band

IF test

Sudan Black stain

PCR

No PCR band

No (typical) colonies

Typical colonies

Identification by fatty acid analysis and/or other biochemical/molecular tests

Pure culture, similar to that used for inoculation

Pure culture

Typical colonies

Diseased plant Screening of inoculated host plant

Final IF or PCR test of reisolated culture

Fig. 7 Example of application of Koch’s postulates in modern phytobacteriology. Diagnosis of potato brown rot caused by Ralstonia solanacearum via 1. Screening using tests such as immuno-fluorescence (IF) staining, simple Sudan Black stain and DNA (PCR) that demonstrate bacteria in tissues; 2. Isolation of the pathogen and identification using biochemical methods; 3. Final confirmation by inoculation into a suitable host plant (tomato); 4. Reisolation from inoculated, diseased plants with a final IF or PCR test on reisolated culture.

6

Chapter I

Table 1

Major domains of living organisms and some of the representative taxa on the basis of comparative sequencing of 16S or 18S rRNA. After Brock (2000)

BACTERIA Aquifex Thermosulfobacterium Thermotoga Green non-sulfur bacteria Flavobacteria Cyanobacteria Gram-positive bacteria Proteobacteria (Gramnegative bacteria

ARCHAEA Marine Crenarchaota Pyrolobus Methanopyrus Thermoproteus Pyrodictium Methanopyrus Thermococcus Methanococcus

EUKARYA Diplomonads (Giardia) Microsporidia Trichomonads Flagellates Entamoebae Slime moulds Ciliates Fungi

Methanobacterium Methanosarcina Thermoplasma Extreme halophiles

Plants Animals Man

Fig. 8 R. Brlansky, Univ. of Florida, CREC, USA

Major lineages (kingdoms) of bacteria as determined by comparison of 16S rRNA sequences. After Madigan et al. (2000)

Fig. 9 Cells of Xylella fastidiosa, a xylem-limited fastidious Gramnegative bacterium (FXLB), causing Pierce’s disease of grapevine. This bacterium is culturable and is persistently transmitted by sharpshooters that feed on xylem fluids.

Introduction to bacteria and bacteriology

7

2. Place of bacteria in the living world Bacteria are micro-organisms, i.e. living organisms that cannot or can hardly be seen with the naked eye. In the living world we find (also see Table 1 and 2):

Macro-organisms

Eukarya or eukaryotes

Animals, man, plants (including algae and certain fungi)

Micro-organisms

Eukarya or eukaryotes

Animals (protozoa, Fig. 10) Plants (algae, Fig. 10) Most fungi

Prokarya or prokaryotes

Bacteria

Cyanobacteria ('blue-green algae')1) True bacteria or eubacteria Rickettsias, FXLB, FPLB and chlamidias2)

Archaea4) (bacteria-like microorganisms living in extreme environments) 1-4

Mycoplasmas, phytoplasmas and spiroplasmas3)

See pages 8 and 10

Eukaryotic organisms have a true nucleus, which is surrounded by a membrane and contains genetic material (DNA + proteins = chromosome). Furthermore these organisms contain mitochondria that play a role in respiration, two types of ribosomes and chloroplasts. They have large ribosomes in the cytoplasm and small ribosomes in the mitochondria. Chloroplasts occur in photosynthetic cells. Cell walls contain cellulose and/or chitin, but never peptidoglycan. Prokaryotic cells are characterized by the absence of a true nucleus with a membrane. The nuclear material (naked DNA) occurs free in the cell. Mitochondria are lacking and ribosomes are small. Chloroplasts do not occur; photosynthetic bacteria contain (bacterio-) chlorophylls in the cytoplasm or in so-called thylakoid membranes. In the case of bacteria the cell wall (when present) contains peptidoglycan or peptidoglycan-like polymers, but never chitin or cellulose. Cell walls of Archaea do not contain peptidoglycan but pseudopeptoglycan, polysaccharide, protein or glucoprotein. Tables 1-4 and Fig. 11 give an impression of the diversity of micro-organisms and bacterial forms in particular.

8

Chapter I

LESSER-KNOWN FORMS OF BACTERIA (A) 1. Cyanobacteria These organisms were formerly called ‘blue-green algae’, because it was assumed that they were plants (algae). They show photosynthesis (photoautotrophy, see Table 3) like plants, but do not contain chloroplasts. In all aspects they clearly belong to the bacteria. Many species are very big (up to 60 µm in diameter, and some filamentous species are many centimetres long). Their morphology is quite diverse. There are unicellular and multicellular species and (branched) filaments may be formed (Fig. 11). Filamentous species may contain specialised cells such as heterocysts, in which nitrogen is fixed and akinetes that are resting spores (see Fig. 11). Cells contain chlorophyll (green) and phycobilins, also necessary for photosynthesis. A main group of phycobilins, so-called phycocyanins, is blue, giving Cyanobacteria a blue-green colour. Cyanobacteria are widely distributed in fresh water, but are also present in soil and seawater. The nomenclature of Cyanobacteria still follows the rules of the Botanical Code (see Chapter I.8).

2. Rickettsias and chlamidias (a); fastidious xylem-limited bacteria or FXLB (b) and fastidious phloem-limited bacteria or FPLB (c) a) Rickettsias are small Gram-negative bacteria that live intra-cellularly (within cells) and they can cause serious diseases in man and animals (e.g. Rickettsia rickettsii causing Rocky Mountain spotted fever). Rickettsias are vector transmitted, usually by ticks. Both rickettsias and chlamidias cannot be cultured on/in artificial media. Chlamidias are very small bacterial pathogens that spread in the air as elementary bodies. These bodies grow into larger reticulate bodies that multiply inside the host cell. b) The fastidious xylem-inhabiting bacteria (FXLB) are rickettsia-like and plant pathogens. They are non-motile, rod-shaped bacteria (0.2-0.5 x 1.0-4.0 µm). The FXLB that are sharpshooter-transmitted have a Gram-negative cell wall and are mostly nonculturable. Leifsonia (Clavibacter) xyli, causing ratoon stunt in Saccharum officinale (sugarcane) is mechanically transmitted, culturable and has a Gram-positive cell wall. Symptoms of sharpshooter-transmitted FXLB are leaf burning, stunting, wilting, or decline. Important diseases include Pierce’s disease of grapevine (caused by the culturable Xylella fastidiosa that also causes alfalfa dwarf, almond leaf scorch, etc. (Table 7, Annex 5 and Fig. 9) and bacterial wilt of clove tree (Syzigium aromaticum), caused by Ralstonia syzigii. Vector insects feed on xylem and transmit the bacteria nonpersistently with no incubation in their body. Bacteria are regurgitated from the insect’s salivary syringe into the xylem upon feeding (also see Purcell and Hopkins, 1996). c) An example of fastidious phloem-limited bacteria (FPLB) is the causal agent of citrus greening or Citrus Huanglongbin disease. Citrus greening occurs in the African and Asian tropics and is one of the most destructive citrus diseases. The non-culturable pathogen with a Gram-negative cell wall is restricted to the phloem of infected plants. Based on 16S rRNA studies strains from Asia are slightly different from those found in Africa and also are transmitted by different psyllid vectors, Diaphorina citri in Asia and Trioza erytreae in Africa. The scientific names of ‘candidatus Liberobacter africanus’ and ‘candidatus Liberobacter asiaticus’ were proposed for the African and Asian isolates, respectively.

Introduction to bacteria and bacteriology

9

Animals are dependent on other organisms to obtain organic material. They are heterotrophic. Plants can synthesize organic materials with energy from light and with CO2 and inorganic salts (photosynthesis). They are autotrophic. Bacteria also show transitions between heteroand autotrophy (Table 3). Plant pathogenic bacteria are chemoheterotrophic.

Table 2

Differences between Prokaryotes (Bacteria and Archaea) and Eukaryotes. After Madigan et al. (2000)

Characteristic

Prokaryotes Bacteria Archaea

Prokaryotic cell structure DNA covalently closed, circular Histone proteins present Membrane-enclosed nucleus Peptidoglycan-based (muramic acid) cell wall Ribosomes Initiator tRNA Introns in most genes Operons Plasmids Ribosome sensitivity to diphtheria toxin RNA polymerases Transcription factors required Sensitivity to chloramphenicol, streptomycin and kanamycin Methanogenesis Reduction of S0 to H2S or Fe3+ to Fe2+ Nitrification Denitrification Nitrogen fixation Chlorophyll-based photosynthesis Chemolithotrophy (Fe, S, H2) Gas vesicles Carbon storage granules of polyhydroxyalkanoates Growth above 80ºC

Eukaryotes

+ + +

+ + + -

+ + -

70S Formylmethionine + + -

70S Methionine + + +

80S Methionine + Rare +

1 (4 subunits)

3 (12-14 subunits)

+

Several (8-12 subunits) + -

+

+ +

-

+ + + +

+ + -

+

+ + +

+ + +

-

+

+

-

+ -

Bacterium:

Different energy and carbon sources used by bacteria. Energy source C source

photoautotrophic

light

CO2

hotoheterotrophic

light

organic compounds

chemoautotrophic

inorganic compounds by oxidation-reduction reactions organic compounds

CO2

Table 3

chemoheterotrophic

organic compounds

10

Chapter I

LESSER-KNOWN FORMS OF BACTERIA (B) 3. Mycoplasmas, phytoplasmas and spiroplasmas These are wall-less, Gram-positive bacteria (free-living protoplasts that have tough membranes containing sterols or lipoglycans and live in protected osmotic-neutral habitats). Most are facultative aerobes (see below). Although there are phytopathogenic forms in this group, they will not be discussed in detail here. Traditionally virologists deal with them, mainly because most cannot be cultured. Spiroplasma citri is a culturable quarantine bacterium, causing stubborn disease of citrus. S. kunkelii causes corn stunt disease in Zea mays and similar diseases in many other hosts. Main symptoms are stunted plants, short internodes, leaf yellowing and mottling. Cells of spiroplasmas are spiralshaped (Fig. 114). S. citri is vector (leafhopper) transmitted (Fig. 112). Phytoplasmas are minute non-culturable bacteria living in the phloem of host plants and causing yellows disease in hundreds of different hosts. The aster yellows pathogen alone infects over 300 hosts, with plant species occurring in 50 families. Symptoms include virescence (loss of flower colour) and green flowers called phyllody, flower sterility, witches’ broom-like shoot proliferation, stunting and yellowing (Fig. 104 left). Some of them are quarantine pathogens. Phytoplasmas have a variable cellular morphology (pleomorphic) ranging from spherical to elongate or filamentous, c. 0.3 to 0.8 µm. They are not seed transmitted, and are usually sensitive to antibiotics of the tetracycline group and to heat treatment. A list of phytoplasmas is given in Annex 4. Phloem-sap-sucking insect vectors, like leafhoppers, plant hoppers and psyllids spread them, but they can also be transmitted by grafting and dodder (Cuscuta spp.). They cannot be cultured, but are identified on the basis of molecular probes such as poly- and monoclonal antibodies, cloned DNA fragments and especially 16S rRNA sequencing. Most phytoplasmas are still named after the diseases they cause, but for some species names have been proposed, such as ‘candidatus Phytoplasma australiense’, The genus Phytoplasma received the ‘candidatus’ status (see Annex 4, Lee et al., 2000 and for the new ‘candidatus’ names http://www.bacterio.cict.fr/candidatus.html. For detection and identification see e.g. Schaad et al. (2001).

4. Archaea Archaea are micro-organisms very much related to bacteria and grow in extreme environments, such as hot springs, salt lakes or deep sea. They have no peptidoglycan in their cell walls, but they have pseudopeptidoglycan or ether-linked lipids or a paracrystalline layer of polysaccharide, glycoprotein and protein in place. The group is very heterogeneous. Many of them produce methane, a major product of biodegradation. They are resistant to lysozyme and penicillin, substances that decompose peptidoglycan. The Crenarchaeota contain extreme thermophylic species, such as Thermofilum. Many methanogenic species, such as Methanococcus and extreme halophylic (enduring high salt concentrations) forms, such as Halobacterium belong to the Euryarchaota.

Introduction to bacteria and bacteriology

EUKARYOTES Fig. 10 Top left: Unicellular protozoan Euplotes. Top right: Unicellular algae Didimella in transparent pellicle. Bottom: Conidia of fungus Fusarium oxysporum.

PROKARYOTES Fig. 11 Top left:

Top right: Bottom:

Cell chain of the cyanobacterium Anabaena flos-aquae, showing nitrogen-fixing heterocyst (HC) and resting spore or akinete (A). Unicellular cyanobacterium Oscillatoria. Cells of a eubacterium, Pseudomonas savastanoi pv. savastanoi, in Gram stain.

11

12

Chapter I

Table 4

Diversity found in the Bacteria

Subdivision Kingdom I: Proteobacteria (Gram-negative bacteria) - Purple phototrophic bacteria - Nitrifying bacteria - Sulfur- and iron-oxidizing bacteria - Hydrogen-oxidizing bacteria - Methanotrophs and methylotrophs - Pseudomonas and the pseudomonads

- Acetic acid bacteria - Free-living aerobic nitrogen-fixing bacteria - Neisseria, Chromobacterium and relatives - Enterobacteraceae - Vibrio and Photobacterium - Rickettsias - Spirilla - Sheathed proteobacteria - Budding and prosthecate/stalked bacteria - Gliding Myxobacteria - Sulphate- and sulfur-reducing Proteobacteria

(also see Annex 1)

Representative genera Chromatium, Rhodobacter, Rhodospirillum Nitrobacter, Nitrosomonas, Nitrosococcus Thiobacillus, Beggiatoa, Thiothrix Ralstonia aeutrophus, Pseudomonas carboxydovorans Methylomonas, Methylobacter Burkholderia, Comamonas, Pseudomonas, Xanthomonas, Ralstonia, Acidovorax, Zymomonas, ‘candidatus’ Liberobacter’, Agrobacterium, Rhizobium, Bradyrhizobium, Mesorhizobium, Azorhizobium, Sinorhizobium, Allorhizobium Acetobacter, Gluconobacter Azotobacter, Azospirillum Moraxella, Acinetobacter Brenneria, Dickeya, Enterobacter, Erwinia, Escherichia, Klebsiella, Pantoea, Pectobacterium, Proteus, Samsonia Salmonella, Serratia, Shigella, Yersinia Rickettsia Spirillum, Campylobacter, Bdellovibrio Sphaerotilus, Lepthotrix Hyphomicrobium, Caulobacter Myxococcus, Stigmatella Desulfovibrio, Desulfobacter, Desulfuromonas

Kingdom II: Gram-positive bacteria - Non-sporulating, low GC, Gram-positive - Endospore-forming, low GC, Gram-positive - No cell wall, low GC, Gram-positive: the Mycoplasmas - High CG, Gram-positive - Filamentous, high GC, Gram-positive: the Actinomycetes

Staphylococcus, Micrococcus, Streptococcus, Lactobacillus Bacillus, Clostridium Mycoplasma, Spiroplasma, Phytoplasma Corynebacterium, Arthrobacter, Mycobacterium, Clavibacter, Rhodococcus, Curtobacterium, Rathayibacter Streptomyces, Frankia, Actinomyces

Kingdom III: Cyanobacteria and Prochlorophytes - Cyanobacteria - Prochlorophytes

Oscillatoria, Nostoc, Anabaena Prochloron

Kingdom IV: Clamydia

Chlamidia

Kingdom V: Planctomyces/Pirella

Planctomyces, Pirella

Kingdom VI: Bacteroides/Flavobacteria

Bacteroides, Flavobacterium, Cytophaga

Kingdom VII: Green sulfur bacteria

Chlorobium

Kingdom VIII: Spirochetes

Spirochaeta, Treponema, Leptospira

Kingdom IX: Deinococci

Deinococcus, Thermus

Kingdom X: Green non-sulfur bacteria

Chloroflexus, Heliothrix

Kingdom XI, XII, XIII: Hyperthermophiles

Thermotoga, Thermodesulfobacterium, Aquifex

Genera with plant pathogenic species Bacterial genera commonly associated with plants (as endophytes, saprophytes or symbiotic nitrogen-fixing organisms)

Introduction to bacteria and bacteriology

13

Bacteria are often thought to be noxious, causing disease or spoilage. Without bacteria, however, life on earth would not be possible. Soil fertility is determined by mineralization activities of bacteria present in the soil. Moreover, there are bacteria that are able to fix atmospheric nitrogen, either free living (e.g. Azotobacter) or in symbiosis with plants (Rhizobium, Frankia), see Figs. 12 and 13. The fixed nitrogen becomes available for plants. Bacteria are necessary for the decomposition of sewage and they are indispensable in the carbon, nitrogen and sulphur cycles on earth. Furthermore they play an important role in fermentation processes in agriculture, e.g. lactic acid fermentation in silage and (food) industry (e.g. butter, cheese, yoghurt, Fig. 13), in production of alcohol, polysaccharides, insulin and antibiotics and leaching of metals out of their ore or decomposition of nonnatural toxic or polluting materials, so called xenobiotics, e.g. decomposition of xylene and toluene. Only a very minor percentage of bacteria is pathogenic to man, animals or plants. Bacteria can be found almost everywhere. In soil their number is dependent on acidity, percentage of humus, O2 tension, humidity and soil cultivation. They are most abundantly present in superficial soil layers; 1 g of this soil may contain c. 108 bacterial cells! Per hectare, live weight of bacteria is c. 100-4000 kg, of fungi 500-5000 kg, of algae c. 700 kg, of protozoa 50 kg. In air many bacteria or bacterial spores are found. Bacteria are often attached to particles, so that dusty rooms contain many bacteria, presenting problems when sterile work is to be performed. Water, especially when polluted, may contain large numbers of bacteria (107 cells ml-1). Their number is dependent on the source of the water, O2 tension and degree of pollution. Pathogens may also be present in water. In non-sterilized food bacteria are almost always present. They can be desired (e.g. for vinegar production and lactic acid fermentation) or non-desired. Some of them cause disease; others cause spoilage or intoxication by production of toxins. On or in living organisms bacteria are always present, e.g. on the skin and in the gut of man and animals, on leaves and on or near roots of plants (Fig. 2).

BENEFICIAL BACTERIA Fig. 12 Left: Right:

Galls, so called rhizothamnia, caused by the nitrogen-fixing bacterium Frankia alni on Alnus glutinosa. Hyphae with nitrogen-fixing vesicles of Frankia alni on root tip. SEM photo.

14

Chapter I

PETIOLE

STOLON

APICAL MERISTEM

TAP ROOT

RHIZOBIUM NODULES

BENEFICIAL BACTERIA Fig. 13 Top left:

Lateral section through a root nodule of a leguminous plant (white clover, Trifolium repens), showing: (A) cortex tissue of root of white clover; (B) vascular tissue of host entering the nodule; (C) cork layer of nodule and (D) living nodule cells filled with nitrogen-fixing, red-stained Rhizobium trifolii bacteria. Top right: Swollen nitrogen-fixing Rhizobium trifolii cells in a Gram-stained smear from a root nodule of Trifolium repens. Centre right: Aerial root nodules on African Sesbania rostrata in symbiosis with Azorhizobium caulinodans. Stem nodules contain chloroplasts and are capable of carbon and nitrogen fixation. Plant is used as cattle fodder. Bottom left: White clover (Trifolium repens) with symbiotic root nodules. Bottom right: Gram-stained smear from yoghurt showing cells of lactic acid-producing Lactococcus (Streptococcus) lactis and Lactobacillus delbrueckii subsp. bulgaricus that will ferment milk to yoghurt.

Introduction to bacteria and bacteriology

15

3. Morphology of bacteria Bacteria have a cellular body with an average diameter of 1 µm. The human eye has a resolving power (ability to observe two very close objects as separate entities) of only c. 1 mm. A light microscope has therefore to be used to make bacteria visible. The resolving power of a normal light microscope is c. 0.2 µm using powerful objectives (usually 100:1 immersion-oil objectives). In transmitted light bacteria are transparent and hardly visible, therefore they are killed, (heat-) fixed and stained in a thin film on a microscope slide. When bacteria are examined under the light microscope, five characteristics can be determined, viz.: 1. Shape of the cell. Bacteria cells can occur as a coccus (spherical cell), coccobacillus (ovoid cell), straight rod, curved rod (vibrio), spiral rod i.e. spirillae (rigid spiral rod) and spirochetes (cell is a flexible rod) or filament (Actinomycetes) (Figs. 14 and 15). Within a culture of some bacteria the shape and size of cells may vary: these are called pleiomorphic bacteria. Sometimes the normal morphology may be changed when bacteria are grown on certain artificial media or during starvation or excessive presence of food. 2. Size of the cell. Generally cocci have a diameter of 0.1-1 µm (Fig. 14), rods usually a length of 1-2 µm (Fig. 14); some, however, can be 10 µm or more. Cyanobacteria (Fig. 11) and sheathed bacteria usually are much larger in size. 3. Combination of cells e.g. diplo-, strepto-, staphylococci, rods in chains, or a chain of cells in a sheath (so-called trichomes of e.g. Cyanobacteria). Also see Figs. 11, 14 and 15. 4. Staining reactions. Staining dyes can divide bacteria into groups. The most well-known example of a differential stain is the Gram stain, discriminating between Gram-positive bacteria (which remain blue-purple stained by crystal violet, even after decoloration with alcohol) and Gram-negative bacteria (which lose the purple stain upon decoloration and which are stained red with a counter stain, such as safranine). The discrimination is based on a difference in cell wall structure and composition (Figs. 19 and 20a). 5. Presence and place of cell organelles and other structures. Using special staining techniques flagella, nuclear material, storage material (granules of glycogen, polyphosphate or poly-ß-hydroxybutyrate), gas vacuoles (in photosynthetic, aquatic bacteria), capsules, loose slime and spores (Fig. 16) can be rendered visible. Flagellar arrangement and absence or presence of flagella is also important in classification of bacteria (Fig. 18). Using an electron-microscope many more details of the simple-looking, but complex bacterial cells or spores can be revealed, such as nuclear material (including plasmids and ribosomes), mesozomes, pili, structure of the cell wall, cytoplasmic membrane and flagella (Fig. 17).

4. Physiology and growth of bacteria Physiological characteristics (used in identification, Chapter II.3.d.) and nutritional requirements are very diverse in the bacterial world. Generally, apart from water, a C and N source, P, S, minerals (Fe, Ca, Mg) and trace elements (Fe, Mn, Co, Cn, Mo, Zn, etc.) are required. C and/or N sources may be sugars and other carbohydrates, amino acids, sterols, alcohols, hydrocarbons, methane, inorganic salts or CO2.

16

Chapter I

BACTERIAL CELL MORPHOLOGY

10 um

Fig. 14 Top left: Top right:

Centre left: Centre right: Bottom left:

Bacterial cell morphology. Large Spirochete (more than 100 µm long!) from surface water. Spirochetes have flagella that fold back from each pole and remain located in the periplasmic space (outer membrane) of the cell and are called endoflagella. These flagella give the cell a cork screw-like movement. Spherical cells in chains of Streptococcus pyogenes, causal agent of sore throat in a Gram-stained smear of sputum. Spherical cells in grape-like bunches of Staphylococcus aureus in a Gram-stained smear from pus in acne disease. Large rigid spiral cell of Spirillum volutans found in surface water. At the polar ends flagellar bundles (FB, red arrows) are visible.

Introduction to bacteria and bacteriology

ACTINOMYCETES, FILAMENTOUS GRAM-POSITIVE BACTERIA, FORMING MYCELIUM EXAMPLE: STREPTOMYCES SCABIEI

Fig. 15 Top left:

Branched filaments (hyphae) of Streptomyces scabiei, causing potato scab, in Gram stain. Top right: Spiral shaped aerial hyphae or sporophores that are formed in an aging S. scabiei colony (see bottom left and right). In the spiral part spore chains are formed (see centre insert). These exospores or conidia are different from resting spores of other bacteria; they have a relatively thin cell wall and dimensions of normal bacteria. Exospores are important for the dispersal of the organism. Centre: Exospores or conidia and spore chain of S. scabiei in Gram stain. Bottom left: 7-day-old colonies of S. scabiei on yeast-malt agar. Start of aerial hyphae formation (white colour). The brown colonies are raised, consist of tough mycelial growth and they are difficult to remove from the agar surface. Bottom right: 14-day-old colonies of S. scabiei on yeast-malt agar. Active spore formation in aerial hyphae that turn into a dusty grey mass.

17

18

Chapter I

Fig. 16 Top left:

Cells of a large rod-shaped bacterium from surface water in a suspension of Indian ink, clearly showing a layer of extracellular polysaccharide slime around the cells. In the cells volutin granules (storage material) can be seen. Top right: Slime capsules (arrow) of Klebsiella pneumoniae in a Gram- stained smear of sputum. Centre left: Thick-walled resting spores or endospores in a Bacillus sp. Because of the thick wall the spores are much more refractive in phase-contrast microscopy than the surrounding vegetative cell. Spores are centrally placed in the cell. Bottom left: Terminally placed resting spores (1) drumstick-shaped in cells of Clostridium tetani, causal agent of tetanus, and a strictly anaerobic bacterium. Individual spores (2) and (3) vegetative cells are also present.

Introduction to bacteria and bacteriology

LONGITUDINAL SECTION THROUGH A BACTERIUM (SCHEMATIC)

Fig. 17 Explanation: C = capsule of extracellular polysaccharides; CW = cell wall, mainly peptidoglycan; CPM = cytoplasmic membrane; F = polar flagellum; FA = flagellar attachment in cytoplasm; FG = fat granule, storage material; Fi = fimbriae; ME = mesosome; N = nucleosome (nucleic acid strand or ‘chromosome’ at one point attached to the cell wall, more than 1000 µm long); P = polyphosphate granule, storage material; PL = plasmid (short strand of closed, circular, free nucleic acid); Ri = ribosomes; Sfi = sex fimbrium or pilus.

19

Chapter I

J. van Vaerenbergh, CLO Merelbeke, Belgium

TYPES OF FLAGELLAR ARRANGEMENT

J. van Vaerenbergh, CLO Merelbeke, Belgium

20

Fig. 18 Top: Types of flagellar arrangement. Centre left: Peritrichous flagella of Erwinia salicis, causing watermark disease of water willow (Salix alba) under the electron microscope. Centre right: Xanthomonas axonopodis pv. begoniae, causing bacterial leaf spot of Begonia showing one polar flagellum under the electron microscope. Lophotrichous flagellar arrangement Bottom: of Pseudomonas savastanoi pv. fraxini, causing bacterial wart disease of ash tree (Fraxinus excelsior) by light microscopy, after silver staining.

Introduction to bacteria and bacteriology

21

DIFFERENCES IN CELL WALL STRUCTURE AND COMPOSITION BETWEEN GRAM-POSITIVE AND GRAMNEGATIVE BACTERIA

Gram-positive cells: Clavibacter michiganensis subsp. sepedonicus, staining blue with the primary dye crystal violet in the Gram stain. Arrow: cells in typical, so-called snapping division.

Gram-negative cells: Pseudomonas savastanoi pv. fraxini, staining red with the counter stain using safranine in the Gram stain.

Fig. 19 Structure of bacterial cell walls and reaction in Gram stain. For description of Gram stain, see text.

22

Chapter I

Structure of cell membrane and phospholipid

Fig. 20a Structure of cell membrane and phospholipid. The cytoplasmic membrane is an 8 nm thick selective barrier around the (bacterial) cell. The membrane consists of a phospholipid bilayer, where fatty acids point inward in a hydrophobic environment and the glycerol/phosphate towards the external hydrophilic environment (A). Phospholipids are complex lipids that form the basis of the cytoplasmic membrane (B).

Fig. 20b

A. Vidaver via M. Scortichini

Bacteriocins are toxic, narrow-spectrum protein metabolites of bacteria that inhibit/kill related bacteria

Test for production of bacteriocin by Clavibacter: inhibition of growth of (most of the) related bacteria tested, visible as a clear halo (no growth) around the colonies of different Clavibacter species.

Introduction to bacteria and bacteriology

23

Bacteria requiring molecular oxygen for growth are called aerobic. Those growing only in the absence of oxygen are called anaerobic, those growing in the presence or absence of oxygen facultative anaerobic and those growing at low oxygen tensions, like some lactic acid bacteria, microaerophylic. Bacteria multiply by division. In addition to cell enlargement that takes place between two divisions, division (asexual reproduction) itself is also called growth. Growth rate of bacteria is dependent on the bacterial species, physical factors (temperature, osmotic value, pH, etc.) and nutritional factors. A typical growth curve of a bacterium growing in an artificial medium is given in Fig. 21. Bacteria that show a higher growth temperature optimum than 45oC are called thermophilic (some of them isolated from hot springs, like Pyrodictium, even have an optimum of 105oC), those with an optimum of 1545oC are called mesophilic, and those with an optimum of 15-0oC (e.g. bacteria of the polar seas) are called psychrophilic. Some bacterial genera can form resting cells, so-called spores. There are two main types of bacterial spores. Firstly, endospores that are thick-walled resting bodies enabling the bacteria to survive adverse conditions such as desiccation, starvation and excessive temperatures. When the adverse conditions terminate the spore can germinate into a vegetative cell again. Endospores are very resistant to heat (therefore temperatures of 115121oC for 20 minutes are usually necessary to kill them), desiccation, radiation, chemical agents, etc. Endospores are mainly found in the genera Bacillus (aerobic spore-forming rods, Fig. 16 centre left) and Clostridium (strictly anaerobic spore-forming rods, Fig. 16 bottom left). The second type of spore is the exospore. These exospores are mainly formed by the hyphae-forming Actinomycetes, including Streptomyces spp. They are not so thick walled as endospores, are not completely dormant and mainly function in dissemination of the organism. Exospores are formed on special hyphae, so-called aerial hyphae (Fig. 15).

5. Metabolism of bacteria Heterotrophic bacteria obtain their energy and building blocks from degradation of organic material. They need energy for growth (chemical reactions in the cell), motility and uptake of nutrients. Complex substances are degraded by enzymes excreted by the bacteria (so-called exogenous enzymes). Low molecular mass substances can pass the cellular membrane and are further decomposed by intracellular enzymes (endogenous enzymes). Some of these enzymes are always present (constitutive enzymes), whereas others are only produced when their substrate is present (inducible enzymes). Aerobic bacteria obtain energy by 1) initial decomposition to a compound functioning in the citric acid cycle; 2) citric acid cycle and 3) electron transport. Anaerobic bacteria obtain energy via lactic acid fermentation, alcohol fermentation, mixed acid fermentation or butyric acid fermentation. Compounds from the decomposition cycles are again rebuilt to amino acids, vitamins, proteins, DNA and cell wall building blocks (carbohydrates, lipids), etc. This rebuilding is regulated via many different, complicated systems. Bacteria can produce toxins (mostly protein substances and often encoded by plasmid genes) that are important for the virulence of pathogenic species, also see Chapter III.1. A special group of secondary metabolites (products formed at the end of the growth phase or stationary phase) are the antibiotics. These substances have an (usually broad-spectrum) antimicrobial action. They are much used in human and veterinarian medicine in the control of infectious diseases but there are also examples in control of bacterial plant disease (see Chapter VI.9). Examples are streptomycin produced by Streptomyces griseus (active against Gram-positive

24

Chapter I

Fig. 21

Typical growth curve of a bacterium (Escherichia coli) growing in a tube with nutrient broth at 37oC. After an initial lag phase, where new molecules are produced and cells show little or no division, the bacterium adapts to the medium and reaches maximum rate of growth and division. After some time growth slows down to no increase in cell numbers due to production of waste and lack of nutrients. Finally the death phase is reached, where the number of living cells is decreasing. After Singleton (1992), 2nd ed., changed.

EXCHANGE OF GENETIC MATERIAL BY BACTERIA THROUGH CONJUGATION

Fig. 22

Explanation: The F+ or donor cell transfers genetic material in the form of an F-plasmid to a recipient F- cell. First the F+ cell makes contact with its pilus with an F- cell (A); the pilus contracts and one plasmid DNA strand is nicked, the other strand unwinds and is transferred to the F- cell (B, C); Subsequently the single strands in the recipient and the parent strain are (re) synthesized to doublestranded plasmids (D, E) and the recipient F- cell becomes an F+ or donor cell too (E).

Introduction to bacteria and bacteriology

25

and -negative bacteria), chlortetracycline by S. aureofaciens (active against Gram-positive and -negative bacteria, rickettsias, larger viruses and some protozoa) and polymyxin by Bacillus polymyxa (mainly active against Gram-negative bacteria). Streptomycetes are very active in production of antibiotics and many antibiotics are now chemically synthesized. Bacteriocins (Fig. 20b) are toxic protein metabolites from bacteria with a very narrow spectrum, i.e. they are inhibitory to or kill only strains of the same species or closely related species. Well studied is the so-called colicin of Escherichia coli. Genes for bacteriocin production are also often located on plasmids. A bacteriocin of Agrobacterium radiobacter is used in biocontrol of the plant pathogenic, tumour-producing bacterium Agrobacterium tumefaciens (see Chapter VI.8).

6. Molecular biology and genetics of bacteria The bacterial chromosome consists mainly of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The subunits of the DNA (so-called nucleotides) in their sequence contain information. Groups of nucleotides containing information for e.g. synthesis of a particular protein are called genes and determine the life of the cell to a high extent by encoding enzymes and RNA molecules. DNA is usually present as a large double-stranded molecule and it is replicated before cell division. RNA is usually present as a single-stranded molecule. DNA can also be present in the bacterial cell as small double-stranded, circular pieces (socalled plasmids). Plasmid DNA is also replicated, but this replication is controlled by the plasmid itself, using the cell’s biosynthetic possibilities. That is why more than one copy of a plasmid can be found in one bacterial cell. Plasmids may encode different functions, such as antibiotic resistance (see below), pathogenicity factors (such as the Ti plasmid of the plant pathogenic Agrobacterium spp., see Chapter III.5.c, Figs. 89 and 90), and genetic transfer from one bacterium to another, see Chapter I.6, Fig. 22 and Panopoulos and Peet (1985). Protein synthesis in the cell takes place on the ribosomes, where the cell uses m(essenger) RNA and t(ransfer) RNA to copy information present on the genes and place amino acids in the correct sequence. The small 70S (from Svedberg sedimentation units) ribosomes of bacteria, present in a high number, consist of two subunits of 30S and 50S. The 30S subunit contains 16S r(ibosomal) RNA molecules and the 50S contains 5S and 23S rRNA (Fig. 25). The rRNA contains stable and variable regions in its sequence of nucleotides and can be used to develop highly specific probes (small pieces of a specific sequence for a particular organism, used for detection and identification, also see Chapter II.3.d.) or (by comparing the sequences) be used for classification of bacteria (see Chapter I.8 and Figs. 8 and 25). Just like other organisms bacteria vary and by this variation they are able to adapt to changing environments. Variations may be determined genetically (genotypic) or by the environment (phenotypic). In the latter case all cells of a culture vary. Genotypic variation is for a large part based on spontaneous mutants (genetically modified bacteria) selected by the environment, which are present in a bacterial population. When for example streptomycin-sensitive bacteria are plated on medium containing streptomycin, generally no growth will take place, because the antibiotic kills bacteria. Sometimes, however, some colonies are found. Bacteria in these colonies contain a gene changing their ribosomes to such an extent that streptomycin becomes ineffective. When streptomycin is used in control of disease, resistant mutants will finally dominate more and more. Streptomycin applications become ineffective and other ways of control have to be used. Often resistance genes against antibiotics occur on plasmids, which can be transferred to other bacteria, mostly of the same species or genus (also see Fig. 149). In this way resistant populations may develop very rapidly. For a review on genomics of plant pathogenic bacteria see Preston et al., 1998.

26

Chapter I

INFECTION OF A BACTERIAL CELL BY A BACTERIOPHAGE (LYTIC CYCLE)

Fig. 23 Explanation: (A) The phage attaches to the cell wall and injects its nucleic acid; (B) phage nucleic acid multiplication using the machinery of the bacterial cell; (C) production of elements of the phage coat; (D) assemblage of new phage particles; (E) cell death and lysis, liberation of phage particles. Free after Salle (1973).

Introduction to bacteria and bacteriology

27

7. Genetic exchange between bacteria As has been stated before bacteria contain genetic material as double-stranded, circular DNA and sometimes also as short double-stranded pieces of circular DNA, so-called plasmids. Both ‘chromosomal’ and plasmid DNA can replicate themselves. Plasmids are not obligate for the existence of bacteria. In nature two mechanisms of genetic exchange (a kind of sexual process) are known to occur in bacteria, viz. transduction and conjugation. In transduction an infection with a bacterial virus (bacteriophage) must take place. The normal infection process by a bacteriophage is shown in Fig. 23. Some phages, called temperate phages, do not kill the bacterium immediately, but their nucleic acid may become integrated in the bacterial chromosome or exist as a ‘plasmid’ and remain dormant for some time (so-called lysogeny, the bacterium becoming lysogenic). During multiplication of the bacterium the integrated phage nucleic acid, called a prophage, is also copied to the new cell. When later the temperate phage starts to multiply and kills the bacterium, among the virus particles assembled, some particles may have a protein coat containing not only viral DNA but (by accident) also bacterial DNA. When such an ‘error’ particle infects another bacterium, bacterial DNA may be incorporated into the genome of the receptor cell, inducing new traits. In conjugation (Fig. 22) parts of ‘chromosomal’ DNA and plasmid DNA may be transferred through thin tubules (so-called pili, Fig. 17) to another cell. For this process are required: a) a donor cell (possessing pili and a special plasmid, containing a so-called F-factor, which encodes the pili and which is necessary for transfer of DNA. This F-factor can be present on the ‘chromosomal’ DNA or on the plasmid. The donor cell is called F+). b) a receptor cell (having no pili and no F-factor, called F-). Usually only a part of the DNA of an F+ cell is transferred to an F- cell. This may be total plasmid DNA or part of the chromosome. Conjugation is rather common and it is responsible for rapid spread of e.g. antibiotic resistance in bacterial populations (Chapter VI.9 and Fig. 149).

8. Taxonomy of bacteria Taxonomy is a scientific activity trying to create order in a complex diversity of organisms. In bacteriology taxonomy comprises: 1) Classification, orderly arrangement of organisms in entities, sub-entities, etc. (Table 6). 2) Nomenclature, giving names or labels to entities defined in 1), in agreement with accepted rules laid down in the International Code of Nomenclature of Bacteria of 1975. 3) Identification of unknown organisms with entities defined and named in 1) and 2). In the taxonomy of bacteria morphological, serological and metabolic (nutritional) characteristics (so-called phenotypic characteristics) are used traditionally. Table 5 presents an example of some of the tests useful in determining phenotypic characteristics for species related to Ralstonia solanacearum. More recently, characteristics of the genetic material itself are also used (genotypic characteristics), such as base composition of DNA, usually G(uanidine):C(ytosine) ratios and degree of homology of total DNA and/or RNA of different bacteria (Fig. 24). DNA and RNA hybridization show how much similarity there is in DNA or RNA sequences of the organisms that are compared. Ribotyping has also been found to be a rapid and specific method for classification and identification. In this technique DNA encoding 16S rRNA is cut into fragments by so-called restriction enzymes. The

28

Chapter I

V + V V + V + 97 +/inf. +

-/V + + + Human

+

-

<1% -

<1.5% -

V

-

(+) (+) + -

-

51 V +Clove tree

36 + +Banana

B. caryophylli

+ + + -

viscid, <5 mm

B. gladioli

+ + +/V +W +/V +

minute

Burkholderia cepacia

-/W +

+ -

Blood Disease Bacterium

fluidal, >5 mm + + + <2% + +/V

Ralstonia syzygii

Colonies on TZC agar Diffusible pigment Motility Growth at 37ºC Growth at 41ºC NaCl tolerance Oxidase Arginine dihydrolase Gelatin hydrolysis Pectin hydrolysis Nitrate reduction Oxidation of: Glucose Sucrose Galactose Glycerol Mannose Ribose Utilization of: Cellobiose Trehalose D-tartrate Mannitol Sorbitol N-propanol β-Alanine Inositol Betaine L-arginine L-lysine Heptanoate D-arabinose D-fucose D-raffinose % C sources utilized Tobacco HR Plant pathogenicity

Ralstonia picketii

Test

Ralstonia solanacearum

Table 5

Differential phenotypic characteristics of Ralstonia solanacearum and some related (plant pathogenic) bacteria

+ +

+ +

+ +

+

V

-

+ + + -/W

V + + -

+ + -

+ + + -

+ + + +

+ + +W -

+ + -/W + + + + + + + + + + V

+ + + + + + + + + + + + -

V + + + +W + + + V + + +

+ Also human

+

+ Dianthus

+ = positive; - = negative; V= variable; W = weak; inf. = infection; HR = hypersensitive reaction

+

29

Introduction to bacteria and bacteriology

fragments are treated with a probe and visualized in a gel. In this way a fingerprint of the bacteria being studied is obtained. For general principles of fingerprinting, see Chapter II.3d. Sequencing of 16S and 23S rRNA and comparing these sequences and fingerprinting of DNA using RFLP, REP-PCR, AFLP and RAPD procedures (Chapter II.3d.) have become extremely important in bacterial classification and have allowed a much better understanding of bacterial relationships than before (Fig. 8). The technique of 16S rRNA sequencing (Fig. 25) involves artificial amplification by the polymerase chain reaction (PCR, for the technique, see Chapter II.3.d) of genes encoding 16S rRNA and subsequent sequencing via standard dideoxy sequencing, so-called Sanger sequencing. For some non-cultivable bacteria (including phytoplasmas), based on the characterization of their DNA and RNA, names have been proposed that receive a so-called candidatus status. Examples are ‘candidatus Liberobacter africanicus’, ‘candidatus Phytoplasma brasiliense’ and ‘candidatus Phlomobacter fragariae’ (also see http://www.isppweb.org/names_bacterial_new2004.asp, http://www.bacterio.cict.fr/index.html and Zreik et al., 1998). Fingerprinting methods based on protein patterns (determined by electrophoretic techniques, see Chapter II.3.d.), fatty acid patterns (determined by gas chromatography, see Chapter II.3.d.), chemical composition of cell walls, or chemical determination of the occurrence of specific sugars, fats, fatty acids, amino acids and proteins are also important for taxonomy.

Table 6

Taxonomic hierarchy for Ralstonia solanacearum (Smith 1896) Yabuuchi, Kosako, Yano, Hotta and Nishiuchi 1996 after Boone et al. (2001)

Taxonomic (sub)division Domain

Name Bacteria

Properties

Method/test for confirmation

Prokaryotic cell

Microscopy, 16S rRNA sequencing, test for peptidoglycan 16S rRNA sequencing, Gram stain, microscopy

Phylum

BXII Proteobacteria rRNA typical for Proteobacteria, Gramnegative cell wall

Class

II. Betaproteobacteria rRNA typical of β group

Order

I. Burkholderiales

Family

II. Ralstoniaceae

Genus

I. Ralstonia

Species epithet

solanacearum

Rod-shaped cells, nonfluorescent

16S rRNA sequencing Microscopy, colony morphology

Typical DNA composition, DNA:DNA hybridization, protein and fatty acid PAGE, FAA pattern Non-fluorescent, PHB granules, oxidative, acid from sucrose, specific 16S rRNA, etc., pathogenic to certain plant hosts

Colony morphology, specific stain, biochemical tests, rRNA sequencing, pathogenicity test

30

Chapter I

THE USE OF DNA:DNA HYBRIDIZATION IN BACTERIAL TAXONOMY

Fig. 24 The use of genomic DNA:DNA hybridization in bacterial taxonomy. In this experiment a strain of Xanthomonas arboricola pv. fragariae shows perfect homology with itself (100%); two strains of the same species and pathovar still show a high homology (80%). A different species in the same genus (X. fragariae) shows only 40% homology with X. a. pv. fragariae and a bacterial species in a different genus (Rhodococcus fascians) shows only 5% homology with X. a. pv. fragariae. In this way genetic relationships between bacteria can be discovered.

Introduction to bacteria and bacteriology

31

Nowadays it is obligatory for the description of a new bacterial species to use as many approaches and methods (both molecular and conventional) to determine its taxonomic position, so-called polyphasic taxonomy (also see Sneath and Sokal, 1973). Research in recent years has led to many changes in classifications and in the past many bacterial names appeared to be non-validly published, either due to insufficient description or incorrect naming. It was decided to reject all invalid names published before 1980 and to publish a list of valid names in the International Journal of Systematic Bacteriology. This so called Approved List was published January 1, 1980. Descriptions and names of new species have now to be published in the International Journal of Systematic Bacteriology (now named: Journal of Systematic and Evolutionary Microbiology) or a summary description thereof taken from the original publication elsewhere. When the Approved List was published in 1980, unfortunately many names of bacterial plant pathogens were lost due to an unsettled taxonomy at that time. To give an example: there are many plant pathogenic bacteria showing much similarity with Pseudomonas syringae and formerly regarded as separate species, for example P. phaseolicola, P. tomato and P. pisi. Names of the latter three bacteria do not occur in the Approved List and are invalid. Therefore a list has been published by phytobacteriologists (International standards for naming pathovars of phytopathogenic bacteria and a list of pathovar names and pathotype strains) (Dye et al., 1980) trying to solve the problem in this way: Pseudomonas syringae pv. (= pathovar, pathogenic variety) syringae = in the old system P. syringae, causal agent of dieback and leaf spot in Forsythia, pear, lilac (Syringa) and other hosts; P. syringae pv. phaseolicola = P. phaseolicola in the old system, causal agent of halo blight of bean, etc. N.B. The naming of a pathovar is free, i.e. not determined by rules of the code. This also holds true for subdivision of a bacterial species or subspecies into pathological races and biochemical, serological or phage varieties (resp. biovars, serovars or phagovars) and genetic groups, see also Annex 5a-c for an example of subdivision of Ralstonia solanacearum into races, biovars and genetic RFLP groups. When new pathovars are described generally the above-mentioned international standards (including the indication of a pathovar type strain) are followed. Current (2004) names of plant pathogenic bacteria and some of their synonyms can be found in Annex 2 and 4. Young et al. published a list of names and synonyms in 1996. The bacteriological code uses the binomial system. The first name beginning with a capital indicates the genus, e.g. Xanthomonas, the second name the species epithet, e.g. citri (no capital). When the exact name has to be mentioned, the author(s) giving the first description of the bacterium with the prefix ‘ex’, and those who made the last taxonomic (re-) classification are also mentioned, together with the year of publication, i.e. Xanthomonas axonopodis pv. citri (ex Hasse 1915) Vauterin, Hoste, Kersters and Swings 1995. When only the genus is known, this is indicated as Xanthomonas sp. (= species). The most commonly used classification and nomenclature of bacteria can be found in Bergey’s Manual of Determinative Bacteriology, now named Bergey’s Manual of Systematic Bacteriology (Boone et al., 2001). Also see Young et al., 1992 and http://www.isppweb.org/names_bacterial.asp . Up-to-date nomenclature can also be found at http://www.dsmz.de/bactnom/bactname.htm of the German Culture Collection of Microorganisms (DSMZ) in Braunschweig, Germany and the site maintained by J.P. Euzéby, http://www.bacterio.cict.fr/.

32

Chapter I

THE USE OF 16S r(ibosomal)RNA SEQUENCING IN BACTERIAL TAXONOMY

Fig. 25 The use of 16S r(ibosomal) RNA sequencing in bacterial taxonomy. In this example the 16S rRNA genes of four different bacteria (A-D) are first multiplied by PCR using 16S rRNA gene-specific primers. After PCR the multiplied 16S RNA genes are separated in a gel, isolated from the gel and purified. In this state the 16S rRNA genes are ready to be sequenced. Sequencing is performed by using e.g. the so-called Sanger method, where the sequence is determined using a radioactive primer. Bacterium B has 5 nucleotides that are different in its sequence of 20 nucleotides compared with Bacterium A, therefore its genetic distance from Bacterium A is expressed as 0.25. Similarly the genetic distance for the other bacteria is determined and with statistical software a genetic distance matrix tree can be created and taxonomic positions clarified.

Phytobacteriology and diagnosis

33

PD

PHYTOBACTERIOLOGY THE STUDY OF BACTERIA THAT CAUSE DISEASES IN PLANTS

Symptoms of fire blight in Photinia (Stranvaesia) davidiana caused by Erwinia amylovora: water-soaked spots along veins and leaf margins, phloem necrosis and bacterial exudates.

Left:

Bacterial blotch symptoms on cultivated mushroom Agaricus bisporus caused by Pseudomonas tolaasii: brown blotches (spots) on the caps. Right: Cells of Xanthomonas populi, causing bacterial canker of poplar tree (Populus species) in Gram stain, average size of cells 1.0 x 1.0-1.5 µm.

34

Chapter II

E. F. SMITH (1854-1938), USA, FOUNDER OF PHYTOBACTERIOLOGY

Fig. 26a Cells of Xanthomonas hyacinthi as drawn by E.F. Smith from microscopic preparations. Source: as in Fig. 26c, page 345.

Fig. 26b Symptoms of yellow disease in hyacinth; compare with drawings of E.F. Smith in Fig. 26c.

Fig. 26c Symptoms of yellow disease caused by Xanthomonas hyacinthi. Plate 19 as it appeared in E.F. Smith, Bacteria in Relation to Plant Diseases, Volume 2, page 334, 1911. (1-3) Hyacinth (Hyacinthus orientalis) leaves inoculated at x, respectively, 23, 23 and 36 days after artificial inoculation, showing glassy discoloration of the vascular tissue. (4) Flower stalk inoculated at x, 27 days after inoculation. (5 and 6) Bulb showing badly diseased plateau with cavities and yellowing of the scales. (7) Same as (6), but base of plateau not yet ruptured. (8) Bulb some hours after cross-section, showing bacterial ooze from diseased vascular bundles. (9) Cross-section of bulb, showing scales very gummy on one side.

Phytobacteriology and diagnosis

CHAPTER II

35

- PHYTOBACTERIOLOGY AND DIAGNOSIS OF BACTERIAL DISEASES OF PLANTS

1. Notes on the history of phytobacteriology Plants, just like man and animals, can be affected by a number of diseases caused by bacteria. For a long time these bacterial diseases did not receive the attention they deserved, as compared to the more numerous fungal diseases of plants. Bacterial diseases, however, can be as disastrous, especially because direct control is impossible in many cases. In human and animal health the situation is reversed. There, fungal diseases form a minority and they are difficult to control. The study of bacterial plant pathogens and of bacterial plant diseases is called phytobacteriology. The Dutchman Jan Hendrik Wakker (1859-1927) was the first to try to prove that bacteria cause plant disease by following Koch’s postulates. This student of the botanist Hugo de Vries and mycologist Anton de Bary, investigated yellow disease of Hyacinthus and its causal agent, which he called Bacterium hyacinthi (now Xanthomonas hyacinthi), giving a description in 1883 (Figs. 26a-c). This was only 7 years after Robert Koch (1843-1910) proved that bacteria could cause disease (1876, experiments with the anthrax bacterium, Bacillus anthracis). Also see Chapter I.1 and Figs. 4-7. Three years before Wakker, in 1880, the American Thomas J. Burrill (1839-1916) had already provided evidence, by microscopic observations and experiments with pieces of diseased tissue, that bacteria could possibly cause fire blight of apple and pear. The founder of phytobacteriology as a science was the American Erwin F. Smith (1854-1938). He described a large number of bacterial plant diseases, including their pathology, isolated the causal bacteria in pure culture, performed inoculations and developed methods (Figs. 26a-c). C. Stapp in Germany, E. Hellmers in Denmark and W. Dowson in England followed in his footsteps. Up to the 1950s phytobacteriology was mainly descriptive. How much this has changed and how sophisticated some parts of it have become is illustrated by the subjects of the Proceedings of the International Conferences on Plant Pathogenic Bacteria (e.g. Lemattre et al., 1994; De Boer, 2000).

2. Phytopathogenic bacteria Bacteria that cause plant diseases are Gram-positive or -negative rods with the exception of Streptomyces spp. that are filamentous. They are found in the genera Acidovorax, Agrobacterium (Rhizobium), Arthrobacter, Brenneria, Burkholderia, Clavibacter, Curtobacterium, Dickeya, Enterobacter, Erwinia, Herbaspirillum, Janthinobacterium, Leifsonia, ‘candidatus Liberobacter’, Nocardia, Pantoea, Pectobacterium, Pseudomonas, Ralstonia, Rathayibacter, Rhizobacter, Rhodococcus, Samsonia, Serratia, Sphingomonas, Spiroplasma, Streptomyces, Xanthomonas, Xylella and Xylophilus (Tables 4 and 7; Annex 2). Most plant pathogenic bacteria are intercellular (living between plant cells) and/or necrotrophic (living on killed cells) pathogens. The group of the fastidious xylem- or phloemlimited bacteria (formerly called Rickettsia-like bacteria and now Phytoplasmas and fastidious xylem- and phloem-limited bacteria (FXLB and FPLB)) will not be discussed in 1)

For more detailed information see Stapp (1956); Fahy and Persley (1983); Starr (1983); Bradbury (1986); Lelliot and Stead (1987); Kleinhempel et al. (1989); Saettler et al. (1989); Goto (1990); Klement et al. (1990); Pérombelon and van der Wolf (1998); Schaad et al. (2001); Griffith et al. (2003); Alvarez, 2004; Kowalchuk et al. (2004);http://www.cabicompendium.org/cpc/home.asp; http://www.eppo.org/

36

Chapter II

Table 7 Genus

Genera of bacteria with plant pathogenic species and main characteristics. Legend: see page 37 Form

Flagella

Acidovorax1)

Gram stain

Metabolism / colour of colony

Main symptoms

rods

polar

-

aerobic/cream

blight

Agrobacterium12) (and Rhizobium) Arthrobacter2)

rods

peritrichous

-

aerobic/ white, cream

tumours, hairy root

coccoid rods

subpolar

+

aerobic/cream

vascular, wilting

rods

polar

-

aerobic/cream

rotting

Clavibacter

rods

none

+

aerobic/cream, yellow

vascular, stunting

Curtobacterium2)

rods

peritrichous

+

aerobic/yellow

vascular

Enterobacter4)

rods

peritrichous

-

facultative anaerobic/cream canker, dieback

Erwinia13) (and Brenneria, Dickeya, Pectobacterium) Herbaspirillum1)

rods

peritrichous

-

facultative anaerobic/ white, cream, yellow

vascular,soft rot, necroses

rods

polar

-

aerobic/cream, mucoid

mottled stripe of sugarcane

Janthinobacterium

rods

polar

-

aerobic/grey-white

soft rot of Agaricus bisporus

pleiomorphic rods rods

none

+

Ratoon stunt of sugarcane

none

(-)

aerobic/non-pigmented, yellow n.d.

short filaments rods

none

+

none

-

Pseudomonas

rods

polar, 1 or lophotrichous

-

Phytoplasmas8) FXLB, FPLB11) Ralstonia6)

rods rods rods

(+) (-) -

Rathayibacter2)

rods

none none polar, 1 or lophotrichous none

+

aerobic/reddish with some white aerial hyphae facultative anaerobic/cream vascular, wilting to orange-yellow aerobic/semi-translucent, leaf spots, necroses, excrescences, cream galls, cankers, vascular; brown blotch of Agaricus bisporus non-culturable various, leafhopper transmitted non-culturable various, leafhopper transmitted aerobic/semi-translucent, vascular, wilting cream aerobic/yellow gumming disease or yellow slime

Rhizobacter

rods

polar and/or peritrichous none

-

aerobic/white,yellowish

galls on carrot (Daucus carota)

+

aerobic/orange

galls

peritrichous

-

facultative anaerobic/white, glistening facultative anaerobic/nonpigmented aerobic/white/yellowish

Erythrina spp. (coral tree)

Burkholderia3) 2)

Leifsonia9) ‘candidatus Liberobacter’ 5) Nocardia 7)

Pantoea

2)

Rhodococcus Samsonia 10)

pleiomorphic rods rods

Serratia

rods

peritrichous

-

Sphingomonas (incl. former Rhizomonas)

rods

1 polar, lateral subpolar or non-motile

-

Spiroplasma

spiral pleiomorphic rods

intracellular fibrils, flexing or rotation

Streptomyces

filaments

none

Xanthomonas

rods

1 polar

Xylella

rods

none

-

aerobic/opalescent, mottled green or red

Xylophilus

rods

1 polar

-

aerobic, pale yellow

no cell wall, facultative anaerobic/tiny, related to transparent fried-egg Gram+ colonies, margin is granular + aerobic/ brown, aerial mycelium white, spore masses grey aerobic/yellow

stunting, greening of fruits, yellowing, psyllid-transmitted galls

Cucurbit yellow vine disease (Cucurbita maxima) corky root of lettuce (Lactuca sativa), S. (Rhizomonas) suberifaciens; Cucumis melo var. inodorus (S. melonis) stubborn disease of Citrus spp., corn stunt, many hosts infected, leafhopper transmitted scab, netted scab, rotting

leaf spots, necroses, cankers, vascular, wilting vascular, leafhopper transmitted necrosis and canker of Vitis

Phytobacteriology and diagnosis

37

Legend of Table 7 + = positive; - = negative; n.d. = not determined; ( ) organisms falling into the group of Gram-positive or -negative bacteria based on genetic analysis only, due to non-cultivability. 1) Pathogens of this genus formerly included in the genus Pseudomonas, such as Acidovorax avenae subsp. avenae from Avena sativa, A. a. subsp. cattleyae from orchids, A.a. subsp. citrulli from Citrullus lanatus, A. konjaci from Amorphophallus rivieri, Herbaspirillum rubrisubalbicans on Saccharum officinarum (sugarcane). 2) Species formerly ranked under Corynebacterium. Examples are Arthrobacter ilicis on Ilex opaca, Clavibacter michiganensis subsp. insidiosus on Medicago sativa, C. m. subsp. michiganensis on tomato, C. m. subsp. nebraskense on Zea mays, C. m. subsp. sepedonicus on potato, C .m. subsp. tesselarius on Triticum, C. xyli subsp. cynodontis on Cynodon dactylon, Curtobacterium flaccumfaciens pv. betae on Beta vulgaris, C. f. pv. flaccumfaciens on Phaseolus vulgaris, C. f. pv. oortii on Tulipa, C. f. poinsettiae on Euphorbia pulcherrima, Rathayibacter rathayi on Dactylis glomerata, R. tritici on Triticum aestivum, Rhodococcus fascians on different hosts. 3) Bacteria of this genus formerly classified as Pseudomonas, such as Burkholderia andropogonis on different hosts, B. caryophylli on Dianthus caryophyllus, B. cepacia on several hosts, B. gladioli pv. alliicola on Allium cepa, B. g. pv. gladioli on Gladiolus spec., B. glumae and B. plantarii on Oryza sativa. 4) Pathogens of this genus formerly included in Erwinia such as E. dissolvens on Sorghum vulgare and Coffea canephora and E. cancerogena on Populus canadensis. 5) Formerly known as ‘Citrus greening bacterium’. The bacterium is non-culturable, but based on 16S rRNA sequencing shown to belong to the Gram-negative alpha group of the Proteobacteria (Annex 1). The genus received the ‘Candidatus’ status (see Chapter I.2, page 8). Two species were described, viz. L. asiaticum, a heat-tolerant form from Asia, and L. africanum, a heatsensitive form from Africa. Causing Huanglongbin (greening) disease of Citrus spp. 6) One pathogen of this genus formerly classified as Pseudomonas, viz. Ralstonia solanacearum on many hosts. 7) Bacteria belonging to this genus formerly classified as Erwinia (Erwinia ananas on Ananas comosus and E. uredovora on Puccinia graminis, now as P. ananas and P. uredovora. Erwinia lathyri on Oryza sativa seed. Erwinia stewartii, now as P. stewartii subsp. stewartii on Zea mays, P. s. subsp. indologenes on Pennisetum, Setaria italica or Pantoea dispersa on different hosts), E. herbicola, now as P. agglomerans as secondary parasite on several hosts. 8) See Chapter I.2, page 8 and 10. 9) Formerly classified as Clavibacter: C. xyli subsp. xyli causing ratoon stunt of sugarcane and C. x. subsp. cynodontis causing Bermuda grass stunting disease. 10) Serratia marescens causing cucurbit yellow vine disease in Cucurbita maxima and C. pepo (Rascoe et al., 2003). Other strains are insect pathogens or opportunistic pathogens of immuno-compromised humans, epi- or endophytes and soil inhabitants. 11) See Chapter I.2, page 8. 12) For reclassification of Agrobacterium spp. in the genus Rhizobium, see Young et al. (2001) and Young (2003). 13) For reclassification of certain Erwinia spp. into genera Brenneria, Dickeya and Pectobacterium, see Annex 2.

Table 8

Some seed-borne and seed-transmitted plant pathogenic bacteria

Bacterium

Main host(s)

Disease

Acidovorax avenae pv. avenae Acidovorax avenae subsp. citrulli Burkholderia glumae Clavibacter michiganensis subsp. michiganensis C. m. subsp. insidiosus C. m. subsp. nebraskensis Curtobacterium flaccumfaciens pv. flaccumfaciens Pantoea stewartii subsp. stewartii Pantoea ananatis Pseudomonas syringae pv. atrofaciens P. s. pv. glycinea P. s. pv. lachrymans P. s. pv. phaseolicola P. s. pv. pisi P. s. pv. tomato Xanthomonas axonopodis pv. phaseoli X. a. pv. phaseoli var. fuscans X. a. pv. malvacearum X. a. pv. vesicatoria X. a. pv. vitians Xanthomonas campestris pv. campestris X. c. pv. carotae X. translucens X. oryzae pv. oryzae X. oryzae pv. oryzicola X. vesicatoria Xylella fastidiosa

oat, rice watermelon (Citrullus lanatus) rice tomato alfalfa corn bean (Phaseolus, Vigna) maize onion cereals soybean cucumber, gherkin bean pea tomato bean (Phaseolus, Vigna) bean (Phaseolus, Vigna) cotton pepper lettuce cabbage carrot cereals rice rice tomato orange (Citrus sinensis)

bacterial blight and brown stripe bacterial fruit blotch bacterial grain rot of rice bacterial canker bacterial wilt bacterial wilt and blight bacterial wilt stewart’s disease, bacterial wilt centre rot leaf spot, basal glume rot bacterial blight of soybean angular leaf spot halo blight of bean bacterial blight of pea bacterial speck common blight of bean common blight of bean bacterial blight of cotton bacterial spot bacterial leaf spot black rot of crucifers bacterial blight bacterial leaf streak, black chaff bacterial leaf blight bacterial leaf streak bacterial spot citrus variegated chlorosis

38

Chapter II

STEPS IN DIAGNOSIS OF BACTERIAL PLANT DISEASES

SYMPTOMS OF BACTERIAL PLANT DISEASES

A

Assessment of symptoms

Leaf spots

B

Isolation of pathogenic bacteria

Excrescences and galls

C

Pure culture of isolated bacteria

Tumours

D Identification of pure culture

Wilting (vascular diseases)

E

Pathogenicity test

Necrosis and cankers

F

Reisolation from inoculated plants

Rotting Bacteria embedded in slime

G Reidentification of reisolate H Diagnosis report

Fig. 27 Top:

Steps necessary in a diagnosis for a bacterial disease and symptoms caused by plant pathogenic bacteria. Bottom: Isolation procedure from symptomatic material. This procedure differs from that used e.g. in mycology, where pieces of tissue are placed on the media. For bacteria, symptomatic material is selected (1), external tissue is briefly disinfected with alcohol and then pieces of diseased tissue at the borderline of healthy and diseased tissue are aseptically removed and placed into a tube (2) with 3-5 ml sterile PBS (so that bacteria can diffuse into the buffer solution). After c. 30 minutes 20-100 µl of the suspension (3) is placed on plate no. 1 of a series of three of a (selective) isolation medium (4) and dispersed on the plate with a sterile spreader. The same spreader is used to further spread bacteria on plate no. 2 and 3 (5, 6). In this way a dilution of c. 1:10 (plate 2) and 1:100 (plate 3) is obtained that yields separate colonies that can easily be checked for typical morphology and purified. Photo shows result of isolation of Erwinia chrysanthemi after 2 days of incubation on YPG agar.

Phytobacteriology and diagnosis

39

detail here, because 1) they are non-culturable and 2) methods to study them are mainly those used in virology or mycoplasmatology. These bacteria have genetic characteristics placing them in the class of the Mollicutes, wall-less, Gram-positive bacteria, that are 0.2-0.3 x 1.02.0 µm in size and are leafhopper transmitted. The exception is the so-called citrus greening bacterium (Liberobacter spp.), causing Huanglongbin disease of Citrus species. This bacterium is non-culturable, but was shown to have a cell wall of the Gram-negative type insitu and is insect (psyllid) transmitted. Several plant pathogenic bacteria can survive or even multiply saprophytically in the natural environment. Bacteria can enter the plant through small wounds (caused by insects, hail or wind-blown sand), stomata, lenticels, hydathodes, nectaries, etc. (also see Chapter III.3). A number of them, especially vascular pathogens, are seed transmitted (Table 8). Distribution of bacteria occurs by wind, splashing raindrops, (sprinkler) irrigation, rubbing plant parts, insects and other animals and by man and his implements (also see Chapter IV). The pathogenicity (based on virulence or aggressiveness of the bacterium) is determined by the ability of the bacterium to maintain itself in plant tissue and to multiply and often by the ability to excrete substances influencing the growth of plant cells (hormones) or that damage or kill cells. Plant pathogenic bacteria can be divided into xylem, phloem or parenchymal parasites, according to their preference for vascular or parenchymatous tissue (Chapter III.1a and b).

3. Diagnosis of bacterial plant diseases Theory and practice of diagnosis, including detection and identification, will be discussed in some detail. Steps that are principally necessary in a diagnosis are mentioned in Fig. 27. It will be evident that these steps are in fact Koch’s postulates (see Chapter I.1 and Figs. 4-7). Diagnosis tries to prove that a bacterium isolated from a diseased plant, and identified, using different techniques, is really the cause of the disease observed.

a. Assessment of symptoms Normally judgement of symptoms (and the complete diagnosis!) in a laboratory depends on the sample sent to the lab. It must consist of plant material showing different stages of the disease (young, old) on different plant parts (leaves, stems, bulbs, roots, etc.). Furthermore the borderline between healthy and diseased tissue must be present. In completely diseased tissue the primary pathogen is hard to find or is replaced by secondary pathogens or saprophytes. To avoid rotting and decay by secondary organisms samples should be wrapped in paper and put in a plastic bag, leaving some space for air. Samples should be accompanied by data as complete as possible, because when a bacterium is found in plant material it does not necessarily mean that this bacterium is indeed the cause of the problem in the field. It is clear that this sample should meet the highest standards. First of all the sample should be (statistically) representative (this is especially important when samples of possibly latently infected plants are taken, see Chapter V.6). Symptoms caused by bacteria can be divided into seven groups (Fig. 27). These types of symptoms, however, can also be caused in a number of cases by fungi, mycoplasmas, viruses or nematodes, due to the fact that plants have a limited number of possible reactions to infection. Visual judgement in most cases is insufficient to make a reliable diagnosis. We can

40

Chapter II

COLONIES OF PLANT PATHOGENIC BACTERIA ON NON-SELECTIVE MEDIA ISOLATION MEDIA NON-SELECTIVE

-

Contain usually water and non-defined nutrients (e.g. peptones, beef extract, yeast extract) or defined inorganic salts and organic nutrients, making them suitable for growth of many bacterial species.

SEMI-(S)ELECTIVE

-

Contain substances to enhance production of pigments and/or substances which can only be used by certain bacteria and/or inhibitors (e.g. antibiotics) for non-desired bacteria.

SELECTIVE

-

Contain nutrients and inhibitors, which by their quantity and quality, allow only one bacterial species to form colonies in/on the medium (in an ideal situation).

Fig. 28

PD

PD

Top left:

Yellow, slow-growing colonies (diameter c. 2 mm) of Clavibacter michiganensis subsp. michiganensis after 5 days’ growth on an isolation plate with yeast-peptone-glucose agar (YPGA). Bottom left: Smooth, fast-growing yellow colonies (diameter c. 3-8 mm) of Pantoea herbicola, after 2 days’ growth on YPGA. Bottom right: Growth of Xanthomonas populi, 10 days after inoculation onto YPGA. X. populi grows extremely slowly and will not form separate colonies on artificial media.

Phytobacteriology and diagnosis

41

only make a preliminary or presumptive diagnosis. When plant material is examined under a light microscope and bacteria are seen to diffuse out of the diseased material, again this is no proof of a primary bacterial infection. One must try to separate the causal agent from the tissue and to grow it in or on an artificial medium for further study: isolation (Fig. 27).

BASIC TECHNIQUE OF OBTAINING PURE CULTURES IN BACTERIOLOGY

Fig. 29 Top: Basic technique to obtain pure cultures. Bottom: Pure culture of Pseudomonas savastanoi subsp. fraxini, after restreaking of a colony from an isolation plate and 3 days’ growth at 27ºC on nutrient agar. Separate colonies allow a check for purity.

42

Chapter II

COLONIES OF PLANT PATHOGENIC BACTERIA ON (SEMI)-(S)ELECTIVE MEDIA Fig. 30 Irregular, slimy, cream-coloured, weakly fluorescing colonies (diameter c. 3-8 mm) of Pseudomonas cichorii after 2 days’ growth on King’s medium B. King’s medium B is elective for fluorescent Pseudomonas species.

Fig. 31 Semi-selective and elective medium for Streptomyces scabiei. This is a so-called minimal medium that is very low in nutrients and not many bacteria will grow on it, but S. scabiei grows well. By adding the amino acid tyrosine, from which S. scabiei can produce a brown diffusible pigment (melanin), its colonies can be easily detected among those of other bacteria.

Fig. 32 Glistening, pulvinate, slimy colonies of Pseudomonas syringae pv. phaseolicola formed after 3 days’ growth on elective 5% sucrose agar. On media with a high sucrose level many plant pathogenic Pseudomonas spp. and Erwinia amylovora form abundant amounts of the extracellular polysaccharide levan.

Phytobacteriology and diagnosis

43

b. Isolation In order to isolate a bacterium usually pieces of tissue taken from the margin of healthy and diseased tissue are briefly disinfected with 70% alcohol and placed in a tube with sterile water or phosphate-buffered saline (PBS, pH 7.2). Tissue is left for 30 min in suspension so that the bacteria can diffuse out of the tissue. Subsequently 20-100 µl of the suspension is plated onto solid media (Fig. 27). When investigating seed samples or material with bacteria present in very low numbers only (e.g. search for latent infections or epiphytic populations) isolation is much more difficult, requiring special (detection) methods, see Chapter II.3.d and Annex 6. Artificial media are solidified by agar-agar, a polysaccharide (i.e. sulphuric acid ester of linear galactan), obtained from red algae of the genera Gracilaria, Gelidium, Pterocladium and Acanthopeltis. Media are sterilized by autoclaving them for 20 min at 115 or 121oC. Most plant pathogenic bacteria grow well in an oxygen-rich atmosphere at 20-27oC. After 2-5 days incubation, separate colonies will form on the isolation plates (Fig. 28). They contain millions of bacteria and show a typical form and colour. Colour is due to pigment production by the bacteria, e.g. the yellow xanthomonadins, produced by bacteria belonging to the genus Xanthomonas and anthocyanins. Sometimes these pigments are water-soluble and will diffuse into the medium, e.g. the brown melanoid pigment of Streptomyces scabiei and Ralstonia solanacearum and the fluorescent pigments (pyoverdins) of many Pseudomonas species. For different pigments of plant pathogenic bacteria, see Table 9 and Figs. 31 and 33. To exclude non-pathogenic bacteria or to enhance the recognition of target bacteria, (semi-) selective or elective isolation media are often used (Figs. 28, 30-32). However, many of the non-desirable bacteria can form colonies that may be very similar to those of the target bacteria. Therefore the selected bacteria must be compared with bacteria known to be pathogenic. In this way one can try to determine the identity of the isolated organism (comparable to use of fingerprints and fingerprint databases by the police).

c. Pure culture From an isolated colony a purified culture should be obtained to ensure that only one type of bacterium is used in further tests. One separate colony from an isolation plate is transferred to a new agar plate. It is assumed that this one colony developed from one cell. If correctly performed, only one type of colony will appear several days after restreaking (Fig. 29). To obtain a pure culture, work should be performed under sterile conditions, usually in a flow cabinet. Contaminating micro-organisms (bacteria, bacterial and fungal spores) float in the air and occur on body, clothes and materials. In many cases the media used by the bacteriologist form a favourable environment for their growth. A pure culture is the basis for all further work in identification and diagnosis!

d. Detection and identification Conventional detection methods Detection is tracing of plant pathogenic bacteria in or on plant material, especially when they occur subclinically (latently), without causing symptoms. It should be clearly distinguished from identification, which is characterization and naming of bacteria. With the detection methods used it should be possible to detect low numbers of bacterial cells, because

44

Chapter II

PHYSIOLOGICAL TESTS IN THE IDENTIFICATION OF BACTERIA

Fig. 33 Production of a weak blue fluorescent pigment by P. s. pv. phaseolicola and a strong greenyellow fluorescent pigment by P. s. pv. syringae after 3 days’ growth on King’s medium B.

Fig. 34

Fig. 35

Sensitivity test for antibiotics. The bacterium (Clavibacter michiganensis subsp. michiganensis) is sensitive to tetracycline (TE), moderately sensitive to streptomycin (S) and erythromycin (E) and insensitive to penicillin (P).

Test for production of toxins by plant pathogenic Pseudomonas spp. In the middle a strain of P. syringae pv. syringae has been streaked and grown for some days. Thereafter the medium was sprayed with a suspension of the fungus Geotrichum candidum. Where the toxin syringomycin has diffused in the agar the fungus is inhibited.

Phytobacteriology and diagnosis

45

these low numbers already may form a threat for a future crop or may cause problems in trade.Traditional detection involves plant tissue that is washed or macerated, filtrated and often centrifuged. Thereafter a small aliquot of the suspension is streaked onto a suitable nonselective or (semi-)selective medium. Often also some of the suspension is inoculated into a test plant, which may act as a selective medium. These methods however are labour-intensive and take a lot of time. Selective media have a fairly high detection level (103-105 cells ml-1). Serological techniques are often used for detection, mainly immuno-fluorescence (IF) and the enzyme-linked immunosorbent assay (ELISA); for a description of these techniques, and also of the immunofluorescence-colony (IFC) staining see ‘Serological characteristics’. Advantages of these techniques are that they 1) are less time-consuming; 2) are simple and robust; 3) have a fairly high detection level (e.g. the IF test has a detection level of 103-104 cells ml-1); and 4) have possibilities for screening many samples and automation. A disadvantage of ELISA is its higher detection level (105-106 cells ml-1). Both IF and ELISA have the disadvantage of showing disturbing cross-reactions of non-target bacteria with antisera used. Some newer reagents have been developed to solve this problem, such as monoclonal antibodies or polyclonal antibodies against specific antigenic determinants (see ‘Serological characteristics’). Cross-reactions, however, cannot be ruled out completely with these new approaches. In some detection methods antibodies are used in combination with nucleic acid methods mentioned below and/or with magnetic capture. In the latter case magnetic beads, which are coated with antibodies, are used to trap the bacteria present in complex extracts of soil, plant tissue, etc (Lopez et al., 2003).

Table 9

Some pigments of plant pathogenic bacteria

Bacterium

Pigment(s)

Colour

Function

Burkholderia glumae

fervenulin, toxoflavin

fluorescent

Clavibacter michiganensis subsp. michiganensis Erwinia chrysanthemi E. rhapontici E. rubrifaciens

carotenoids

yellow, non-water soluble

chlorosis, inhibition of leaf and root elongation ?

indigoidine proferrorosamine rubrifacine

blue, water-soluble pink, pink

Fluorescent Pseudomonas pyoverdins spp.

green or blue fluorescent, diffusible

Streptomyces scabiei

melanin

Ralstonia solanacearum

melanin

Xanthomonas spp.

xanthomonadins

brown, watersoluble brown, watersoluble yellow, waterinsoluble

? iron-chelating inhibition of electron transport binding and transport through cell wall or membrane of Fe, so-called siderophores ?, on tyrosine-containing media ?, on tyrosine-containing media ?, unique for Xanthomonas

46

Chapter II

BIOCHEMICAL TESTS FOR IDENTIFICATION OF BACTERIA

Fig. 36 Classical biochemical test used to determine ability to decompose carbon compounds and check for fermentative/oxidative metabolism of glucose. In the two tubes on the left Pseudomonas syringae was inoculated. After 48 h of growth only the far left tube shows a slight change of pH in the medium (from green to yellow, because a pH indicator in the medium, bromothymolblue, changes in colour due to acid formation by the bacterium under aerobic (oxygen present) conditions. P. syringae is a strictly aerobic species. The tube sealed with paraffin (no oxygen) remained unchanged. The two tubes on the right were inoculated with Erwinia carotovora, a facultative anaerobic species. Glucose has been actively decomposed in both unsealed and sealed tubes: total yellow colour change of the pH indicator.

Fig. 37 The API System of miniaturized tests for carbon source utilization. The database of the system is mainly directed towards medically important bacteria.

Fig. 38 Demonstration of pectin hydrolysis by pectinolytic enzymes of Erwinia carotovora on the double-layer medium of Perombelon after 48h of growth. The top layer is pure pectin and by hydrolysis the bacteria form pits in the surface.

Phytobacteriology and diagnosis

47

Conventional identification methods In identification of a pure culture using conventional methods the following phenotypic characteristics can be determined: - Morphology of the bacterial colony. Form, colour and smell of a colony may be an indication of a bacterial pathogen, but many other non-pathogenic bacteria may form similar colonies. Diffusible pigment production is also of importance in the first screening of a bacterium (Figs. 29-32 and Table 7). - Morphology of the bacterial cells (Chapter I.2 and Figs. 14-18). Plant pathogens are Gram-positive or -negative rods or they are filamentous (Streptomyces spp.). Again many other non-pathogenic bacteria may show a similar morphology and further tests are necessary. - Physiological characteristics. Determination can be growth at different temperatures, e.g. 4oC or 37oC; thermal death point (for plant pathogenic bacteria usually 50-55ºC, when kept for 10 minutes at this temperature in liquid medium); growth at different levels of NaCl, e.g. 2, 5 and 7%, etc. The pattern of antibiotic resistance may also be determined (Fig. 34). Testing sensitivity to an antibiotic is important when this antibiotic is used to control a particular bacterium in the field (e.g. streptomycin against Erwinia amylovora). Furthermore screening for toxin production (Fig. 35 and Chapter III.1) and ice-nucleation activity (Chapters II and IV.1) will yield additional characteristics useful in the identification of plant pathogenic bacteria, especially those belonging to the Pseudomonas syringae group and Clavibacter spp. Detection of these substances nowadays often takes place via molecular detection of the responsible genes (Bender et al., 1999). - Biochemical characteristics. In biochemical tests the expression of the genetic material by the diverse enzyme systems of the bacterium is determined. By offering the bacteria nutrients in a culture tube or agar plate, the action of these enzyme systems can be checked. Tests have been developed to determine if bacteria are able to decompose certain C sources (e.g. sugars, alcohols, organic acids, glycosides) and/or N sources (e.g. amino acids). This may be done by inoculation of tubes containing a sole C or N source in minimal medium, including a pH indicator. If the bacterium is able to decompose the C or N source to acid or alkali products, the pH indicator (and therefore the colour of the medium) will change. When inoculating two tubes with glucose as the C source and covering the medium in one of them with sterile paraffin the oxidative (O) and/or fermentative (F) metabolism of glucose can be determined. This is an important test for differentiation between genera (e.g. Pseudomonas, Xanthomonas O+ F-, Erwinia O+F+, see Table 6 and Fig. 36). Some commercial applications of testing for acid formation from C and N sources use miniaturized test tubes and the results can be obtained in 24-48 h (e.g. API system strips, see Fig. 37). Decomposition and utilization of a C or N source can also be determined by checking if growth or no growth takes place after spot-inoculation on a solidified minimal medium, containing the C or N source. Again other systems use oxidation-reduction reactions to determine C and N utilization in a miniaturized (ELISAplate) format (BIOLOG system). Other biochemical tests determine the formation of certain end-products by the bacteria, e.g. formation of H2S from cysteine, indole from tryptophane, NO2 or NH4 from nitrate (NO3). Effects of enzymes can also be visualized directly or after staining on certain (agar) media containing the substrates for the enzymes (also see p. 49), e.g. hydrolysis of pectin (Fig. 38), gelatine, starch and casein by pectinases, gelatinase, amylases and casease, respectively, or hydrolysis of cellulose by cellulases or fats/fatty acids (Fig. 39) by lipases (esterases), respectively.

48

Chapter II

+ d d -

+ + + -

+ + -

+ + + + + -

+ + + -

-

-

-

-

-

+ + +

+ + d

+ + d d

+ + d

+ + -

d + d + + + d + + d +

d + d + + d d d d + + + -

+ + + + d d d + + + d + + +

+ + + + + + + + + d +

+ + + + -

E. uredovora (P1. ananas)

+ + + -

+ + + d d + + d + + + + + -

E. stewartii (P1. stewartii)

+ w +

E. nigrifluens

+ + + +

E. cypripedii

+ + + +

E. chrysanthemi

E. salicis

+ + + +

E. carotovora

E. quercina

+ w + -

E. rhapontici

E. rubrifaciens

+ w + +

E. herbicola (P1. agglomerans)

E. mallotivora

Motility Anaerobic growth Growth factors required Pink diffusible pigment Blue pigment Yellow pigment Mucoid growth Symplasmata (cell aggregates) Growth at 36ºC H2S from cysteine Reducing substances from sucrose Acetoin Urease Pectate degradation Gluconate oxidation Gas from D-glucose Casein hydrolysis Growth in KCN broth Cotton seed oil hydrolysis Gelatine liquefaction Phenylalanine deaminase Indole test Nitrate reduction Growth in 5% NaCl Deoxyribonuclease Phosphatase Lecithinase Sensitivity to erythromycin (15 µg/disk)

E. tracheiphila

Cultural, physiological and biochemical characteristics of the species of the genus Erwinia

E .amylovora

Table 10

+ + + d d + -

+ + + d + + + + + + + +

+ = positive; - = negative; d = doubtful; w = weak; 1 = Pantoea

Fig. 39 Hydrolysis of fat, demonstrated on a medium containing tributyrin, seen as clear zones around colonies of Xanthomonas axonopodis pv. begoniae (7 days after inoculation).

Phytobacteriology and diagnosis

49

When conventional biochemical tests have given a certain pattern, this pattern is compared with those of plant pathogens described earlier (Table 10). Furthermore it may be necessary or fruitful to compare the isolated bacteria with a living reference culture of the pathogen in the tests performed. Some typical tests used for biochemical characterization of plant pathogenic bacteria are mentioned below: Arginine dihydrolase Test for enzymes that enable certain otherwise aerobic bacteria (e.g. pseudomonads) to grow anaerobically. The enzymes generate ATP by decomposition of arginine to CO2 and NH3. The change in pH due to NH3 formation is determined by a colour reaction in a semisolid agar medium from light orange to cherry red. Catalase The catalase enzyme decomposes hydrogen peroxide (H2O2) to water and O2. Hydrogen peroxide is very toxic for bacteria. Bacteria are smeared in a drop of hydrogen peroxide and checked for formation of oxygen gas bubbles. Most bacteria are catalase positive. Pectin hydrolysis Pectic enzymes are important for degradation by bacteria of middle lamellae and cell walls of plants. Media with sodium polypectate have been developed (single or double layer) that allow detection of pectinolysis in the form of pits in the medium (Fig. 37). Pectic enzymes have different pH optima. Pseudomonads usually produce low pH pectin lyases (PL), whereas erwinias usually produce high pH pectin methylgalacturonases (PMG). Esterase activity (fat hydrolysis)Media with Tween 80 (polyoxyethylenesorbitamonooleate polymer) or tributyrin (glyceryl tributyrate) can demonstrate fat or fatty acid esterase activity, especially useful in characterization of xanthomonads. An opaque zone with crystals (Tween 80) or a clear zone (tributyrin) around the colonies shows esterase (lipolytic) activity (Fig. 39). Gelatine hydrolysis Liquefaction of gelatine is easily demonstrated in tubes with a gelidified gelatine-nutrient broth medium and is especially helpful in identification of pseudomonads. Hydrogen sulphide production from tryptophane or cysteine Volatile hydrogen sulphide production from organic sulfur compounds can be demonstrated by lead acetate paper (5%) strips placed above inoculated culture medium. Oxidase test This test demonstrates the presence of cytochrome c respiratory enzyme by oxidation of tetramethyl-ρ-phenylenediamine. Some bacterial growth is rubbed into a drop of the chemical on a filter paper. In a positive reaction a deep purple substance is formed. The test is especially useful for identification of pseudomonads and erwinias and is also available commercially. Nitrate reduction and denitrification Obligate aerobic bacteria can use nitrate instead of oxygen in anaerobic conditions as an electron acceptor. They reduce nitrate to nitrite and then to N2. Bacteria are grown in medium with KNO3, whereafter nitrite is demonstrated with sulfanilic acid and αnaphtylamine. For further information see: Klement et al. (1990); Madigan et al. (2000).

50

Chapter II

PRINCIPLE OF SEROLOGY AND CROSS-REACTIVITY OF POLYCLONAL ANTISERA

Fig. 40 Principle of serology and cross-reactivity of polyclonal antisera.

Table 11

Effect of antiserum dilution on the occurrence of crossreactions with an antiserum against Clavibacter michiganensis subsp. sepedonicus

BACTERIUM Target bacterium C. m. subsp. sepedonicus Cross-reactive bacterium 1 Cross-reactive bacterium 2

10

20

40

+

+

+

+ +

+ +

ANTISERUM DILUTION 80 160 320 640 +

+

+

+ + + + + DANGER ZONE

+

+

1280 +

2560

5120

+ ± (titre) ± DILUTIONS PREFERRED

Phytobacteriology and diagnosis

51

- Serological characteristics. Serology is an important tool, by providing additional information in the process of identification, i.e. a bacterium cannot be identified on the basis of its serological behaviour alone!1). Antibodies (defence proteins in the blood serum called immunoglobulins) are formed by animals against compounds introduced in their body (these compounds are called antigens), in our case a plant pathogenic bacterium or parts of it. Antibodies and antigens can also react outside the body (in vitro) with each other. The reaction can be visualized in several ways (see below and Fig. 40) and is usually specific; even antisera against flagellar proteins can be prepared and used for refined identification of bacterial strains (Fig. 45, Malandrin and Samson, 1999). The following methods and techniques are mostly used: a. Agglutination test on a microscopic slide. If antibodies (in antiserum) and a suspension of bacteria are mixed in a certain concentration they will clump together and an agglutination reaction takes place (Fig. 41). In the so-called latex agglutination test antibodies are coated to sensitized latex beads. In this way the antibodies are enlarged so to say, and the reaction is more readily visible, making the test more sensitive than slide agglutination. Unfortunately the agglutination test is often liable to disturbing cross-reactions. New is the development of so-called lateral flow kits (e.g. by CSL, York, UK) where an agglutination test can be performed with a kit in the field on symptomatic material. b. Precipitation test. In this test only certain soluble antigenic proteins or polysaccharides of the bacteria react with the antibodies. Bacteria, often killed and disrupted by phenol, are placed in wells in the agar plate. Soluble antigens and antibodies diffuse in the agar. Where they meet in a certain concentration they bind, form flakes and precipitate, visible as a white line (Fig. 41). How the lines of two different bacteria meet each other in the agar can say something about their serological relationship. The precipitation test is very insensitive. c. Immunofluorescence (IF). This is a very sensitive and robust serological test (detection level of c. 103-104 cells ml-1 of plant extract) because the primary reaction of antigen and antibody is made visible. Binding reactions can be observed at very high titres (titre = highest dilution of the antiserum where a clear reaction is still visible) of antiserum. In the IF test antibodies are marked with a chemical dye that fluoresces in blue light, mostly fluorescein isothiocyanate (FITC). For IF a light microscope fitted for epi-fluorescent light is necessary with the suitable excitation and barrier filters for FITC. In so-called direct IF antiserum against a certain plant pathogen is already labelled with FITC. In indirect IF (Figs. 41 and 42) the bacteria are first treated with a pathogen-specific rabbit or mouse antiserum (against the target bacterium). After incubation and washing, a second, labelled anti-rabbit or anti-mouse serum, prepared in another animal (e.g. goat), is applied. This anti-rabbit or antimouse serum is called the conjugate. Only the antibodies bound to the bacteria will fluoresce, while the others are removed by washing. Indirect IF is slightly more sensitive and less specific than direct IF (Anonymous, 1998). 1)

It is stressed here that modern (serological) kits, available commercially, can only be used for presumptive identification in a presumptive diagnosis. Because these systems use only one characteristic or one set of similar characteristics no definite identification can be obtained. There are too many risks of errors (cross-reactions, background, etc.). Up till now only following Koch's postulates leads to satisfactory identifications and a correct diagnosis!

52

Chapter II

SEROLOGICAL TECHNIQUES USED IN DETECTION AND IDENTIFICATION OF BACTERIA

CONJUGATE + SPECIFIC SERUM + BACTERIUM

Fig. 41 Serological techniques used in detection and identification of bacteria. tag = target antigen; ntag = non-target antigen. Also see text.

Phytobacteriology and diagnosis

53

Immunofluorescence can also be combined with dilution plating in a detection technique called immunofluorescence colony staining (IFC). Bacteria are grown in or on a suitable agar medium and (micro)colonies treated with FITC-labelled antiserum. With a low-power objective colonies are screened for fluorescence and transferred to obtain a pure culture (Van Vuurde, 1987). The advantage is that the bacteria are still living, but on the other hand the method uses low serum dilutions for trapping and is therefore more liable to cross-reactions. Subculturing after serology in this method is often difficult. d. Enzyme-linked immuno sorbent assay (ELISA). This method can be compared to a large extent to IF. The marker, however, is not a fluorescent dye but an enzyme. Basically antibodies are first adsorbed (coated to) in the wells of a plastic ELISA plate. Bacterial antigens in buffered sample solution are subsequently pipetted in the wells and trapped by the coated antibodies. After incubation and washing an enzyme-labelled antiserum (conjugate) is added. Only wells where antigens reacted with the enzyme will change in colour after incubation with a suitable enzyme substrate (Fig. 43). The enzyme is usually alkaline phosphatase and substrate p-nitrophenylphosphate. A disadvantage of ELISA is that in many cases only products of bacteria and not the entire cells are detected. Cells are washed out due to insufficient binding capacity of coating antibodies. Moreover there is no information on the morphology of the cells and the sensitivity is lower than that of IF (c. 105 cells. ml-1).

PD

PD

Fig. 42

Fig. 43

Application of antiserum to bacteria (or plant extract containing bacteria) fixed to wells on a microscopic slide for use in the indirect immunofluorescence (IF) test. After incubation with a specific antibody, slides are washed and incubated with a second antibody labelled with a fluorescent dye (called conjugate). Positive reactions between antibodies and bacteria are made visible under a fluorescence microscope (Fig. 44).

Plastic plate as used in the Enzyme-linked immuno sorbent assay (ELISA). Wells are coated with antibodies and after incubation with plant extract or bacterial suspension a second serum labelled with an enzyme is used. After incubation of this second antiserum, positive reaction between antibodies and antigens is made visible through enzyme-substrate reaction (yellow colour)

54

Chapter II

PRINCIPLE OF FLUORESCENCE MICROSCOPY

Fig. 44 Top:

Path of light in an (epi-)fluorescent microscope. A fluorescent compound like FITC as used in the IF test needs a specific excitation wavelength (blue light of 450-490 nm), from a powerful (mercury) lamp to start its fluorescence and also a specific fluorescence emission wavelength (yellow-green light of 520-550 nm). Through a combination of filters the correct wavelengths are allowed to enter the preparation and eyepiece, resulting in the picture as shown below, right Below left: Modern fluorescence microscope (Zeiss Axio). Below right: View of IF-positive cells of Erwinia chrysanthemi under the fluorescence microscope.

Phytobacteriology and diagnosis

55

In serology specific antisera are preferred, reacting with the target bacterium or its products only. Sometimes sera are too specific, missing certain strains of the pathogen. Often, however, sera are not specific enough. They may cross-react with (non-) pathogenic bacteria possessing some of the antigenic determinants of the target bacterium in common, especially at lower serum dilutions (Table 11, Fig. 40). Elimination of these cross-reactions can be achieved by: a) dilution of the antiserum, cross-reactive strains usually do not react at higher dilutions, while the pathogen still does (Table 11); b) absorption of the antiserum with the cross-reactive strains or its antigens. In many cases, however, the titre of the antiserum drops dramatically; c) production of monoclonal antibodies (see Newer Detection Methods); d) production of polyclonal antisera against specific antigenic determinants of the bacteria, the latter being isolated and purified first. This method combines the strong reaction of a polyclonal antibody with the high specificity as reached by monoclonal antibodies. Bacteriophages. Specific bacterial viruses (bacteriophages, see Fig. 23) have sometimes been used in identification. Phages, however, are usually too specific or not specific enough. An example where bacteriophage typing for strain identification was successful is described by Du et al. (1982). Three bacteriophages of Xanthomonas arboricola pv. pruni isolated from soil under diseased plum trees were used to type six South African and four other X. a. pv. pruni isolates which were previously shown to be serologically closely related. According to patterns of lysis and efficiency of plaque formation by the phages, the bacterial isolates comprised three distinct groups. Isolates from South Africa, the USA and Argentina were lysed by the three phages with a high degree of efficiency, whereas two isolates from New Zealand showed no lysis and lysis with a low degree of efficiency, respectively. Both New Zealand isolates were lysogenic, liberating phages spontaneously or following UV irradiation. Moreover phage typing provided evidence that peach, apricot and plum trees in South Africa were infected by the same strain of X. a. pv. pruni. In exceptional cases bacteriophages are also used in the control of plant pathogenic bacteria (Balogh et al., 2003, also see Chapter VI.8).

Fig. 45 Use of immunofluorescence for localizing cell-wall (or O-) antigens and flagellar (or H-) antigens and differentiation of serogroups within Erwinia chrysanthemi. Left: Right:

Cells of strain PD482 from potato (biovar 5, O-group 1, H-group 1 or O1:H1), with serum prepared against an O-group 1, H-group 2 or O1:H2 strain. Reaction with (homologous) O1 antigens only, flagella are not visible. Cells of strain 82/21 from chicory (biovar 5, O1:H1), with serum prepared against a O1:H1 strain. Reaction with homologous O1 and H1 antigens. Cells and flagella are visible.

56

Chapter II

DNA DOT/SLOT-BLOT HYBRIDIZATION

Fig. 46 In A) DNA is extracted from the bacteria; in B) DNA strands are separated and fixed on a filter; in C) labelled probe is added, which hybridizes in D) to homologous sequences of target DNA; after washing away nonhybridized probe in E) the hybridized DNA can be visualized on an X-ray film (see Fig. 47).

Fig. 47 X-ray film showing dark spots (dots) where DNA of Clavibacter michiganensis subsp. sepedonicus (C.m.s) is present in a dilution series of suspensions of pure cultures (1-3) and in potato extracts (5-9) hybridized with a probe that consists of a plasmid-derived sequence of this bacterium. Extracts 7-9 were negative, 4 and 10 are empty buffer rows, 11 is positive control DNA of C.m.s. The probe was labelled with biotin (to avoid radioactive label) and biotinylated probe detected with a PhotoGene (GIBCO BRL) detection kit.

Phytobacteriology and diagnosis

57

Newer detection methods Monoclonal antibodies (MA) When a plant pathogenic bacterium is injected into a rabbit, different cell types of the rabbit produce normally many types of antibodies. The antiserum taken from such a rabbit is called a polyclonal serum. In the MA technique one antibody-producing cell is selected (B-lymphocyte from a mouse) which produces only one type of antibodies against one antigen (or epitope) of the bacterium. The B-lymphocyte is then immortalized by fusion with a cancer cell (plasma cell) of the mouse. In this way a so-called hybridoma is produced. With these hybridomas antisera can be produced with a very constant quality and of a high specificity. Reactivity of monoclonal sera is usually lower than that of polyclonal sera, because only one epitope of the bacterium will show the binding reaction. To enhance reactivity of monoclonal sera mixtures of monoclonals can be made, if available (De Boer, 1987).

DNA/RNA (dot/slot-blot) hybridization This is a technique that has become less common for detection after the introduction of the PCR technique (see Fig. 49). In so-called blot hybridization antibodies are not used to bind with the target bacterium, but genetic material (DNA, RNA) of the bacterium itself. In principle this is a very specific reaction. Due to its high specificity the DNA or RNA hybridization can also potentially be used simultaneously for detection and identification. A sample of plant tissue suspected of bacterial contamination is treated in such a way that as much as possible non-DNA material is removed. Thereafter DNA cleavage takes place under low pH conditions. The now single-stranded (target) DNA is fixed on a nitrocellulose filter and then a so-called DNA probe is added. This probe contains a large number of specific nucleic acid molecules of the target bacterium, which have been multiplied before in another bacterium, mostly using a virus as a vector. The nucleic acid probe binds to the complementary parts on the single strands present on the filter (if present of course!), a process that is called hybridization (Fig. 46). After washing, the hybridized DNA or RNA or RNA:DNA complexes may be visualized in different ways, using either radioactive or non-radioactive labelled nucleic acid probe (Figs. 46 and 47). The detection level of this technique is still rather high (c. 105 cells ml-1), equal to or higher than the enzyme linked immuno sorbent assay (ELISA). This may be enhanced by application of enrichment culture. Useful applications are: 1) detection of organisms in diseased tissue or on agar plates (colony blotting) and 2) verification of identity of PCR products (see below) and in RFLP studies (see below). In the latter case target nucleic acids are transferred from a gel to a membrane in order to be analysed (so-called blotting). Fluorescent in situ hybridization (FISH) using rRNA (rDNA) oligonucleotide probes In this method short (20-30mers) oligonucleotide probes against 16S or 23S r(ibosomal)RNA/DNA are used. These oligonucleotide probes can be used for in situ hybridization, because they are able to diffuse through the cell wall of micro-organisms that are present in thin tissue sections or in plant or soil extracts fixed on a microscopic slide. For Gram-positive bacteria sometimes a lysozyme step (the enzyme lysozyme decomposes the peptidoglycan layer of the cell wall) is necessary to enhance penetration of the probe into the cell. Sensitivity depends also on the metabolic activity of the cells, although killed cells show a positive reaction for a considerable period of time. When probes have been labelled with a fluorescent dye or a gold label the micro-organisms can be visualized by incident light

58

Chapter II

FLUORESCENT IN SITU HYBRIDIZATION (FISH)

Fig. 48 Top: Bottom left:

Bottom right:

Principle and procedure of the FISH test, also see text. Fluorescent microscopic view (using FITC filters) of potato extract with brown rot bacteria (Ralstonia solanacearum, Rs) hybridized with 1) an FITC-labelled 23S rRNA probe hybridizing with all eubacteria (both Rs and saprophytic cells (two cells indicated in white rectangle) visible as green cells) and 2) with a specific, CY3-labelled 23S rRNA probe against Rs. Same preparation and microscope slide as bottom left, but now using CY3/rhodamine filters: only the Rs bacteria are visible as red cells.

Phytobacteriology and diagnosis

59

(fluorescence) microscopy (Fig. 48). Specificity of the method is quite high, usually much higher than in serological methods, but false-positive reactions are still possible due to homology of RNA of non-target organisms with the specific probe sequence (Moter and Göbel, 2000; Wullings et al., 1998).

Polymerase chain reaction (PCR) With this method a specific part of the nucleic acid of the target bacterium is artificially multiplied before detection takes place. This multiplication (Fig. 49) is reached by repeated cycles of: 1) Denaturation (melting) of nucleic acid, usually at 95ºC. 2) Annealing of short (specific) strands of nucleic acids, so-called primers (these primers have been artificially assembled and consist of a sequence known to be specific for the target organism), at c. 68ºC (depending on the primers used). 3) Extension of nucleic acid strands at 72ºC in the presence of free nucleotides and a thermostable nucleic acid polymerase (usually Taq polymerase, originally isolated from the hot-spring thermophylic bacterium Thermus aquaticus, but also available as an artificially synthesized product). After multiplication the PCR products can be visualized on an ethidium bromide-stained agarose gel via electrophoresis (Fig. 49). Theoretically the sensitivity of PCR is very high: one copy of target DNA in a sample can be detected. In practice usually a lower sensitivity is reached, e.g. due to inhibitory substances in the plant extract. To verify identity of PCR products, hybridization with probes via blotting is often applied. When specific restriction sites are present in the product, restriction enzyme analysis (REA) can equally well be performed (Fig. 49), also see under Restriction Fragment Length Polymorphism (RFLP) Analysis. As in DNA dot/slot-blot hybridization this method can be used simultaneously for detection and identification, if enough is known about the specificity of the primers used. Fulfilling Koch's postulates, using PCR, is not possible anymore! It is rather difficult to interpret a negative PCR result; it could be caused by e.g.: a) Absence of the target or target sequence in the sample, e.g. when non-toxigenic strains are important under field infections and the PCR uses a sequence of DNA coding for the toxin, the pathogen will not be detected (Rico et al., 2003). b) Degradation of the target DNA in the sample. c) Inhibition of the PCR reaction in the sample due to wrong experimental conditions or inhibitory compounds. A check on the right conditions for PCR can be obtained by including an internal control such as primers against plant DNA that will multiply this DNA (which is always present in the plant extract) also in the same PCR experiment. Unfortunately false-positive reactions in a bacteriological context are still possible due to homology of non-target organism DNA with the primers thought to be specific for the target. PCR is also performed in combination with pre-enrichment in (selective) liquid medium, immuno-magnetic capture and fluorescent labels in order to increase sensitivity and/or specificity. In some tests an ELISA plate set up is achieved (Lopez et al., 2003). Real-time (TaqMan®) PCR Quantitative real-time PCR has become possible by the development of detectors (Sequence Detection Systems, SDS) that can measure fluorescence that is emitted during the PCR cycle. The method is based on the 5'-->3' exonuclease activity of the Taq DNA polymerase, which

60

Chapter II

POLYMERASE CHAIN REACTION (PCR) FOR DETECTION 1. Boil sample (with or without NaOH) to destroy cells and to separate DNA strands or extract DNA with a commercial DNA extraction kit 2. Include positive and negative controls and internal control when available 3. Place a few µl of extract in PCR thermocycler and run PCR

4. Place a few µl of PCR product extract on agarose gel and perform electrophoresis 5. Analyze products 6. Perform restriction analysis prior to electrophoresis when necessary

Fig. 49 Result of a PCR amplification of DNA of Ralstonia solanacearum in potato extract (1-4) and from colonies on an isolation plate (5-8). The typical product (based on a DNA sequence coding for 16S rRNA) has a size of 288 base pairs. In this experiment it was shown that dilution of plant extract sometimes yields negative results, due to diluting the DNA too much (samples 2 and 4) or sometimes, to the contrary, yields positive results due to excessive DNA or too many inhibitory compounds in the undiluted extract (sample 3). NCW = negative control sample with water only; NCM = negative control with master mix (primers, Taq polymerase and nucleotides); PC = positive control DNA from a pure culture of R. solanacearum. The DNA ladder is included to determine the size of the PCR product obtained.

Phytobacteriology and diagnosis

61

results in cleavage of fluorescent dye-labelled TaqMan® probes during PCR. The principle for the so-called TaqMan® technique (using laser light) is as follows (see Fig. 51): • Optical fibres direct light to the sample tube and excite fluorescent molecules in the extract. The emitted fluorescent light returns through the optical fibre and is detected and measured by a spectrograph connected to a CCD camera. More than one wavelength can be detected, allowing also for internal controls to be added to the reaction mixture. • In Taqman® PCR one or more specially developed Taqman® probes are used (Fig. 51A). • To the probes two fluorescent dyes, a reporter and a quencher are attached using a special TaqMan® PCR Reagent Kit. The reporter dye can be e.g. 6-carboxyfluorescein (FAM) and the second fluorescent dye (quencher) e.g. 6-carboxy-tetramethyl-rhodamine (TAMRA). The 3' end of the probe is blocked, so it is not extended during the PCR reaction (Fig. 51B). • When both dyes are attached to the probe, fluorescence of the reporter dye is inhibited (quenched) due to proximity of the quencher dye. During each extension cycle, the probe is displaced at the 5' end by Taq DNA polymerase (Fig. 51C). • Taq DNA polymerase degrades the probe and separates the reporter dye from it via its 5'-->3' exonuclease. When the reporter is a certain distance away from the quencher, it can emit its characteristic fluorescence that can be measured. The amount of fluorescence is a measure of the amount of PCR product (Fig. 51D and E). The result of a typical TaqMan® PCR using a dilution series of Ralstonia solanacearum is shown in Fig. 50. An advantage of real-time TaqMan® PCR is that the multiplication of DNA during early stages can be made visible, so that positive samples can be detected before inhibitory compounds in the extract block the PCR reaction. Moreover it could be used for (rapid) detection. This holds probably true for detection in the field using a portable machine (Schaad and Frederick, 2002) or when used with more than one probe for reliable detection of statutory (quarantine-) organisms (Schaad et al., 2003). It is not yet known how far other socalled molecular beacon techniques will be promising in this field (Van der Wolf et al., 2004).

109 108 107 106 105 104 103

102

Fig. 50 TaqMan® PCR amplification result of samples from a dilution series (cells ml-1) of Ralstonia solanacearum. Normalized reporter signal is plotted against cycle number (logarithmic amplification plot). The threshold cycle is the PCR cycle at which a significant increase in reporter fluorescence (blue lines) above the baseline is detected, and is marked by the red horizontal line.

62

Chapter II

TAQMAN REAL-TIME PCR

Fig. 51 Principle of TaqMan® PCR; for description see text.

Phytobacteriology and diagnosis

PROTEIN ELECTROPHORESIS (1)

PD

A. PURE CULTURE

B. PROTEIN EXTRACTION

Fig. 52a Principle of whole cell protein electrophoresis; for description see text.

63

64

Chapter II

PROTEIN ELECTROPHORESIS (2)

PD

C. POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE): OBTAIN PROFILE

Separation of (classes of) proteins in bands, after migration through the PAM gel matrix, in an electric field

PD

PD

Electrophoresis equipment with power supply and water bath

Computer with GelCompar software and gel scanner: digitizing protein profiles and identification and/or taxonomic analysis

PAGE gel with protein profiles of several bacteria after electrophoresis and staining

D. SCANNING, IDENTIFICATION AND CLUSTERING WITH SOFTWARE

Peak pattern of one digitized protein profile

Cluster diagram of protein profiles of different bacteria after cluster analysis

Fig. 52b Principle of whole cell protein electrophoresis.

Phytobacteriology and diagnosis

65

Newer identification methods In recent years completely new identification techniques have been developed. This is mainly due to the major developments in molecular biology, unravelling the structure and function of micro-organisms. Knowledge of the composition and the place of structural elements in micro-organisms has made the development of real fingerprinting methods possible, such as: Special serological techniques, eventually in combination with others - Immuno-electrophoresis. - Monoclonal or polyclonal antibodies against specific components (e.g. a protein or a polysaccharide) of the bacterium, so-called monospecific antibodies. - Immuno-gold labelling. Separation of bacterial components (proteins, nucleic acids) in an electric field: electrophoresis - Polyacrylamide gel electrophoresis (PAGE), one- or two-dimensional plasmid electrophoresis, Figs. 52a and b. - Immuno-electrophoresis. - Native protein electrophoresis (enzyme detection). - Restriction fragment length polymorphism (RFLP) analysis, Fig. 54. - Random amplified polymorphism DNA (RAPD) analysis. - Repetitive sequence-based (REP) fingerprinting PCR (REP, ERIC or BOX), Figs. 55-57. - Amplified fragment length polymorphisms (AFLP) analysis, Figs. 58 and 59. Separation of bacterial components using chromatography - Amino acids and cell wall carbohydrates (paper chromatography, chromatography). - Isoprenoid quinones (thin-layer, high-performance liquid chromatography). - Fatty acids, fats (gas chromatography), Fig. 53.

thin-layer

Some of these techniques will be treated in more detail:

Polyacrylamide gel electrophoresis (PAGE) of whole cell proteins With PAGE usually total protein profiles of bacteria are compared. Approximately 20-40 different protein classes can be made visible. The proteins have first to be extracted and denatured with a detergent (sodium dodecyl sulfate, SDS) and mercaptoethanol. The proteins lose their 3-dimensional structure and are negatively charged. Subsequently the mixture of the denatured, negatively charged proteins is loaded on a porous polyacrylamide gel in an electric field. The proteins will migrate to the positive pole, where the smaller ones will migrate faster than the bigger ones (less resistance in the gel). After separation the proteins are stained and the protein bands can be compared. (Fig. 52a and b). Comparison is visual or by computerbased analysis. In the latter case gels are scanned and banding patterns digitized into peak patterns. The laboratory can then construct databases. No standard libraries are available, due to the difficulty of interlaboratory standardization (also see under Fatty acid analysis). Gelscan and gel-analysis programs often applied are GELCOMPAR or BIONUMERICS. They are produced by AppliedMaths, Kortrijk, Belgium. These software packages allow sophisticated data handling, library generation and statistical analysis for identification and

66

Chapter II

GAS CHROMATOGRAPHIC FATTY ACID ANALYSIS USING THE MIDI INC. MICROBIAL IDENTIFICATION

Chromatograph (automatic sampler and injection tower), used for the MIS system

2

1

3

Fig. 53 Principle of whole cell fatty acid analysis.

2-Dimensional plot of a principal component analysis of fatty acid data obtained from strains of Pseudomonas savastanoi: clear differentiation in clusters of strains from Fraxinus (1, pv. fraxini), Olea europaea (2, pv. savastanoi and Nerium oleander (3, pv. nerii)

Phytobacteriology and diagnosis

67

taxonomic purposes. Whole cell protein electrophoresis is discriminative at a low taxonomic level (below species level) and there is a good correlation with RNA/DNA:DNA hybridization data (see Kersters et al., 1994 and Chapter I.8).

Gas chromatographic Fatty Acid Analysis (FAA) In this technique the total fatty acid profiles of bacteria are compared. Bacteria contain lipids in concentrations of 0.2 to 50% of the dry weight, usually 5%, mainly in their membranes. The lipids important for the technique are those containing esterified fatty acids, such as a) phospholipids, present in the cell membrane, b) lipid A in lipopolysaccharide of Gram-negative bacteria and c) ß-polyhydroxybutyrate, present in storage material. Bacterial fatty acids contain 10-20 carbon atoms and they can be linear or branched (iso and ante-iso), saturated or unsaturated, containing hydroxyl and cyclic groups. Generally a bacterium contains a certain number (10-30) of different fatty acids, which may differ quantitatively and qualitatively from those of other bacteria. When bacteria are grown under standardized conditions, discrimination can even take place below species level. Analysis of the total fatty acid profile is performed by gas chromatography (Fig. 53), with a special system (Microbial Identification System, MIDI Company, Newark, DE, USA). The bacteria are lysed by boiling, the fats saponified to release the fatty acids, the fatty acids methylated to make them more volatile, and extracted. After gas chromatography the profile obtained can be compared (matched) with a large number of reference profiles present in the database (library) of a connected computer. The computer identifies the fatty acids, determines deviations from a reference fatty acid mixture and presents the identification of the bacterium and the percentage of similarity. With this method identification is achieved using a large and very stable part of the bacterial phenotype (fatty acids) with little work in a very short time, (after obtaining a pure culture 48-72 h) with a great accuracy. The databank of the system is at present one of the largest in the world and includes libraries for aerobic and anaerobic bacteria, clinically important bacteria, actinomycetes, mycobacteria and yeasts. A library for fungi is in preparation. Moreover the system includes library-generating software, enabling inclusion of own (unknown) strains and statistical software to perform taxonomic research. There is a good congruence with RNA/DNA:DNA hybridization data. Also see Janse, 2004a; Stead et al., 1992.

Restriction fragment length polymorphism (RFLP) analysis Patterns obtained by RFLP analysis are based on mutations in the DNA sequence that change the recognition sequence of a restriction enzyme (that is, an enzyme that cuts the DNA strand at a specific site). Total DNA can be analysed and fragments visualized in an agarose gel after staining with ethidium bromide (ethidium bromide stains DNA by intercalating in the DNA strand and under UV light the stained DNA fluoresces). However, fragments obtained in this way are usually (too) numerous and overlap. Due to this problem probes are used which render visible the fragments that hybridize to the probe (Fig. 54). Both randomly cloned and specific probes may be used. To obtain a pattern, DNA is blotted after agarose gel electrophoresis onto a filter (so-called Southern blotting) and hybridized. After hybridization visualization of the pattern can be achieved as described under DNA slot/blot hybridization. RFLP analysis is discriminative at low taxonomic level, often strain level and is now often combined with PCR, e.g. digestion after PCR of the 16S rRNA gene (Coenye et al., 2001).

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Chapter II

Fig. 54 Restriction fragment lengh polymorphism (RFLP) analysis; for explanation see text.

Fig. 55 Principle of repetitive extragenic palindromic (REP) fingerprinting technique; for explanation see text.

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Random amplified polymorphic DNA (RAPD) analysis With the RAPD method patterns are obtained by PCR-amplification of genomic DNA with arbitrary, short (c. 10 bp) primers (this means that in this case the primers are nonspecific and will bind at arbitrary places on the DNA strand) at a permissive annealing temperature of 36-45ºC. The pattern of amplification products is usually discriminative at low taxonomic level, often strain level. No prior sequence information about the target, no probe, no blotting and hybridization and no restriction are necessary, making it a very fast method. However RAPD appears to be sensitive to colony age and standardization is difficult (Clerc et al., 1998).

Repetitive extragenic palindromic (REP)-PCR REP-PCR is a fingerprinting technique that uses repetitive sequences (mostly of unknown function, that are interspersed throughout the DNA) present in the genomic DNA of bacteria. For example, repetitive palindromic units are present at about 500-1000 copies in the DNA of Escherichia coli and Salmonella typhimurium. Whole cells can be used or DNA extracted from a pure culture of the bacteria, usually using a commercially available DNA extraction kit. Primers are used that specifically anneal to these sequences and multiply the DNA between two repetitive sequences (Figs. 55-57). The primers are rather long (18- to 22mers) and, therefore, higher annealing temperatures that enable greater stringency and less variation can be used. Approximately 10-40 PCR fragments are generated ranging in size from 200 bp to > 6 kbp. These fragments are separated by size in electrophoresis on a gel and visualized in ethidium bromide-stained gels. The band patterns can be scanned, digitized and used for identification and environmental (epidemiological) and taxonomic studies, just as in protein electrophoresis (Figs. 52a and b). REP-PCR uses 35-40 bp repetitive extragenic palindromic sequences, BOX-PCR uses the 154 bp so-called BOX element and ERIC-PCR uses 124-127 bp enterobacterial repetitive intergenic consensus sequences. More primer sets against different repetitive elements have been developed. Which method yields the most discriminative patterns has yet to be determined empirically. REP-PCR is discriminative at very low taxonomic level, usually strain level, and is very useful in epidemiological and environmental studies (tracking of strains, e.g. in hospital infections). It is plasmid-DNA independent. For identification purposes libraries have to be developed by individual laboratories, since interlaboratory standardization is not easy (Louws et al., 1994). Amplified fragment length polymorphisms (AFLP®)-PCR The AFLP® fingerprinting method is usually applied for discrimination at low taxonomic level, i.e. to detect genetic variation among closely related species, varieties (e.g. potato varieties) or even individuals of a species. The AFLP® fingerprints (Figs. 58 and 59) are a variation of the RFLP fingerprints (Fig. 54) visualized by selective PCR amplification of DNA restriction fragments. Basically, genomic DNA from a sample is digested to completion, typically with two different restriction enzymes in order to produce a large number of fragments. Specific oligonucleotide adapters (these are complementary to the restriction sites) of 25-30 bp are ligated to the restricted DNA fragments. Oligonucleotide primers that anneal to these adapters are used, however, to impart additional selectivity, the primers vary at their 3'-end, such that they will amplify only a subset of the restricted DNA fragments. The specificity of AFLP®-PCR is based on the 3' extensions of the oligonucleotide primers. These 3'-end primer extensions must match the target sequence for amplification to occur. The amplified DNAs are separated on a denaturing polyacrylamide gel and the amplified fragments are visualized by autoradiography. Usually a high number of bands is obtained (higher than with REP-PCR), most of which may be present in both of the samples being compared (depending on the nature of the samples). Different (polymorphic) bands can

70

Chapter II

Fig. 56 BOX-PCR results for several subspecies of Clavibacter michiganensis. Non-numbered lanes 1 kb DNA ladder; lanes 1-9 and 10-12 C. m. subsp. michiganensis; lanes 13-15 C. m. subsp. nebraskensis; lanes 16-18 and 19-24 C. m. subsp. insidiosus; lane 25 positive control C. m. subsp. sepedonicus; lane 29 negative control, ultra pure water. A, B and C = misidentified strains, strain C tested twice; DS = deviating strain of C. m. subsp. michiganensis, probably a mislabelled strain of C. m. subsp. nebraskensis (compare profiles!).

Fig. 57 Dendrogram from data shown in Fig. 56, using Bionumerics software (AppliedMaths, Kortrijk, Belgium). There is a clear separation between the subspecies of Clavibacter michiganensis.

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be excised from the gel and sequenced. This allows for specific PCR primers to be developed, if necessary. The method is highly discriminative, also useful in population studies, but expensive (partly because it is patented) and labour intensive (Rademaker et al., 2000).

Perspectives and pitfalls of molecular methods Molecular methods have now an important place in diagnostic bacteriology. But when one studies the literature carefully it is apparent that conventional methods have not disappeared. There are several reasons, which are discussed below. Many advantages of molecular methods have been advocated. Those ones that have proven to be often true are mentioned on page 73. However, in many cases new methods have proven e.g. to be time consuming, insensitive and expensive. It is sometimes suggested that molecular methods, being genetic, are of a higher and therefore better level than conventional methods, which are phenotypic. However, there is no such contradiction. After purifying bacteria and isolating their nucleic acids, these nucleic acids no longer function. They are immobilized in gels, cut into pieces, etc. and their analysis is a purely phenotypic one, too. Living organisms are able to switch genes on and off and where they seem to be similar in the lab, they behave totally different in the field. One should avoid a shortsighted, reductionistic view of molecular phenotypes (i.e. organism equals DNA), which will inevitably lead to problems in detection, identification and classification (Figs. 62 and 63). Modern methods should be seen as a welcome addition to already existing ones. They should be carefully checked for specificity and reproducibility and their limitations should be realized (Figs. 60 and 61 and page 73). This is especially true for bacteriology, where false positive reactions with unknown organisms are also easily possible with these methods. In how far nucleic acid probes can be used in so-called micro-arrays for reliable detection of multiple pathogens in a sample, only time will tell (Fessehaie et al., 2003). The great advantage of molecular methods is that they are or can be made specific at a very low taxonomic level, even at the strain level.

Fig. 58 UPGMA (= unweighted pair group method with arithmetic mean) cluster analysis of 33 AFLP® fingerprints of Xanthomonas species, including three strains (PD 2696, 2780, 3164) of the strawberry blight pathogen X. arboricola pv. fragariae, showing the taxonomic position of these strains and their relatedness to other pathovars of X. arboricola (light and dark green boxes) and no relatedness to the usual strawberry pathogen, namely X. fragariae (red box). From: Janse et al., 2001.

72

Chapter II

Fig. 59 Principle of AFLP® fingerprinting; for description, see text.

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POSSIBLE PERSPECTIVES OF MOLECULAR BIOLOGICAL METHODS •

Rapid, sensitive and cost effective

•

Integration into certification/inspection schemes

•

Commercially available, standardized test kits

•

Non-culturable

organisms

such

as

phytoplasmas

can

be

in

the

analysed •

Genetically

modified

organisms

can

be

traced

environment more easily •

Less sensitive to mutation or variation

•

Discrimination at low taxonomic level, often strain level

POSSIBLE PITFALLS OF MOLECULAR BIOLOGICAL METHODS •

Specificity, sensitivity and reproducibility unknown or only tested to a limited extent (not validated)

•

Negative

influence

of

conditions

and

biochemicals

(experimental error) •

Impossibility to discriminate between viable and non-viable cells and free nucleic acid in a sample

•

False negatives and false positives difficult to verify, Koch’s postulates cannot be fulfilled

•

Changing probes/primers/enzymes/methods/chemicals may yield different (conflicting) patterns or no patterns at all

•

Only small part of structural elements of an organism used (sampling error)

•

Answers from automated identification systems are as good as standard libraries and present-day taxonomy

•

Points of reference usually determine choice of patterns

74

Chapter II

A

EXPERIMENTAL ERROR B

C

D

Fig. 60 Example of experimental error. Dot-blot hybridization of identical filters containing 16S rRNA of target Clavibacter michiganensis subsp. sepedonicus (row 1 and 2, green rectangle, shown in D only), related Clavibacter and Curtobacterium species (row 3) and non-related (serological) cross-reacting bacteria (row 4, 5, 6). In (A) a non-specific eubacterial probe was used, where it was shown that on all spots bacterial rRNA was present. By lowering the temperature during washing in 1xSSC, 0.1% SDS buffer by 5 (C) or 20ºC (B) from the probe-specific temperature, 60ºC (D) the probe becomes increasingly less specific, yielding false-positive reactions with related bacteria (red rectangles). After Mirza et al., 1993.

EFFECT OF DILUTION OF EXTRACT IN PCR TARGET C.m.s. DNA 502 BP

INTERNAL CONTROL PLANT DNA 377 BP

Fig. 61 Result of a PCR amplification of DNA of Clavibacter michiganensis subsp. sepedonicus (C.m.s.) in potato extract (samples A and B 1:1) and in potato extract 1:10 diluted (A and B 1:10). The typical DNA product has a size of 502 base pairs. In this experiment it was clearly shown that dilution of plant extract sometimes yields better results, due to diluting inhibitory compounds in the extract (red arrows), in sample A 1:10 the reaction is even stronger after dilution. The internal control that is used in this PCR consists of plant DNA and shows the normal dilution effect, namely a weaker reaction (green arrows). NCW= negative control sample with water only; NCM=negative control with master mix (primers, Taqpolymerase and nucleotides); PC=positive control DNA from a pure culture of C. m. subsp. sepedonicus. The DNA or molecular weight ladder (MWL) is included to determine the size of the PCR product obtained.

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Molecular methods are: no magic bullets, but welcome additional tools to study organisms, especially at a low taxonomic level. It should be realized that an organism (Fig. 62) is more than (a small part of) its DNA or RNA, which has been extracted, immobilized and visualized on a filter or gel (Fig. 63). Organisms also switch genes on and off differently. Otherwise: risk of reductionism and scientific error.

Fig. 62 Papilio machaon (swallow-tail).

Fig. 63 DNA on a filter in dot-blot hybridization.

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Chapter II

THE CONFIRMATORY PATHOGENICITY TEST IS STILL VALID, STILL GOING STRONG AND IMPORTANT TO FULFIL KOCH’S POSTULATES

Fig. 64

Fig. 65

Tomato (Lycopersicon esculentum) inoculated with Ralstonia solanacearum .Wilting symptoms, 6 days after inoculation with a hypodermic syringe of 107 cells ml-1 bacterial suspension into the vascular tissue.

Chicory (Cicorium intybus) inoculated with Pseudomonas viridiflava causing soft rot, 5 days after inoculation of a 106 cells ml-1 bacterial suspension into the parenchymal leaf tissue.

Fig. 66 Hypersensitivity test on tobacco (Nicotiana tabacum) cv. White Burley. A dense, milky (108 cells ml-1) suspension is infiltrated in the mesophyll between the two epidermi of the tobacco leaf using a small hypodermic syringe. In a positive reaction the infiltrated area (arrow) will become necrotic within 24-48 h.

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Confirmation by the pathogenicity test – Koch’s postulates fulfilled When on the basis of tests mentioned before a bacterium has been named (= identified), in a number of cases a pathogenicity test is required. This is due to the fact that some bacteria that are almost identical in the tests performed may cause different diseases. Moreover there are saprophytic bacteria that resemble pathogens in phenotypic or genetic characteristics (almost) completely. Possibly in the future, methods (like PCR) will be developed so far that they can discriminate without the tedious and time-consuming pathogenicity test. However, that is still not possible in many cases due to insufficient knowledge about specificity of primers and probes. For legal purposes therefore the pathogenicity test cannot be missed.

e. Pathogenicity test Final proof and confirmation that a bacterium isolated from a plant with or without symptoms really is causing disease can be proven by a pathogenicity test using a host of the suspected pathogen. Especially in critical cases (e.g. when a pathogen is detected or described for the first time, in legal disputes between government and grower or im- or exporter and between countries) this test is still indispensable and obligatory in many official testing and diagnostic schemes. An example is the diagnostic scheme including detection of latent infections, identification pathways and the confirmatory pathogenicity test for Ralstonia solanacearum (as laid down in an official EU and EPPPO testing scheme) as presented in Annex 6. Plants can be artificially inoculated by injection of a bacterial suspension in buffer using a hypodermic needle (Figs. 64 and 65). In order to reproduce leaf spot diseases, leaves are first rubbed with carborundum powder to make small wounds; subsequently the bacterial suspension is smeared or sprayed onto the leaf surface. For xylem-inhibiting bacteria, like Xylella fastidiosa, it is necessary to infiltrate the xylem by applying a suspension of the bacteria under vacuum to the (cut) root, stem or leaf. A specific plant test which discriminates between most (fluorescent) plant pathogenic Pseudomonas spp. and some Xanthomonas and Erwinia spp. on one hand and non-pathogens on the other is the so-called hypersensitivity test on tobacco (Fig. 66). A dense bacterial suspension (c.108 cells ml-1) is infiltrated in the mesophyll of a tobacco leaf. A pathogenic bacterium will cause a hypersensitive (HR) reaction: the cells leak and collapse within 24 h after infiltration, rendering the tissue glassy, and later necrotic. Infiltration with non-pathogens only causes some yellowing after several days or no reaction at all. In a pathogenicity test a negative control (plants inoculated with sterile buffer solution only) should always be included. Where necessary also a positive control (using a known pathogenic strain of the pathogen) should be included. Positive and negative controls should be well separated from each other in the greenhouse, to avoid any contamination from controls or samples to each other! When dealing with quarantine organisms pathogenicity tests should be performed in a special quarantine greenhouse, where insects are excluded and a special quarantine protocol and regime for workers is in place.

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FOR COMPARISON WELL PRESERVED AND DOCUMENTED REFERENCE CULTURES AND THEREFORE CULTURE COLLECTIONS ARE INDISPENSABLE Preservation of bacterial isolates In order to use bacteria that were isolated from diseased material as reference material and for further study they have to be stored and preserved properly. Most plant pathogenic bacteria can be stored on agar slants (preferably yeast-glucose-chalk agar, or Wilbrink’s medium) in screw-capped bottles for some time (months). However, bacteria easily perish or change due to mutation and loss of pathogenicity. Some bacteria like Erwinia carotovora and Ralstonia solanacearum can be very well kept in sterile tap water (unless it contains too much chlorine) at room temperature for many years. Storage is also possible in 15% glycerol or commercial equivalent cryoprotectants at -20º or better -80ºC in screw-capped bottles. The best way of preservation is by freeze-drying (lyophilization) using a commercial lyophilization apparatus, where viability and pathogenicity/virulence are best preserved for many years. Usually bacteria are freeze-dried in ampoules or small screw-capped bottles (Fig. 67). Culture collections

PD

When isolates are designated as reference strain (especially when they function as the type strain for a certain species or pathovar, see Chapter I.8) they should be deposited in one of the official culture collections such as: - ATCC (American Type Culture Collection, Rockville MD, USA) - CNBP (Collection National de Bactéries Phytopathogènes, Angers, France) - ICMP (International Collection of Micro-organisms from Plants, DSIR Auckland, New Zealand) - NCPPB (National Culture Collection Plant Pathogenic Bacteria, CSL, York, UK) - PD (Culture Collection Plant Protection Service, Wageningen, The Netherlands).

Fig. 67 Some lyophilization ampoules and bottles used by culture collections to preserve bacteria for long periods of time.

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79

Reference cultures, to be used in (pathogenicity) tests, should be kept in a reference collection, preferably in the following way (also see page 78): 1. Screw caps containing a rubber stopper, in the refrigerator at 4oC. 2. In sterile water at room temperature. Cultures stored as described in 1) and 2) can be kept without transfer for 1 month to several years depending on the species. However, this way of storage does not prevent change by mutation or loss of pathogenicity. 3. Frozen at -20 or preferably at -80oC on sterile beads covered with a thin film of cryoprotectant fluid such as glycerol or in 15% glycerol. There is little or no risk of mutation or loss of pathogenicity. 4. Lyophilized. Bacteria are grown for 2-3 days on nutrient-agar slopes, suspended in a cryoprotectant (e.g. in nutrient broth with 7% sucrose or in horse serum), frozen at c. -35oC, whereafter water is extracted from the cells under vacuum via sublimation. Survival under these conditions may be for more than 25 years. There is little or no risk of mutation or loss of pathogenicity (Fig. 67).

f. Reisolation To be sure that the inoculated bacteria really caused the symptoms observed in test plants, reisolation from tissue at a certain distance from the inoculation place is necessary. When typical colonies are obtained in (almost) pure culture and some rapid confirmative tests (serology, PCR) are positive, a conclusive diagnosis can be made.

g. Reidentification In important cases, e.g. when it is presumed that a bacterium is found for the first time in a country or imported material, reidentification should be performed completely to fulfil Koch’s postulates to the very end. In these critical cases the original sample, sample extract, pure culture, and other test material obtained should be retained for reference purposes.

h. Diagnosis report How long it will take before a diagnosis can be made depends on the bacterial species and the methods available for its identification; it may vary from 3-50 days. A diagnosis report usually is a letter sent to the original correspondent with the following information: - Origin and description of the sample. - Name of the bacterium isolated. - Name of disease caused by this bacterium and description of its symptoms. - Nature (biology, epidemiology) of the pathogen. - Nature of the damage. - Advice on preventive or control measures.

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Chapter II

EXAMPLE OF A DIAGNOSIS OF RALSTONIA SOLANACEARUM BIOVAR 2, RACE 3 AS THE CAUSE OF A WILTING DISEASE IN PELARGONIUM In 2000 a wilting disease was observed in Pelargonium cuttings produced for the European market in Africa. The bacterium isolated proved to be Ralstonia solanacearum. Since infections of this bacterium in Pelargonium had only been reported a few times previously, a thorough identification and diagnosis were performed, using both potato and Pelargonium strains and different techniques to identify species, biovar and race. Pelargonium strains proved to be biovar 2, race 3. A confirmatory host test on Pelargonium and tomato using biovar 2, race 3 strains from both hosts proved pathogenicity (see Janse et al., 2004).

Phytobacteriology and diagnosis

81

82

Chapter II

UPGMA dendrogram obtained from BOX-PCR fingerprint as shown above. Pelargonium strains clearly cluster with biovar 2 strains of solanaceous hosts and isolated from surface water.

Phytobacteriology and diagnosis

83

84

Chapter II

Disease and symptoms

85

CHAPTER III - DISEASE AND SYMPTOMS CAUSED BY PLANT PATHOGENIC BACTERIA1) Plant pathogenic bacteria cause irritation and pathological changes (disease) in host plants. To differentiate these changes from those caused by other pathogens is not easy, because the plants have a limited number of possible reactions to infection. To clarify the relationship between bacteria and host the following subjects will be treated: 1. What is a pathogenic bacterium and what can it do in the plant? 2. What factors determine a plant to be a host for a bacterium? 3. What is the interaction between bacteria and plant on a molecular and cellular level (pathogenesis)? 4. How are symptoms formed and what kind of symptoms can be distinguished?

1. The pathogenic bacterium Most bacterial plant pathogens are necrotrophic parasites (living from and in plant cells, that were first killed by the bacterium); a few are biotrophic parasites (living (initially) from and in living plant cells), like some phloem-inhibiting phytoplasmas and a bacterium causing mummy disease in the mushroom Agaricus bisporus. The ecological basis for the interaction of the gall-forming Rhodococcus fascians and the tumour-forming Agrobacterium tumefaciens is not yet understood. Properties that determine phytopathogenicity are: a. Toxigenicity. This is the ability to produce toxic substances, such as exotoxins, excreted by living bacteria in the tissue (e.g. glycoproteins, lipoproteins and polysaccharides) and endotoxins. The latter are mostly parts of the bacterial cell wall, which are only liberated after death of the bacteria. Toxins of plant pathogenic bacteria are generally non-hostspecific and usually there is a direct relation between the toxin produced and a particular symptom. Examples of toxins produced by plant pathogenic bacteria are: 1. Chlorosis-inducing dipeptides (yellowing, i.e. decomposition of chlorophyll or inhibition of its formation, Figs. 68 and 69) such as tabtoxin, coronatine, phaseolotoxin, tagetitoxin, produced by Pseudomonas syringae pv. tabaci, P. s. pv. coronofaciens, P. s. pv. phaseolicola, P.s. pv. tagetis, and some other pathovars. 2. Cyclic lipodepsipeptide compounds (LDPs) such as syringomycins, syringotoxins and syringostatins, causing necrosis, produced by P. s. pv. syringae. Also tolaasin, produced by P. tolaasii that attacks the mushroom Agaricus bisporus, belongs to this group. These toxins form ion channels in the cell membrane, causing leakage of cells. 3. Scab-inducing toxins such as the cyclic dipeptide thaxtomin A and B, produced by Stretomyces scabiei, causing common scab of potato. Demonstration of these toxins can also be used for identification (Kinkel et al., 1998). 1)

More detailed information on pathogenesis and its molecular basis can be found e.g. in Smith (1905, 1911, 1914); Stapp (1956); Starr (1983); Goodman et al. (1986); Collmer et al. (1987); Kleinhempel et al. (1989); Leigh and Coplin (1992); Sigee (1993); Lee et al., 1995; Ream and Gelvin (1996); Bender et al. (1999); Schell (2000); Vanneste (2000); Crosa and Kado (2002); De Boer (2003); Greenberg and Yao (2004); Puhler et al. (2004); Nester et al. (2005).

86

Chapter III

Table 12

Ability to produce and excrete plant growth-stimulating substances (hormones) in vivo by some plant pathogenic and nitrogen-fixing bacteria of different genera. After Kleinhempel et al. (1989).

Bacterial species or pathovar

ß-Indole Cytokinins acetic acid (IAA, auxin)

Agrobacterium tumefaciens A. rhizogenes

+ +

Frankia spp.

+

+ +

Gibberellin

Abscisic acid (ABA)

+ +

+ +

Erwinia carotovora subsp. carotovora Pantoea agglomerans (also pvs. betae, gypsophilae and milletiae) Pseudomonas savastanoi pv. savastanoi Pseudomonas savastanoi pv. fraxini P. syringae pv. cannabina P. syringae pv. phaseolicola P. syringae pv. sesami

Ethylene

+

+

++

+

- or w

+ + +

Ralstonia solanacearum

+

+

Rhizobium spp.

+

+

Rhodococcus fascians

+

+

Streptomyces scabiei

+

Xanthomonas citri X. vesicatoria

+

+

+

Fig. 68 Large white, necrotic spots caused by the toxin of Clavibacter michiganensis subsp. michiganensis. Isolations from such leaves are usually negative, because the bacteria are only present lower in the plant. The low molecular weight toxin diffuses throughout the plant and causes necrosis.

PD

For correct diagnosis and isolation of the bacterium it is therefore necessary to include the stem or at least the basal part of the stem in a diagnostic sample.

Disease and symptoms

87

4. Glycoproteins, such as those produced by C. m. subsp. michiganensis (causing white necrotic spots on leaves, Fig. 68) and C. m. subsp. sepedonicus. 5. Extracellular polysaccharides (EPS), such as the EPS of P. syringae and Xanthomonas pathovars (Rudolph et al., 1989), and amylovorin (a bacteriocin) of Erwinia amylovora. Amylovorin consists of 98% galactose and causes wilting symptoms in shoots and necrosis of plant cells in callus cultures (Vanneste, 2000). 6. Wilt-inducing 7-azapteridine antibiotic (toxoflavin, produced by Burkholderia glumae (Jeong et al., 2003). Furthermore the production by plant pathogenic bacteria of growth-influencing plant hormones, such as indole acetic acid (IAA) and cytokinins may play a role in symptom (gall and excrescence) formation as has been established e.g. for P. savastanoi pv. savastanoi, Rhodococcus fascians, Agrobacterium tumefaciens and Pantoea agglomerans pv. milletiae (Table 12). Genes for hormone production may be located on plasmids or on the chromosome (see Costacurta and Vanderleyden, 1995; Surico and Iacobellis, 1992). b. Virulence/Aggressiveness. This is the ability to penetrate, to establish and multiply in a host plant and it is determined by: 1. Protective factors, such as extracellular slime (EPS), preventing desiccation by its water-holding capacity and preventing immobilization of bacteria on cell walls of the host. EPS induces and maintains watersoaking and is also important in transfer of plant carbohydrates to the bacteria and restriction of water movement in the plant. Examples are levan and amylovoran produced by E. amylovora (Vanneste, 2000; Maes et al., 2001), stewartan of Pantoea stewartii (Langlotz et al., 1999), alginate produced by Pseudomonas syringae pvs. (Koopmann et al., 2001) and xanthan gum produced by Xanthomonas campestris pv. campestris. Xanthan is important in the food industry as a gel and stabilizer in e.g. cosmetics, ice creams, salad dressings and toothpastes and also as a drilling lubricant in oil wells (Kennedy and Bradshaw, 1984). 2. Virulence factors or aggressins, compounds (primarily enzymes) which make plant tissue and cells accessible for bacteria, such as pectinases, cellulases, proteases, lipases, amylases and ribonucleases (Tables 13, 14 and 17). Also pili (hrp-pili and type 4 pili, tfp) can be important as a virulence factor (Tfp are also important for adherence to leaf surfaces, biofilm formation and resistance to UV, see Kang et al., 2002) 3. Antigenic components that enable recognition between host and bacterium and other factors, e.g. Avr-proteins, determining host specificity (see Chapter III.3). 4. Histo- or organotropism: the ability to establish and multiply in certain tissues or organs.

Fig. 69 PD

Infection of Xanthomonas campestris pv. campestris, in a cabbage leaf, through the water pores or hydathodes (one hydathode infection indicated by red arrow) at the leaf margin. Symptoms are blackening of the small veins and yellowing due to action of a toxin produced by the bacteria (also see Fig. 74).

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EFFECT OF NUTRITION ON STRUCTURE OF PLANT AND VULNERABILITY TO DISEASE: MAN OFTEN FACILITATES DEVELOPMENT OF DISEASES

POOR NUTRITION (low N)

RICH NUTRITION (high N)

LARGE INTERCELLULAR SPACES

ABUNDANT LIGNIFIED TISSUE

Fig. 70 Transverse sections through two different tomato (Lycopersicon esculentum) stems. Left: weakly vegetative stem. Right: highly vegetative stem, due to high N uptake. Ca = cambium; col = collenchyma; en = endodermis cells; ep = epidermis; i and o ph =. inner and outer phloem cells; i and o pcl = inner and outer pericycle cells; pi = pith; xy 1 and xy 2 = xylem. From this figure it is clear that the highly vegetative plant has large intercellular spaces in the pith and less lignified tissue in cortex and xylem. Such plants are in fact weakened and are easily attacked by bacteria, which cause so-called pith necrosis (Pseudomonas corrugata and others) on newly sterilized soils with high N applications under humid conditions. After Hayward (1938).

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The extent to which bacteria possess the properties mentioned and to what extent they can express them in the host determine for a large part pathogenicity and virulence and therefore symptom formation. Especially in the Erwinia bacteria causing soft rot, pectic enzymes play an important role in pathogenicity, but they are found also in many other plant pathogenic bacteria. In Erwinias we find the following pectinases: pectate lyase, pectin lyase, pectin transeliminase, pectin methylesterase and polygalacturonase. Pectic enzymes may cause maceration of tissue, due to dissolution of the middle lamellae of plant cells and damage to plant cells due to disruption of the cell wall and cell membrane (Figs. 78-80). c. Ice-nucleation activity. Some plant pathogenic bacteria, such as Pseudomonas syringae pathovars, Pantoea stewartii, Pseudomonas viridiflava and Xanthomonas translucens, have certain proteins in their cellular membrane that enable them to act as an ice-nucleus. This means that water with soluble compounds on or in plants or intercellular fluid, which due to their composition will not freeze below 0ºC (-1ºC to -10ºC), will freeze in the presence of these ice-nucleation-active (INA) bacteria. In this way plant tissue is damaged by frost and the damaged tissue forms a further easy port of infection and multiplication of the bacteria (Lindow et al., 1989). Frost damage and the concomitant bacterial infection is important in infections of P. syringae pv. syringae in poplar, apricot and peach and in infections of P. s. pv. pisi in pea. Ice-nucleation genes are similar in different bacterial species.

Table 13 Plant structure

Plant surface and cell structures, building blocks serving as nutrients for bacteria, and degradative enzymes of plant pathogenic bacteria degrading these structures. Building Degradative enzyme block/nutrient

Cutin (cuticle) Suberin (cork layers)

Fatty acid peroxides Fatty acid polyesters

Cutinase Suberin esterase

Cellulose (cell wall) Hemicelluloses (cell wall) Proteins (cell wall)

Glucose monomer β-1,4-linked xylans Polypeptides

Cellulases, C1, C2, Cx, β-glucanase Xylanases Proteases, proteinases

Pectic substances (cell wall and middle lamellae)

Galacturonans

Proteins (cytoplasmic membrane, CM) Phospholipids (CM) Phosphatidyl compounds (CM)

Polypeptides

Pectate lyase, oligogalacturonase, pectin methylesterase, pectin lyase, polygalacturonase Proteases, proteinases

Phospholipids Phospholipids

Phospholipase Phosphatidases

DNA RNA

3-deoxy polynucleotides Deoxyribonucleases Ribopolynucleotides Ribonucleases

90

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Optimal calcium incorporation in the plant cell wall and middle lamellae makes plants less susceptible to bacterial (enzymatic) attack Effect of Ca nutrition on brown rot (Ralstonia solanacearum) incidence in tomato when treated with different amounts of essential nutrients (P and K Table 14 constant and optimal). Clear positive effect of Ca (treatment 2 and 6). Mg seems to have a negative effect. Nutrient added in grams per kg air-dried soil. After Chellimi et al. (1997).

Treatment

CaO

Ca(NO3)2

NH4NO3

MgO

Disease incidence and SD

1

-

-

3.0

-

85 (0.15)

2

2.1

-

3.0

-

17 (0.03)

3

-

-

3.0

1.5

35 (0.35)

4

1.0

4.4

1.5

-

25 (0.25)

5

-

8.8

-

1.5

25 (0.05)

6

-

8.8

-

-

5 (0.05)

SD = standard deviation; - = no dosage.

Fig. 71 Diagrammatic longitudinal section of a potato stem, showing the course of vascular bundles in main stem and petiole bases. Vascular pathogenic bacteria can easily move through these bundles to all plant parts, including seeds and roots. Large vascular bundles: red. Small vascular bundles: yellow. After Hayward (1938).

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91

2. The host plant The susceptibility and sensitivity of a host, and therefore the occurrence of symptoms are determined by: a. Structure of the plant. Cutin, suberin and lignin are not or with great difficulty decomposed by plant pathogenic bacteria (cutinase was detected in Pseudomonas syringae pv. tomato and it was found to be of some importance in the infection process, see Bashan et al., 1985). Therefore older potato tubers with a high percentage of suberized lenticels are less sensitive to blackleg. A difference in structure between stomata of mandarin (Citrus nobilis) and grapefruit (C. grandis) appeared to determine field resistance of mandarin for Xanthomonas axonopodis pv. citri. b. External factors and condition of the plant.Factors are damage of tissues, water potential (bacteria need free water for their development), light, temperature, soil properties, density of the crop, availability of nutrients and actual uptake by the plant (Table 13), and other diseases present. c. Ability to react, including defence mechanisms.The plant is, as far as is known now, rather limited in its possible reactions upon infection by pathogens (Fig. 73). The following occur in bacterial infections: Hypoplasia - Incomplete development of organs (e.g. formation of small, malformed stem bulbs of Lilium, under the influence of Rhodococcus fascians), reduction in cell number or thickness of cellwall, dwarf growth caused by phloem-inhabiting fastidious bacteria. Metaplasia - Enhanced production of protein, starch, gum, thickened cellulose walls, lignification and suberization of cells. Hypertrophy - Enlargement of cells. Hyperplasia - Abnormally increased cell division. Defence mechanisms - Reactions directed against the pathogen or against tissue damage caused by the pathogen, e.g. the production of phytoalexins, polyphenoloxidases, cell wall lectins and other agglutinins, accumulation of gum and phenolic compounds and formation of cork and callus tissue. Phytoalexins are low molecular weight toxic products produced by the plant following infection or a HR reaction. They will rapidly kill the bacterial population. In the case of soft rot caused by Erwinia spp. in potato, phytoalexins (rishitin, phytuberin) develop only in the presence of sufficient oxygen. This may be one of the reasons that soft rot rapidly increases in a potato lot when anaerobic conditions are prevalent. The phytoalexins kievitone and phaseollin, produced in kidney bean after infection by Pseudomonas syringae pv. phaseolicola are highly toxic to Gram-negative bacteria in general. Lectins and other agglutinins are proteins or glycoproteins that immobilize nonpathogenic or saprophytic bacteria in plant tissue and they have been demonstrated in many plant species. Lectin in potato or tobacco extracts for example could agglutinate

92

Chapter III

Fig. 72

A. Visser

PD

Cassava (Manihot esculentum) showing three different symptoms, caused by one bacterium, Xanthomonas axonopodis pv. manihotis. 1) Wilting due to systemic infection and blocking of transport vessels by masses of bacteria embedded in slime; 2) yellowing, due to action of a toxin, and 3) leaf spots, due to infection through stomata or wounds.

Fig. 73 Similarity in symptoms (and risk of wrong diagnosis in the field) due to limited possibilities of reaction of the host plant and similarities in pathogenic activity of the pathogen. Fungal leaf spots, caused by Micosphaerella pinodes (left) and bacterial leaf spots, caused by Pseudomonas syringae pv. pisi (right). The yellow halo is usually more pronounced in fungal infection, whereas the water soaking is typical for the bacterial infection.

Disease and symptoms

93

avirulent, but not virulent isolates of Ralstonia solanacearum. Capsules and extracellular slime (EPS) produced by virulent pathogens inhibit this agglutination because they cover the binding sites. Non-virulent strains lack EPS and are trapped by the plant. Resistance against bacterial plant pathogens may be based on a gene-for-gene relation, e.g. between a resistant cultivar and a virulent strain. In this case there is an interaction between avirulence genes (so-called avr genes) of the bacterium and resistance genes of the plant, causing a so-called hypersensitivity reaction (HR). A HR takes place very rapidly: the tissue collapses within 8-12 h after inoculation, becomes necrotic and the bacteria are trapped and die. The HR can be differentiated into three stages, viz. 1) induction, 3-4 h, 2) latent period, 4-5 h and 3) tissue collapse and necrosis, 1-2 h. HR is also involved in interactions between non-hosts and plant pathogenic bacteria and between hosts and avirulent strains of plant pathogenic bacteria. Many plant pathogenic bacteria that cause necrotic spots can induce the HR in incompatible hosts. Not only does vertical gene-for-gene resistance (mainly between cultivars and pathogenic varieties of the bacteria, often called races) occur, but also horizontal resistance, based on many genes. This type of resistance is usually longer lasting, but not complete and its genetic basis has not yet been fully elucidated. Vertical resistance has been established e.g. for cultivars of soybean and pathogenic races of P. s. pv. glycinea, kidney bean and P. s. pv. phaseolicola, rice and X. oryzae, tomato and X. vesicatoria, pea and P. s. pv. pisi and cotton and races of X. c. pv. malvacearum. For interactions and reactions between bacteria and plants see Table 15 and Figs. 76-82

Table 15

Interactions and reactions between bacteria and plants (after Kleinhempel et al., 1989, changed)

Bacterium

Plant

Saprophyte

any plant

-

-

Potential pathogen

non-host plant

+

-

Pathogen (virulent)

Non-pathogenic mutant

susceptible host resistant host resistant host susceptible host susceptible host

+ + + + (-)*

+ -

Soft rot bacterium

host and non-host

- (+)*

+ (-)*

Pathogen (avirulent)

Reaction of plant HR Symptoms

HR = hypersensitivity reaction; - = no reaction; + = positive reaction; * both reactions occur, the more uncommon between brackets.

94

Chapter III

PATHWAYS OF INTRODUCTION OF BACTERIA INTO PLANTS

NATURAL OPENINGS: HYDATHODES

Fig. 74 Left: Section through a hydathode or waterpore of Primula sinensis. E = epidermis; GC = guard cell, always open; HE = thin-walled tissue, epithem; NLP = normal leaf parenchyma; XY = xylem vessels. After Belzung (1900), changed. Right: Small part of a cabbage leaf with hydathodal infections, caused by Xanthomonas campestris pv. campestris. From the infected hydathodes (IH) the bacteria have progressed into smaller (IVL) and larger (IV) veins that are stained black. HV = healthy veins. After Smith (1905), changed.

WOUNDS: SMALL WOUNDS AT THE PLACE WHERE NEW LATERAL ROOTS PUSH THROUGH MAIN ROOT CORTEX

Fig. 75 Longitudinal section through the root of Polinisia uniglandulosa at the place of lateral root formation. E = epidermis of the new rootlet; EN = endodermis; RC = root cortex; SV = spiral vessel. After Belzung (1900).

Disease and symptoms

95

3. Molecular basis for interaction between a pathogenic bacterium and a (non-) host: pathogenicity, (a) virulence, HR reaction and resistance Once the bacterium has gained entrance into the plant, either the bacterium will cause disease (compatible interaction) or the host will recognize the pathogen and induce a resistance response (incompatible reaction). On a molecular level genes code for virulence factors such as toxins, hormones, EPS and cell wall degrading enzymes. But the ability to invade and colonize plant tissue (pathogenicity) and to induce the hypersensitive reaction (HR) by phytopathogenic bacteria appears to a large extent to be conferred by two sets of genes: 1) a cluster of genes of c. 22-25 kb (in 6-8 transcriptional units), named hrp (from HR and pathogenicity) and 2) avirulence (avr genes). Also see Fig 76. The hrp genes form a specialized and tightly regulated secretion pathway for necrosis eliciting proteins (so-called harpins) and avirulence (Avr) proteins. Hrp secretion proteins show homology to secretion pathway proteins in Yersinia and Salmonella, implying that hrp clusters function to secrete proteins that interact with plants, in a manner analogous to the export of animal pathogenesis determinants. Hrp genes play a direct role in recognition between bacterium and host and appear to be essential for pathogenicity. But they are also necessary for the incompatible (non-host) interaction. The plant host recognizes the secreted proteins, or their action, and induces a HR involving gene-for-gene type interactions, which leads to resistance (reactions) in the plant (Table 16). Avr genes are often located in the vicinity of hrp genes. Thus the hrp and hrp-dependent avr genes also contribute to specificity of the pathogen and limit the host range. Hrp proteins are involved in: a. Sending signals to the plant b. Receiving signals from the plant c. Coordinating regulation of hrp, avr and other pathogenesis genes The hrp box (= conserved sequence motif, GGAACCNA-N14-CCACNNA) may be involved in transcriptional regulation. A regulatory connection of hrp with virulence determinants and with some avr genes has been established. This indicates how central and interrelated these may be in overall pathogenicity (Boucher et al., 1992; Alfano and Collmer, 1996; Preston et al., 1998; Collmer et al., 2000; Quirino and Bent, 2003; Abramovitch and Martin, 2004). Hrp genes have now been detected in most bacterial plant pathogens and they can be divided into two groups: 1) those occurring in Ralstonia solanacearum and Xanthomonas spp. and 2) those of Erwinia spp. and Pseudomonas syringae pvs. Some hrp gene clusters are located on plasmids (R. solanacearum), others on the chromosome (E. amylovora, P. syringae and Xanthomonas spp.). Some of the well-conserved hrp genes are now named hrc genes. In Fig. 76 a model for the hrp/avr interaction is given. Some hrp genes produce outer membrane proteins (hrp A in Fig. 76) that form a pilus. Others produce outer membrane lipoprotein (hrp C in Fig. 76), others produce inner membrane-associated proteins (RSTUV in Fig. 76), and there are hrp genes that produce cytoplasmic protein (hrp N in Fig. 76) and ATPase. The avr genes encode products that interact (directly or indirectly) with host plant resistance (R) gene products to induce defence reactions. Avirulence genes are positive determinants that are specifically recognized by the (resistant) host, leading ultimately to the limitation of disease. Race-specific resistance is often genetically specified by dominant single loci in the host that correspond to specific, dominant avr genes in the pathogen (gene-for-gene interaction). The lack of either member of the gene pair usually results in a compatible (disease) interaction. Thus to produce disease a pathogenic bacterium should not contain avr

96

Chapter III

Fig. 76 Model for the function of so-called hrp genes and of their products. Products are the proteins A, RSTUV, C, J, N and ? (unknown protein) in this case. The interaction is shown between a pathogenic bacterium and a resistant (top) and susceptible (bottom) host. The hrp gene expression is triggered by environmental factors such as pH and possibly by plant signal molecules, where after proteins are produced and transported through a tube (pilus) also formed by hrp genes. Harpins appear not to enter the cell protoplast. Top: In the resistant reaction avirulence (avr) signal molecules or elicitors are transported to the plant cell and recognized by proteins produced by resistance loci of the host (receptors) after the avr protein has eliminated the RIN4 protein, where after the HR takes place, the bacteria are killed and the plant remains healthy. Bottom: In the susceptible reaction a virulence factor (molecule causing necrosis) is transported and also avr molecules. In the absence of resistance genes the avr proteins eliminate the RIN4 protein that protects the basal defense of the cell. The basal defenses are reduced and the plant becomes diseased. Hrp gene products are not enough to cause disease; other virulence factors (aggressins) such as toxins, pectolytic enzymes and EPS are necessary. After Leach and White (1996), Vanneste (2000), Mackey et al. (2003), and others.

Disease and symptoms

97

genes that are recognized by genetically resistant host plants (Hutchinson, 2001). When the Rgene is lacking, avr proteins apparently function as virulence factors that can stimulate the production of a protein in the plant that blocks the so-called RIN4 protein that is important in the plant basal immune system (Mackey et al., 2002). Several avr and R genes from bacteria have also been utilized to confer resistance to plants expressing the corresponding resistance gene. For example: HR and disease resistance occur when P. syringae pv. tomato with the avirulence gene avrPto infects tomato plants carrying the resistance gene Pto. Avr genes are often located on plasmids (Vivian et al., 2001). Genetic study of the determinants of pathogenicity has been facilitated in recent years by the possibility of sequencing the complete genome of plant pathogenic bacteria. This hard work has been performed e.g. for Agrobacterium tumefaciens (Goodner et al., 2001); Erwinia carotovora subsp. atroseptica (Bell et al., 2004); Pseudomonas syringae pv. tomato (Buell et al., 2003), Xylella fastidiosa (Simpson et al., 2000), Xanthomonas campestris pv. campestris and X. axonopodis pv. citri (Da Siva et al., 2002), X. oryzae pv. oryzae (Lee et al., 2005) and also for Ralstonia solanacearum. In the latter case a race 1 strain from tomato (GM11000) was used. The genome was found to consist of 5.2 megabases (Mb). It is organized into two replicons: a 3.7 Mb chromosome and a 2.1 Mb mega plasmid. Many genes playing a role in pathogenicity have been identified, including those coding for effector proteins (Salanoubat et al., 2002). For updates on complete genome sequences of (plant pathogenic) bacteria see http://www.ebi.ac.uk/integr8/ (European Bioinformatics Institute, EMBL-EBI).

Table 16

Gene-for-gene relationship between pea (Pisum sativum) cultivars and races of Pseudomonas syringae pv. pisi, causing pea blight. The pea variety Kelvedon Wonder is susceptible to all known races and no resistance against race 6 of P. s. pv. pisi has been found. After Vivian and Gibbon (1997).

Cultivar

Resistance gene no.

Races 1 Avirulence 1,3,4,6? genes

2

3

4

5

6

7

2

3

4

2,4,5?,6?

nil

2,3,4

+

+

+

+

+

+

+

+

Kelvedon Wonder nil

+

Early Onward

2

+

Belinda

3

+

Hurst Greenshaft

4, 6?

+

Partridge

3, 4

+

Sleaford Triumph

2, 4

Vinco

1, 2, 3, 5?

Fortune

2, 3, 4

+

+ +

+

+ + +

+

+ +

+ = pathogenic reaction, symptom formation; ? = race/resistance gene not fully elucidated.

+ +

98

Table 17

Chapter III

Occurrence of plant tissue degradative enzymes in different plant pathogenic bacteria. After Kleinhempel et al. (1989).

Bacterial species or pathovar

Cellulase

Pectate lyase endo

Clavibacter michiganensis subsp. michiganensis C.m. subsp. sepedonicus

+

Erwinia carotovora E. chrysanthemi E. cypripedii E. amylovora

+ + +

Pseudomonas fluorescens* P. marginalis pv. marginalis P. viridiflava

+

Ralstonia solanacearum

+

Streptomyces scabiei**

+

+

+

+

Xanthomonas arboricola pv. pruni X. axonopodis pv. citri X. a. pv. malvacearum X. a. pv. vesicatoria X. campestris pv. campestris X. oryzae pv. oryzae Xylella fastidiosa

exo

+

Polygalacturonase endo exo +

Pectinesterase

Protease

+

+ + + +

+ +

+ + +

+ + +

+

+ +

* some strains only; ** S. scabiei also produces suberinase.

+

+

+ +

+

+

+ + + +

+

+ + + +

+ +

+ +

+ +

+ +

Disease and symptoms

99

4. Phases in pathogenesis In pathogenesis four phases can be distinguished, namely infection, incubation, appearance of symptoms and progression or cessation of the disease process followed by eventual recovery. Infection. The penetration of a bacterium in the host is dependent on a complex interaction between pathogen, host and abiotic and biotic factors in the environment. Bacteria cannot, as many fungi do, enter the plant directly through the epidermis. Therefore the place of infection already determines symptom formation to a large extent. Bacteria are dependent on high humidity, free water and temperatures conducive for their growth. Bacteria can enter the plant via: a. Hydathodes water pores that are always open, e.g. infections of Xanthomonas campestris pv. campestris in cabbage (Fig. 69 and 74). b. Nectaries also extra floral nectaries (e.g. infections of Erwinia amylovora). c. Stomata respiration pores that can be closed (Fig. 77). d. Trichomes hairs, e.g. infections of Clavibacter michiganensis subsp. michiganensis in tomato. e. Leaf scars shortly after leaf fall when no cork layer has yet been formed. f. Lenticels respiration pores in woody plants. g. Surface wounds such as caused by insects, frost, hail, wind-blown sand, rain or mechanical damage, or damage by lateral root formation (Fig. 75). Incubation. During incubation bacteria establish themselves in the tissue, multiply, spread and exercise their first influence on the plant. Most plant pathogenic bacteria can only spread via the intercellular spaces and kill, destroy and penetrate plant cells from there (Figs. 78-80). The vascular pathogens can multiply and spread selectively in the xylem or phloem. Apart from that, the latter also multiply at first intercellularly, e.g. X. oryzae in the hydathode-epithem. The reverse may also be true: Erwinia amylovora, not primarily a vascular pathogen, can establish itself first in xylem vessels of a leaf trace and colonize the parenchyma from there in a later stage. Phloem-colonizing phytoplasmas as well as root-nodule N-fixing bacteria spread intracellularly via living cells. As far as is known bacteria are spread passively in the plant and they lose their flagella in plant tissue. In the substomatal and intercellular spaces sufficient humidity (free water) must be present for bacterial development. Therefore bacterial infections almost always take place under humid conditions. Necrotrophic bacteria are able to maintain these humid conditions because they excrete membrane-damaging substances (pectinases and extracellular polysaccharide or EPS). Due to the action of these substances water and nutrients leak into the intercellular spaces. Due to this leaking phenomenon many bacterial diseases show a typical water-soaked, glassy zone at the margin of healthy and diseased tissue. In this water-soaked area dying or dead plant cells, but only a few bacteria, are found (Figs. 79 and 84). After these initial stages hydrolysis of middle-lamellae (by pectinases) and cell walls (cellulases and other hydrolytic enzymes) can proceed: necrotrophic bacteria typically produce cavities. In these cavities masses of bacteria embedded in slime and remnants of degraded cells are found (Figs. 78-80, 89). Vascular pathogens behave in a similar way in the vascular parenchyma in later disease stages. Appearance of symptoms. In the case of necrotrophic pathogens this may be caused by disturbance of the metabolism, leakage of cells, cell death, degradation of tissue, first defence reactions (Figs. 78-80) and obstruction of water movement. In the case of gall- and tumourforming bacteria there is a visible multiplication of plant cells (Fig. 89).

100

Chapter III

PENETRATION OF BACTERIA INTO STOMATUM (NATURAL OPENING IN PLANT SURFACE)

Fig. 77 Section through a stomatum (3-dimensional) AB = saprophytic bacterium attached to cell wall; B = bacterium on cuticle; CU = cuticle; E = epidermal cell; FB = free bacterium (in water!); GC = guard cell; HY = fungal hypha; PP = palisade parenchyma; SA = stomatal aperture.

EARLY STAGE OF INFECTION: MULTIPLICATION OF BACTERIA IN INTERCELLULAR SPACES AND DISSOLUTION OF MIDDLE LAMELLAE

Fig. 78 Transverse section of bark parenchyma of Fraxinus excelsior infected by Pseudomonas savastanoi pv. fraxini. Bacteria are seen as masses of small dots in intercellular spaces and in killed parenchymal cells. Also dissolution of middle lamellae is seen (yellow arrows).

Disease and symptoms

101

Progression or cessation of the disease. This is dependent on virulence factors of the bacteria, sensitivity, age and defence reactions of the host and physiological and climatological circumstances. In the case of potato scab or certain leaf spot diseases, infection may stop within the season. Very often, however, especially in the case of vascular diseases and necroses, an organ or a plant is killed completely.

Fig. 79a Transverse section of bark parenchyma of Fraxinus excelsior. Similar infection as in Fig. 78, at the junction of healthy and diseased tissue. Lower right: b = bacteria in an enlarged intercellular space. Upper right: b = bacteria in cells, protoplast degraded, s = starch, still present. Left: various stages of necrosis, bacteria lacking, np = necrotic protoplast Electron microscope photograph.

P N B

Fig. 79b Cells (B) of Pseudomonas savastanoi pv. fraxini in an intercellular space. Similar infection as in Fig. 79a. Cells surrounded by electron-dense material, most probably slime and plant cell products (P). Plasma of adjacent plant cells collapsed and necrotic (N). Electron microscope photograph. Bar represents 1 µm.

102

Chapter III

LATER STAGES OF BACTERIAL INFECTION IN THE BARK OF ASH TREE (FRAXINUS EXCELSIOR)

Fig. 80 Transverse section of bark parenchyma of Fraxinus excelsior showing progressive stages of infection by P. savastanoi pv. fraxini. The plant reacts by cork (periderm) formation. Enclosure of bacteria often fails and new cork layers have to be formed - the results are necrotic excrescences; see lower left and right photographs. After Janse (1981). bc = bacterial cavity; co = collenchyma; dc = dead collenchymal cells; gc = gum cell; p = periderm; pa = bark parenchyma; pf = pericycle fibre; st = stone cell.

RESULTING EXCRESCENCES ON BRANCHES AND TRUNK OF THE ASH TREE

Disease and symptoms

INTERACTIONS BETWEEN BACTERIA AND PLANTS

Fig. 81 Growth in water-soaked bean leaf tissue of a homologous (pathogenic for bean, Pseudomonas syringae pv. phaseolicola), heterologous (pathogenic, but not for bean, P. syringae pv. syringae) and saprophytic bacterium (Pantoea agglomerans = Erwinia herbicola). The pathogen will cause disease and symptoms, the pathogen in a non-host will start to multiply but is then inactivated through a HR reaction of the plant and the saprophyte will be immobilized by agglutinins. After Young (1974).

Fig. 82 Respiration of potato tubers after infection by Erwinia carotovora subsp. atroseptica at a wound depth of 15 mm at two different temperatures. The temperature of the rotten tissue also rises due to the disease interactions, turning it into a self-perpetuating process, leading to severe losses in a short time. After Kleinhempel et al. (1989).

103

Chapter III

PD

104

Fig. 83 Water-soaked spots on leaves and pods of pea (Pisum sativum), caused by Pseudomonas syringae pv. pisi.

Fig. 84 Water-soaked spot, typical of bacterial infections, on leaf of Cattleya orchid, caused by Acidovorax avenae subsp. cattleyae A = water-soaked margin, where cells are leaking, but bacteria are not yet present; B = yellow, chlorotic ring due to action of bacterial toxin; C = necrotic area, where also secondary pathogens and saprophytic organisms can be found.

Fig. 85 Histoid galls of Pseudomonas savastanoi pv. savastanoi on olive (Olea europaea).

Disease and symptoms

105

5. Symptoms Symptoms which may be caused by the different bacterial genera are presented in Table 7, page 36. The following classes of symptoms are distinguished:

a. leaf spots b. excrescences and galls c. tumours d. vascular diseases and wilt e. necroses and cankers f. rotting g. bacteria embedded in slime It should be remembered that one bacterial pathogen may cause different symptoms (Fig. 72), e.g. Pseudomonas syringae pv. mors-prunorum causing leaf spots, necroses and cankers and X. c. pv. campestris causing a vascular blackening and leaf spots. Furthermore two bacteria or a bacterium and another plant pathogenic organism may cause almost identical symptoms (Fig. 73). Secondary pathogens may change symptoms drastically and combined infections may be confusing. The classes of symptoms will now be treated separately:

a. Leaf spots Bacterial leaf spots can often be distinguished from those caused by other organisms by a chlorotic halo which is formed under the influence of toxins, followed by a water-soaked zone formed by EPS, a brown to black necrotic part and a greyish to brown papery dry centre (Figs. 83 and 84). When leaf spots are bordered by larger, lignified veins they are angular in the case of dicotyledonous plants and longitudinal in the case of monocotyledonous plants; for the rest they are circular or irregular. When leaf spots coalesce larger areas of the leaf lamina are killed. Development of leaf spots often stops when the weather becomes dry. Bacterial slime is pressed out of the plant under humid conditions via stomata and ruptures (Figs. 99, 100 and 102, compare with Fig. 102 right). The slime can be observed as a thin silvery film under dry conditions.

b. Excrescences and galls The filamentous bacterium Streptomyces scabiei causes excrescences, called scab, on potato (Fig. 88), (sugar)beet, radish and carrot. The bacterium often penetrates the plant via young, not yet suberized lenticels or wounds. S. scabiei produces pectinases, enabling the hyphae to grow between cells. After plant cells have been killed, intracellular growth also takes place. Because S. scabiei does not produce toxins that rapidly kill plant cells, cork layer formation can take place in tissue at a distance from the lesions. The plant tries to localize the infection. The cork layer meristem (phellogen) sometimes forms many other parenchymal cells apart from cork cells, in that case so-called raised scab will develop.

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Fig. 86

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Organoid galls (cauliflower-like galls) on Gladiolus corms, caused by Rhodococcus fascians.

Fig. 87 Organoid galls (malformed stem bulbs) on Lilium sp., caused by Rhodococcus fascians.

Fig. 88 Histoid galls (common potato scab), caused by the bacterium Streptomyces scabiei.

Fig. 89 Transverse section through a histoid gall caused by Pseudomonas savastanoi pv. savastanoi. P = bacterial pocket in centre of gall; C = ring of cork cells; G = small, parenchymatous gall cells, formed under the influence of plant hormones produced by the bacterium.

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Fig. 90 Twenty-five-days-old tumour on Pelargonium zonale, caused by Agrobacterium tumefaciens. After Gäumann (1951). e = epidermis; p = healthy parenchyma; r = giant cell = hypertrophy; tp = tumour parenchyma; x = xylem (tracheids).

Large tumour, developed on a wound that resulted from grafting on Ficus sp., caused by Agrobacterium tumefaciens.

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TUMOUR FORMATION BY AGROBACTERIUM TUMEFACIENS

Fig. 92 Tumour formation by Agrobacterium tumefaciens and schematic representation of Ti -plasmid. Also see text. Bottom: Tumour (leaf on the left), malformed organs (teratomata, i.e. the outgrowth in the axil on the right and the abnormal stem root formation) on Kalanchoe daigremontiana inoculated with a virulent strain of Agrobacterium tumefaciens.

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When the processes as described for potato scab persist for a number of years, excrescences are formed as are found with the bacterial knot disease caused by Pseudomonas savastanoi pv. fraxini (Fig. 80). Bacterial galls are tissue swellings generated by hypertrophy and hyperplasia, caused by the hormone-balance disturbing influence of the bacterium. The growth of the gall only takes place when the pathogen is present (in contradiction to tumour growth, see below). One can distinguish histoid, little-differentiated galls, such as caused by P. savastanoi pv. savastanoi on olive trees. These galls consist of many newly formed parenchymal cells with spontaneously formed islands of xylem (vessel) tissue and bacterial cavities surrounded by cork layers (Fig. 89). The galls develop under the influence of hormones indole acetic acid (IAA) and cytokinins, excreted by the bacteria. The plant cells are not transformed as in the case of tumours (see below). The other type of gall is called organoid, such as those caused by Rhodococcus fascians. This bacterium stimulates resting meristematic tissue in buds and (stem) bulbs, by excreting cytokinins and IAA, to abnormal growth and sprout formation (Figs. 86 and 87). No bacterial cavities are found in this case. Witches’ broom symptoms, dwarfing and deformation occur in diseases caused by phloem-inhabiting fastidious bacteria and phytoplasmas (Figs. 103 and 104).

c. Tumours Malignant swellings, comparable to human or animal cancer, are caused in plants mostly by the bacterium Agrobacterium tumefaciens (Figs. 90-94). The host range of this bacterium is extremely wide, including more than 600 species of (mainly) dicotyledon plants. A. tumefaciens is a wound parasite, which can only change (transform) plant cells to cancer cells when there are living, dividing cells at the margin of the wound (Fig. 92). Virulent strains carry large Ti (tumour-inducing) plasmids with a size of 150-250 kb. These plasmids contain, apart from tumour genes, virulence genes, and genes for production of growth hormones, production and utilization of amino acid derivatives (opines) such as nopaline, agropine or octopine, replication and transfer of the plasmid and susceptibility to a bacteriocin (agrocin 84), produced by non-plasmid-containing strains, usually named A. radiobacter (also see Chapter VI.8 and Fig. 147). A. vitis, producing tumours on grapevine, has a narrow host range and a different Ti -plasmid. This bacterium is systemic in its host (Fig. 94), causing extensive tumour formation all over the plant, leading to serious damage and crop loss. The bacterium first becomes attached to the outer cell wall. In this case attachment does not inactivate the bacterium as is the case with other plant pathogenic bacteria and it is determined by several virulence genes located on the Ti -plasmid (Fig. 92). The Ti -plasmid exchange between bacteria via conjugation, and therefore the infectivity of the A. tumefaciens population can increase under the influence of so-called ‘quorum sensing’ where opines from the transformed plant cells in the tumour and proteins from the bacterium (TraI, TraR and TraM) serve as signal molecules for conjugation at high bacterial population densitities. Quorum sensing is also involved in the infection process of Erwinia carotovora spp. where it stimulates production of protease, cellulase, pectinase and exopolysaccharide (Henke and Bassler, 2004; Newton and Fray, 2004). The attached bacterium initiates the transfer of T(umour)-DNA under influence of phenolic compounds such as acetosyringone, produced by wounded plant cells. Subsequently A. tumefaciens transfers plasmid DNA (from the Ti -plasmid) into the living cell and part of the plasmid coding for tumour formation (Ti -region) is integrated in the genome of the plant. This process, which takes + 24 h, is called induction (Fig. 92). Usually three copies of TDNA can be detected in the plant genome. The transformed cell divides continuously under the influence of hormones (indole acetic acid and cytokinins). Genes for these hormones are

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TRANSFORMED AGROBACTERIUM TUMEFACIENS USED FOR GENETIC MANIPULATION OF PLANTS: GENETIC ENGINEERING

Fig. 93 Transformed Agrobacterium tumefaciens used for genetic manipulation of plants: genetic engineering of plants.

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present in the Ti -region, and also acquired by the transformed cell. The transformed cells produce opines that can be used by the Agrobacterium cells as nutrients. The presence of the bacterium is no longer necessary. Therefore it is sometimes difficult to isolate the bacterium from older tumours and bacterial cells generally occur in outer layers of the tumour. In tumours, as in galls, islands of lignified (xylem) tissue occur (Fig. 90). On certain tumours malformed organs, called teratomatas, are sometimes formed (Fig. 92). A comparable pathogenic process leading to a disease syndrome of abnormal root proliferation, called ‘hairy root’ is caused by the related Agrobacterium rhizogenes. The Ti-comparable plasmid is called Ri (root-inducing) plasmid. Both Agrobacterium spp. are subject to intensive genetic and molecular biological research and both are used as vectors in genetic manipulations of plants (genetic engineering). In this case the Ti - or Ri -plasmid has the sequences responsible for tumour formation deleted and replaced by other sequences (resistance genes, etc.). Then it is placed again in the Agrobacterium, which transfer the new sequences into the plant cell, where integration in the chromosome and transformation and (often) expression takes place. More specifically in a socalled ‘binary vector’ system two plasmids are used to transform plants (see Fig. 93). The plasmid (vector) contains elements from a plasmid of Escherichia coli and of A. tumefaciens. First the target genes for transformation (e.g. genes coding for resistance to a herbicide) are integrated into a plasmid in E. coli. Thereafter this plasmid is transferred to A. tumefaciens by conjugation. The target DNA and a kanamycin resistance marker can be mobilized by the Ti plasmid of A. tumefaciens, which has had the T(umour) –genes deleted. After integration and recombination of the target DNA with the plant chromosome, the foreign DNA can be expressed, giving the plant new properties (herbicide resistance in our example). Production of transgenic plants with Agrobacterium tumefaciens or A. rhizogenes has been successful mainly in dicotyledon plants. Examples are glyphosate (herbicide) resistance in soybean (Glycine max), introduction of Bacillus thuringiensis toxin (insecticidal) genes in a number of plants, introduction of virus coat protein genes for protection against virus infection, and introduction of genes encoding the production of interferon and human antibodies, useful in human medicine. An example for bacterial plant pathogens is the incorporation of cholera toxin, subunit A from Vibrio cholerae in tobacco, producing resistance against Pseudomonas syringae pv. tabaci (Lorito and Scala, 1999).

Fig. 94 Severe systemic tumour formation in grapevine (Vitis vinifera), leading to death of infected plants, caused by Agrobacterium vitis.

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Fig. 96

Fig. 95 Tomato (Lycopersicon esculentum) stem showing yellow-brown vascular discoloration, due to infection of Clavibacter michiganensis subsp. michiganensis.

Wilting of tomato (Lycopersicon esculentum) due to blocking of vascular tissue, caused by Ralstonia solanacearum.

Fig. 97 Left: Right:

Black discoloration of vascular bundles of a cabbage (Brassica oleracea) stem, due to infection of Xanthomonas campestris pv. campestris. Cells of Ralstonia solanacearum present in a small spiral vessel of potato (Solanum tuberosum). Gram stain of a smear of infected vascular tuber tissue.

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d. Vascular disease and wilting Vascular diseases may be caused by infection through roots or stolons by (soil) pathogens such as Ralstonia solanacearum, causative organism of brown rot in potato, tomato, tobacco, etc., Clavibacter michiganensis subsp. sepedonicus, causing ring rot of potato and C. m. subsp. michiganensis causing bacterial canker of tomato (Figs. 95-97). Other possibilities are infection through infected seed or hydathodes at the leaf margin, such as in infection caused by Xanthomonas campestris pv. campestris or X. hyacinthi (Figs. 26, 69 and 74). Vascular pathogens can excrete toxins (glycoproteins), which diffuse more rapidly than the bacteria, causing wilting, yellowing and sometimes large glassy, later necrotic spots. In these spots no bacteria are found (Fig. 68). Because the bacteria degrade non-lignified parts of vessels and walls of neighbouring parenchymal cells, vascular tissue is damaged, transport is disturbed and bacterial cavities are formed. EPS and reaction products or degraded products of the plant may cause further blocking and wilting. In the case of bacterial ring rot and brown rot the degradation of vascular tissue in the tuber can be clearly seen as a slimy ring in the rest of the still-healthy tissue. Xylem-inhabiting fastidious bacteria like Xylella fastidiosa do not degrade the xylem. They cause yellowing, wilt, ‘burning’ of the leaf margins and death of the plants (Figs. 103 and 104).

e. Necroses and cankers When bacteria spread rapidly through the tissues and kill them, large areas of sunken, dead, brown to black necrotic tissue are formed. Bark, leaf and internal necroses can be distinguished. Bark and leaf necroses are caused by E. amylovora (the necrosis possibly caused by extracellular polysaccharide slime (EPS), Fig. 98 top left), P. avellanae (Fig. 98 top right) and P. syringae pv. syringae for example. Internal necroses are formed by E. rubrifaciens in walnut, so-called phloem necrosis and by P. corrugata in tomato, so-called pith necrosis (Fig. 98 lower right). When a perennial host tries to limit bark necroses, where the cambium is locally damaged, by callus formation, then over the years cankers develop. A canker is an open wound of the wood, caused by local cambium and wood destruction, surrounded by rims of callus tissue, formed by the undamaged cambium. Cankers are formed by P. syringae pv. morsprunorum in stone-fruit trees and Xanthomonas populi in poplar trees for example (Fig. 98 lower left).

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Fig. 98 Top left:

Rapid necrosis (fire blight), caused by Erwinia amylovora in hawthorn (Crataegus sp.).

Top right:

Rapid necrosis caused by Pseudomonas avellanae in Corylus avellana.

Lower left:

Tree canker (killing of the bark up to the wood and callus formation), due to Xanthomonas populi in Populus sp.

Lower right:

Pith necrosis in tomato (Lycopersicon esculentum), caused by Pseudomonas corrugata.

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f. Rotting Necrosis of tissue can occur in combination with exudation of fluid and a bad smell, the typical symptoms of rotting. Several plant pathogenic bacteria can cause rotting by a rapid degradation of the middle lamella (maceration, Fig. 101) and cell content by pectolytic, cellulolytic and proteolytic enzymes. Examples are soft rot caused by E. carotovora subsp. carotovora, brown rot of orchids caused by E. cypripedii and soft rot caused by E. chrysanthemi in a large number of hosts, including corn, Dahlia, Dieffenbachia, Philodendron and Begonia.

g. Bacteria embedded in slime Bacteria do not form fructifications on or in the host as fungi do. They are only visible as masses embedded in slime, which protrude from the tissue, either spontaneously (Figs. 99100 and 102) or after cutting or wounding of the tissue. For some diseases, like fire blight or brown rot of potato, this slime may have diagnostic value. Bacterial slime may be present as very thin threads, so-called strands (Figs. 99 and 100). The colour of the slime is often grey, but also yellowish or orange.

Fig. 99

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Strands of bacterial slime (that contain bacteria and can be dispersed by wind) on water-soaked lesions caused by Pseudomonas syringae pv. porri on leek (Allium porrum).

Fig. 100 Bacterial strands of Erwinia amylovora on a twig of Crataegus (hawthorn).

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Fig. 101 Left: Right:

Soft rot, due to rapid dissolution of middle-lamellae of plant cells and leaking of cells, caused by Erwinia carotovora subsp. carotovora. Some human and animal pathogenic bacteria can exceptionally also cause disease in plants and vice versa. An example is Pseudomonas aeruginosa (an opportunistic pathogen causing a number of infections in man and animals), causing soft rot of onion and potato (Plotnikova, 2000). The photograph shows a strain from sheep causing rot in potato, 7 days after artificial inoculation (Janse et al., 1992). Strains of the plant-associated Agrobacterium radiobacter have been isolated from clinical specimens (blood, urine, pleural exudates, sputum, wounds, etc.).

Fig. 102 Left: Right:

Droplets of bacterial slime on water-soaket spots as result of infection by Pseudomonas syringae pv. phaseolicola, causing halo blight of bean (Phaseolus vulgaris). Slime drops are not always caused by bacteria! In this case it is slime containing fungal spores (ergot, Claviceps africana on Bulrush millet, Pennisetum typhoideum).

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h. Symptoms of fastidious, (non-)culturable bacteria, including Xylella fastidiosa, phytoplasmas and spiroplasmas Symptoms caused by the Gram-negative, fastidious xylem-limited bacteria (FXLB) are foliar burning (Fig. 103), stunting, wilting and/or decline. Phytoplasmas mainly cause yellows diseases. Symptoms include yellowing and chlorosis or bronzing of foliage, stunting (shortening of internodes, reduction of leaf size), proliferation of axillary buds often resulting in a witches’ broom effect, virescence (greening), proliferation of secondary roots, abnormal fruit and seeds, and sterile flowers. Important examples are aster yellows, coconut lethal yellowing, stolbur of tomato (Fig. 104), elm yellows (phloem necrosis), Paulownia witches’ broom, pear decline, tomato big bud and peach X (also see Annex 4).

M. Scortichini

Symptoms of spiroplasmas include stunting, chlorosis and yellowing, distorted fruits and reduced fruit size (Fig. 104), reduction of leaf and flower size, necrosis, and wilting.

Fig. 103

M. Scortichini

Leaf burning symptoms of Xylella fastidiosa on grapevine (Vitis sp.).

Fig. 104 Left: Right:

Symptoms (distorted inflorescence and buds) of stolbur, caused by a phytoplasma in tomato (Lycopersicon esculentum). Typical symptom (lopsided and small fruits on the left) of stubborn disease of Citrus sinensis, caused by Spiroplasma citri.

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COMBINATION OF HIGH HUMIDITY AND TEMPERATURE FAVOURABLE FOR BACTERIAL DISEASE DEVELOPMENT Table 18

Influence of soil humidity and soil temperature on the occurrence of soft rot in potato. After Gäumann (1951). Soil temperature 6-10 oC

Humidity %

Fig. 105 Top:

20 oC

Diseased tubers %

Diseased tissue %

Diseased tubers %

Diseased tissue %

Diseased tubers %

Diseased tissue %

0 0 0 0 100

0 0 0 0 50

0 10 50 100 100

0 1 20 60 40

0 20 60 100 100

0 1 23 73 73

J. van Vaerenbergh

25 50 75 100 125

15 oC

Difference in disease development in Chrysanthemum cv. Sunny Mandalay plants up to 48 days after inoculation with Pseudomonas cichorii, when kept under low (55-70%) and high (85-95%) relative humidity conditions. After Janse (1987). Bottom: Severe rotting of button mushroom (Agaricus bisporus), caused by Janthinobacterium agaricidamnosum, stimulated by very a high (relative) humidity (88-91%) that is necessary during cultivation of the mushroom. The bacterium is easily spread by water and contact.

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CHAPTER IV - EPIDEMIOLOGY1) 1. Environmental effects and disease development Occurrence and epidemic development of disease are dependent on the presence of susceptible host plants and virulent bacteria and are determined by environmental conditions (Fig. 106). Generally speaking high humidity (free water, soil humidity and relative humidity of the air) is one of the most important environmental factors positively influencing disease development. Furthermore humidity in combination with temperatures in the range of the optimum growth temperature of the bacterium is necessary (Fig. 105, Table 18). For example in early spring in The Netherlands under conditions of high humidity (and damage of plants due to hail or frost) and temperatures of 10-20oC infections by Pseudomonas syringae pv. syringae are very common. This bacterium has a rather low growth temperature optimum in vivo (20oC). Infections by Erwinia amylovora are only found much later in spring or in early summer for the first time. This bacterium has a higher growth temperature optimum (2830oC). Frost, hail, strong winds and wind-blown sand may cause wounds and in this way stimulate infections by bacteria. Infections by pathovars of Pseudomonas syringae are often found in combination with frost. These bacteria (and others like Pantoea stewartii, Pseudomonas viridiflava and Xanthomonas translucens) are ice nucleation active (INA). This means that the bacteria (in fact proteins in their membranes) function as so-called ice nuclei, causing quick freezing of plant cells (and concomitant ice crystal formation and death of cells) at temperatures where plants normally do not show frost damage (also see Chapter II.1b). Soft rot Erwinia species are strongly dependent on wounds, in addition to the combination of high humidity and high temperature (Table 18). Also the soil type, pH of the soil, microbial (antagonistic) populations in the soil, etc. may influence disease occurrence by influencing survival of the bacteria, multiplication of the bacteria in the rhizosphere and condition of the host. R. solanacearum is able to grow and/or survive in the rhizosphere or in micro-lesions of many (non-)host plants, such as cabbage, bean, corn and Polygenum spp. Most bacterial pathogens are sensitive to a low and a high pH and prefer a neutral pH for optimal growth (Fig. 107). Plant nutrition and often more generally human production technology are other important factors that may influence disease development. Generally plants high in N and low in Ca and K are weakened and may be attacked by bacterial plant pathogens more easily (Fig. 70 and Table 20, McGuire & Kelman, 1984). Bacterial stem blight in chrysanthemum, caused by P. cichorii, is a big problem under conditions of close planting, high humidity and high N fertilization. It is not a problem at all under more ‘old-fashioned’ conditions. Overhead sprinkler irrigation and tidal systems of watering/feeding plants may be disastrous in greenhouses when a pathogen is present. Furthermore planting or sowing time (Table 21), manipulations during planting, the growing period, harvesting and storage may enhance disease as well. Cutting of planting material without disinfection of the cutting knife or machine is a very efficient way of spreading the pathogen and increasing disease. This has been observed e.g. in the development and spread of bacterial ring rot (Clavibacter 1)

For more information see Leben (1965); Van der Plank (1982); Hirano and Upper (1983); Henis & Bashan (1985); Beattie and Leben (1999); Denny (1999); Mühldorfer and Schäfer (2001); Lindow and Brandl (2003); Goodman (2004).

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Fig. 106 Life cycle model for plant pathogenic bacteria In the three main phases (I-III) that can occur in the life cycle of plant pathogenic bacteria, one can discriminate the following: Bacteria survive and locally multiply epiphytically, endophytically or in the rhizosphere (A, C, I) and serve as a source of inoculum. They can survive on or in dead (symptomatic) plant material (H) and free in the soil (B) and from these places spread to other hosts (C, D, E). They can multiply in vascular tissues (I, K, F) and spread to seeds (L), tubers and roots (I) and can be excreted in the rhizosphere, free soil, on stems and leaf scars (J, K) and dispersed to other hosts for shorter or longer distances. Bacteria can also multiply in parenchymatous tissues on plant parts above and under the ground from where they can spread to other hosts and soil (E, F, G, I). Bacteria can also be transferred from one plant to another via root contact (C).

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michiganensis subsp. sepedonicus) in the USA, when potato seed was cut (Table 19) and also for Ralstonia solanacearum when leaf cutters were used during the tobacco harvest. Other pathogens may enhance or decrease disease spread or severity. It was found that root knot nematodes increase the incidence of brown rot (R. solanacearum). There was a negative effect on disease development of ring rot of potato (C. m. subsp. sepedonicus) when simultaneous infection by Verticillium or Erwinia chrysanthemi took place.

2. Survival Plant pathogenic bacteria do not form endospores and cannot therefore be as persistent in nature as spore-forming bacteria. Still, bacterial plant pathogens are often able to survive in the environment quite well. This may be in the form of active cells (that appear to be reduced in size due to starvation, see Monier and Lindow, 2003a) in the epiphytic state (on plants, for a review on epiphytic colonization see Beattie and Leben, 1999) or in the rhizosphere (on roots), where they even multiply, or more or less active cells in the plant (endophytic, latent state). In particular, vascular pathogens are able to survive from season to season in a latent form (Fig. 106). Because in the latent state no symptoms develop, these bacteria are hard to detect and form a threat for spreading disease. That is why for these bacteria special detection methods have been developed. Bacteria can also survive in a more dormant state, encapsulated and protected by their exopolysaccharides on any surface and in seed. Survival time is influenced by environmental (climatic) conditions (Fig. 106 and Tables 22 a and b, 23 and 24). Thus it has been established that C. m. subsp. sepedonicus can survive for several months on metal of machinery or burlap sacks. Some bacteria that may survive in seed are C. m. subsp. michiganensis, Curtobacterium flaccumfaciens pv. flaccumfaciens, P. s. pv. lachrymans, P. s. pv. phaseolicola, P. s. pv. pisi, X. c. pv. campestris, X. a. pv. malvacearum, X. a. pv. phaseoli and X. vesicatoria (also see Table 8). Survival as free cells in the soil is short for many bacterial pathogens. There is a rapid decline in population, most probably because they compete poorly with the soil microflora and due to lack of appropriate nutrients. Exceptions are A. tumefaciens and R. solanacearum, which may survive in the soil for a very long time in the absence of hosts. Other bacteria survive only when they are protected by host tissue (which may be of microscopic dimensions!). For soft rot Erwinias and R. solanacearum it has been found that they can survive in surface and irrigation water for months, even during wintertime (Figs. 119-121).

Fig. 107 Incidence of potato scab and relative growth of its causative bacterium, Streptomyces scabiei, as influenced by pH of the soil. After Alexander (1977).

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EFFECT OF HUMAN CULTURAL PRACTICES ON DISEASE DEVELOPMENT AND CROP LOSSES Table 19 Seed

Distribution of ring rot (Clavibacter michiganensis subsp. sepedonicus) by cutting of seed. After Perrault (1948). % Diseased plants derived from tubers Non-cut seed Cut seed

A

3

14

B

8

47

5.5

30.5

Mean

Table 20

Influence of fertilization (nutrition) on the occurrence of Erwinia soft rot in stored potato. After Gäumann (1951).

Fertilization

Spontaneously rotting tubers %

Normal

5.9

Nitrogen Surplus as calcium cyanamid Surplus as ammonium sulphate Shortage

17.5 20.8 2.8

Potassium Surplus as kainite KMg (SO4)Cl.H2O Surplus as 40% potassium salt Shortage

8.4 11.4 12.1

Phosphoric acid Surplus as super phosphate Surplus as basic slags Shortage

7.1 6.8 6.9

Table 21

Sowing date 15th of July 5th of August 15th of August

Influence of sowing date on development of Erwinia soft rot in Chinese cabbage. In this case early sowing was a high risk due to favourable weather conditions for the pathogen! After Kleinhempel et al. (1989).

Diseased plants % 90.0 61.5 15.4

Yield (kg/10 acre) 452 1336 2859

Weight per plant (kg) 0.754 0.947 1.132

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SURVIVAL OF BACTERIA INFLUENCED BY: HUMIDITY AND SURFACE

Table 22a

Survival (in months) of Clavibacter michiganensis subsp. sepedonicus, causing ring rot of potato, on different materials. After Starr (1947).

Material

Survival in months

Jute, plastic, paper

High RH

Low RH

<7

> 24

Rubber, concrete

> 10

Metal

<4

Clavibacter michiganensis subsp. sepedonicus survives very well under dry conditions on materials with rough surfaces

INFLUENCED BY: ATMOSPHERIC CONDITIONS, RADIATION

Table 22b

Survival % on jute bags of Clavibacter michiganensis subsp. sepedonicus, causing ring rot of potato, in days. After Starr (1947).

Location of jute bags 10

Duration of experiment in days 30 20

40

South side building (outside)

70

49

20

10

Within building, dark

95

95

66

70

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SURVIVAL OF BACTERIA INFLUENCED BY: TEMPERATURE AND LIGHT

Table 23

Survival (in days) of Ralstonia solanacearum biovar 2, race 3, causing bacterial wilt, in different substrates at room temperature in daylight and at 4ºC in the dark. After Janse et al. (1998).

Substrate

Survival in days Low temperature (4ºC), Room temperature, light dark

Surface water

17

33

Ditch mud

6

24

Sewage sludge, potato industry

23

53

Chicken manure

23

30

Cow manure

7

11

INFLUENCED BY: SURFACE AND STATE OF BACTERIA (INCLUDING PROTECTION BY SUBSTRATE)

Survival (in days) of Ralstonia solanacearum biovar 2, Table 24 race 3, causing bacterial wilt, on different materials. After Janse et al. (1998).

Material

Survival in days Inoculum: rubbed Inoculum: pure culture naturally infected potato from nutrient agar tuber tissue

Wood

4

4

Metal

14

14

Rubber

55

87

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RISK ASSESSMENT SYSTEMS BASED ON DEVELOPMENT OF (EPIPHYTIC) POPULATIONS CAN BE OF IMPORTANCE FOR DISEASE PREDICTION AND CONTROL EXAMPLE: FIRE BLIGHT (ERWINIA AMYLOVORA) Predicting disease may help growers to obtain more effective control and reduced costs for the pesticides applied. In the case of blossom blight a good prediction system requires a general understanding of the biology of Erwinia amylovora (Ea), the bacterium that causes fire blight, and the susceptibility of the blossoms of a host plant. Disease development is dependent on conditions that will favour multiplication of the bacterium. In the case of fire blight bacteria colonize the surface of the stigma in the flower (Figs. 108-110) when petals are open. As many as 10,000 bacteria can be present on one stigma. Multiplication can occur between 4-32ºC, but most rapid multiplication occurs between 24-29ºC. Nectaries (Fig. 110) must be open and functional (before petal fall) for infection to occur. They contain organic acids and sucrose that can be used by bacteria for multiplication. Flowers have an expected lifetime and are susceptible to infection for about 4 days. Furthermore disease development is dependent on: •

Wetting (rainfall, dew, spraying) and (soil) moisture

•

Susceptibility of the tree variety, age, vigour, and the number of blossoms present

•

Inoculum sources in the area: overwintering cankers, new disease, colonized flowers

•

Potential for bacterial growth in blossoms

•

Presence of insects

•

Dry winds (spread of bacterial strands)

•

Temperature (especially high temperatures during bloom)

Prediction is based on monitoring of maximum, minimum, and average daily temperature, wetting events in the form of rain, dew or spraying, the presence of blossoms and the weather forecast for the next couple days. Both in the USA and Europe several systems, such as MaryblytTM (Steiner, 1990), BIS (Billing’s integrated system, Billing 1999) have been developed. MaryblytTM e.g. predicts potential risk of blossom blight infection by E.amylovora based on the occurrence of certain conditions in sequence: •

Presence of open blossoms

•

Temperatures that induce multiplication of bacterial cells after colonizing stigmas

•

A wetting event with dew or 0.025 mm of rain after the heat units are accumulated. If 0.25 mm of rain or more falls the day before the heat units are accumulated, bacteria from the stigma can be washed down into the nectaries where the cells can start to multiply and cause disease

•

To facilitate further multiplication of cells after entering the nectaries, the average daily temperature should be at least 15.5ºC

These disease-forecasting systems have helped in fine-tuning spraying schemes with copper and streptomycin and have helped in avoiding development of resistance. They are rather accurate for prediction of blossom blight in apple and pear in many areas, but less reliable for fire blight in other hosts and shoot blight.

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EPIPHYTIC POPULATIONS (BACTERIA LIVING ON DIFFERENT PLANT PARTS IN A SAPROPHYTIC FORM) PLAY AN IMPORTANT ROLE IN SURVIVAL AND DISEASE DEVELOPMENT Fig. 108 Epiphytic colonization of ‘Jonathan’ apple (Malus) blossoms during a rainless period as detected by stigma imprints in 1996 and 1997 in a 2.5 ha orchard. Oozing cankers provided natural inoculum. Incidence of colonized flowers increased from near 0 to 100% in only 2 days. Fire blight occurred 10 days after the first detection. After Vaneste (2000).

Fig. 109 Flower of apple (Malus sp.).

Fig. 110 Anatomy of a flower of apple (Malus sp.). Erwinia amylovora can enter through the nectaries and start multiplication even in the high sucrose content of these glands, from where they start to spread in the plant. Bacteria can also colonize the stigma. See Vaneste (2000).

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3. Dissemination and transmission of the pathogen and epidemiological cycles The dispersal of bacterial diseases is by the bacteria themselves (either embedded in slime or not) or by diseased plant parts containing bacteria. For dispersal of bacteria wind, splashing rain (e.g. Roberts, 1997), surface water, irrigation water, insects, mites, other animals and humans (and their implements) are important (Bashan, 1985). Bacteria may spread from the epiphytic or resting state or from active lesions (Fig. 106). Plant parts important in disease dispersal are true seeds (this way of dispersal is especially important for vascular pathogens, which may infect the seed externally and internally, see Table 8, but also non-vascular pathogens that contaminate and infect the outer seed coat, such as P. s. pv. pisi, glycinea and tomato), tubers (e.g. Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum), bulbs (e.g. X. hyacinthi), transplants (e.g. R. solanacearum, vegetatively propagated material (Agrobacterium tumefaciens), tissue culture material and plant residues after harvest. Insect transmission is usually non-specific, but in some cases it is specific for the fastidious bacteria (leafhoppers). Examples of specific transmission are that of the xylem-limited Xylella fastidiosa causing Pierce’s disease of grapevine by sharpshooters such as Dhomolodisca coagulata and Oncometopia nigricans; Spiroplasma citri by the beet leafhopper Circulifer tenellus and S. kunkelii, causing corn stunt, by Dalbulus maidis (Figs. 111-113). S. citri colonizes the salivary glands and adjacent muscle cells of the vector. The bacterium can also be found in the midgut epithelial and salivary gland cells. The vector acquires the bacteria through endocytosis (Kwon et al., 1999). Pollinating insects (mainly honeybees) are important in transmission of E. amylovora (Fig. 124). Transmission of Pantoea ananatis, causing centre rot of onion, by the tobacco thrip, Frankliniella fusca, was demonstrated (Gitaitis et al., 2003). Transmission of pathogenic bacteria can take place from vegetation period to vegetation period and within a crop from primary infected plants to healthy plants, and also by root contact. The transmission cycle can be continuous or discontinuous, i.e. interrupted by a saprophytic, epiphytic or resting phase (see Fig. 106). Bacteria are able to bridge the gap between vegetative growth of their hosts in several ways: − Infection or contamination of basic planting material (most bacterial plant pathogens, Fig. 137). − Infection or contamination of the seed (Table 8, Figs. 116-118 and 137). − Dispersal to and infection or contamination of permanent crops, especially pathogens that occur in tropical/subtropical areas. − Dispersal to (perennial) other hosts, including weeds. − Epiphytic or rhizosphere colonization of non-hosts, including weeds. Many bacterial plant pathogens (especially those causing leaf spot diseases) can survive in the epiphytic stage, under hydric stress also in so-called biofilms, where bacteria adhere closely to each other (Jacques et al., 2005; Monier and Lindow, 2003b). Examples of an epiphytic stage in the epidemiological cycle are that of Erwinia amylovora in flowers of hosts (Figs. 108110), Pseudomonas syringae pv. syringae of fruit trees and P. syringae subsp. savastanoi on olive (Olea europea) (Fig. 115). Rhizosphere colonization of non-hosts such as bean, corn and many weeds is known for R. solanacearum, especially race 1. − Survival in plant debris and volunteer plants (most bacterial plant pathogens). − Survival in vectors (Pseudomonas syringae pv. lachrymans in insects (Diabrotica spp.) and Clavibacter tritici and C. rathayi in the nematode Anguina spp.). E. amylovora is efficiently spread by honeybees to flowers of distant trees, up to 5 km. Biofilms are important for survival in insect vectors also (Newman et al., 2004)

A. Wayadande, Oklah. State Univ. USA

Chapter IV

A. Wayadande, Oklah. State Univ. USA

128

Fig. 112

Corn leafhopper Dalbulus maidis that transmits corn stunt Spiroplasma kunkelii .

The beet grasshopper Circulifer tenellus that transmits the wall-less Spiroplasma citri (causal agent of stubborn disease) in a propagative manner. The bacterium colonizes cells of the salivary glands and the midgut of this grasshopper. The bacterium has a negative effect on the longevity of the insect. Grasshoppers are also vectors of phytoplasmas.

R. Brlansky, Univ. of Florida, CREC, USA

Fig. 111

Fig. 113

Fig. 114 Spiroplasma kunkelii, causing corn stunt, in the phloem of an infected corn plant (Zea mays).

R.E. Davis, Mol. Plant Pathol. Lab., USDAARS, Beltsville, MD, USA.

Sharpshooter Oncometopia nigricans becomes systemically infected with Xylella fastidiosa, causing Pierce’s disease, and transmits the bacterium to grapevine.

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Survival in a saprophytic or resting state in the soil (Agrobacterium tumefaciens, R. solanacearum, Clavibacter sp.) or water e.g. Erwinia spp. (see McCarter-Zorner et al., 1984) and R. solanacearum. For R. solanacearum an intimate relation between some riparian weed hosts, e.g. bittersweet (Solanum dulcamara) and water contamination has been established. When the host is perennial (such as bittersweet) the bacterium may be found in the water even during cold (winter) periods. Survival in water without host present was shown to be not more than c. 30 days and sometimes even shorter due to antagonistic action of other organisms such as algae and cyanobacteria (Elphinstone et al., 1998; Wenneker et al., 1999, Fig. 119 and Table 23). Water contamination and subsequent irrigation usually leads to dramatic epidemiological development of disease (Fig. 123) as has also been shown for some medically important pathogens, such as Vibrio cholerae (Fig. 122). Dispersal via water can also be very important in modern hydroculture systems of plant production in greenhouses. It is unclear to what degree so-called viable but non-culturable bacterial cells (VBNC) exist in water and soil in an active resting state or as slowly dying populations (Grey and Steck, 2001; Van Elsas et al., 2001; Caruso et al., 2005). − Aerial transmission has been clearly established for some bacterial pathogens, e.g. Erwinia spp. (Quinn et al., 1980) and Xanthomonas albilineans, which causes one of the most devastating diseases (leaf scald) of sugarcane, Saccharum spp. (Daugrois et al., 2003). − Survival on machines, tools, stands in greenhouses, stores, packing material and other materials (Tables 22a, 22b and 24). Spread of diseases via contact is usually much less dramatic than via water (Fig. 122). Survival of bacteria on legs of migrating birds has been shown to play a role in the distant spread of Erwinia amylovora. Some disease cycles (both continuous and discontinuous) of plant pathogenic bacteria are presented in Figs. 124-128. Studying these cycles will reveal many obvious factors important for bacterial pathogen dispersal and survival.

Fig. 115 Development of epiphytic populations of Pseudomonas savastanoi pv. savastanoi on different organs of the olive tree (Olea europaea cv. Paesana) during the year (after Lavermicocca & Surico, 1987). Population expressed as average number of cells per cm2 (twigs or leaves) per g of fresh weight (drupes).

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PLANT PATHOGENIC BACTERIA ARE OFTEN (TRUE) SEED TRANSMITTED WHEN THE BACTERIUM IS DETECTED IN THE SEED, THIS DOES NOT NECESSARILY MEAN THAT THE DISEASE WILL APPEAR IN THE CROP! Fig. 116

A. Visser

Increase (left to right) in severity of symptoms of seed infection by Pseudomonas syringae pv. pisi in pea (Pisum sativum). Water soaking and white necrosis of seed coat. Usually many pea seed lots can be found to be contaminated, but epidemic occurrence of the disease is rare and is heavily dependant on climatic factors.

M. Schortichini

Fig. 117 Cauliflower seedlings (Brassica oleracea var. botrytis) with severe symptoms caused by Xanthomonas campestris pv. campestris, due to infection of the seeds. Seeds were sown in a greenhouse for transplants to be used in the field.

Fig. 118

M. Schortichini

Cotyledons of a seedling of cucumber (Cucumis sativus) with symptoms caused by Pseudomonas syringae pv. lachrymans that occurred in/on the cucumber seed. Actually a seedling test (where seedlings are produced in a greenhouse under optimal conditions for disease development) can be used to detect the bacterium in seed lots.

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131

IMPORTANCE OF WATER IN DISTRIBUTION OF BACTERIAL PATHOGENS IS OFTEN UNDERESTIMATED Fig. 119 Left:

The shadouf was used for irrigation along the River Nile in Egypt in pharaonic times, and is still used for that purpose along with modern engine-driven pumps. When surface water is contaminated with plant pathogenic bacteria all forms of irrigation using this water present a risk in the dispersal of disease. Center: Graph showing number of contaminated surface water locations (out of 20) in relation to water temperature in a waterway, where latently infected bittersweet (Solanum dulcamara) was present and the water was contaminated by the bittersweet with Ralstonia solanacearum. The drop in population numbers in autumn and rise in springtime were related to the minimal growth temperature of the bacterium of c. 15ºC (see lower left). The drop in population during summertime was related to algal bloom and subsequent massive death (green and black arrow). Lower right: Growth temperature curve of R. solanacearum (after Gäumann, 1951). Compare with graph shown in center, where 15ºC is a key temperature in growth of the population in water.

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Fig. 120 Right

Monitoring of populations of Ralstonia solanacearum in traditional production areas in the Nile Delta in Egypt yields important data for pathogen control and knowledge of its distribution. (top and bottom) Pivot irrigation as in desert areas of Egypt, especially when using deep soil water, is an ideal way of producing disease-free potatoes and other crops.

NIVAP, The Hague, NL

Left:

Fig. 121 When surface water is checked for contamination with bacterial plant pathogens, industrial and municipal sewage plants deserve special attention.

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133

CHOLERA EPIDEMIC IN HAMBURG, 1892-93: CLASSIC EXAMPLE OF THE IMPORTANCE OF (DRINKING) WATER IN DISPERSAL OF PATHOGENS AND OUTBREAKS OF DISEASE

Fig. 122 An epidemic outbreak of cholera (caused by Vibrio cholerae) occurred in 1892 in the city of Hamburg, Germany. The graph shows: a) Typical water epidemic curve (red): very sharp rise when the organism appears in the water and very steep decline when the organism is eliminated, with some longer remaining contact epidemic cases afterwards (red blocks). At that time Hamburg was taking drinking water without filtration from canals of the River Elbe a little above the town and it was shown that through tidal movement in the river, (V. cholerae-contaminated) harbour water could reach the inlet providing drinking water. In addition, passing ships could have contaminated this water, leading to the catastrophic epidemic. b) Typical contact epidemic curve (yellow and yellow blocks) in the suburb Altona, where water played no role, thanks to a separate drinking water system. In Altona, therefore, people became infected only through contact when they moved to Hamburg city, or later in Altona itself. After Kolle & Hetsch (1916).

Chapter IV

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134

Fig. 123 Top

Dispersal of Xanthomonas malvacearum, causing bacterial blight of cotton (Gossypium malvacearum) by irrigation water in a pivot system. On May 12 irrigation was locally applied and 10 days later the first infections observed (A). On May 22 the whole field was irrigated, starting from the top (B) and on June 2 the whole field below the first infection foci was infected (more than 50% of the plants). After Gäumann (1951). Lower right:So-called ‘bird’s eye’ symptom on tomato fruits caused by stomatal infection by Clavibacter michiganensis subsp. michiganensis after sprinkler irrigation in a greenhouse where primary infected wilting plants were present. Lower left: Bacteria can easily be spread in the rows by hands and tools during manipulations such as pruning from primary infected plants. Wilting tomato, infected by Ralstonia solanacearum.

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135

DISEASE CYCLE OF FIRE BLIGHT (ERWINIA AMYLOVORA) ON APPLE, PEAR AND SOME OTHER ROSACEOUS HOSTS

Fig. 124 In (A) honeybees transmit bacteria to flowers, where they subsequently penetrate nectarthodes (nectaries) and leaves through wounds and stomata and start multiplication and spread through tissues (B, C). The infected flowers become black, shrivel and die (D) and the infection spreads to other flowers, leaves and twigs of the same tree (E). Disease becomes extensive (F) and cankers are formed on branches and stems (G). Eventually whole twigs or trees are diseased and killed (H, I). Bacteria may overwinter at margins of old cankers (J) that may enlarge and girdle trees (K). Bacteria in exudates on cankers can be spread by insects and rain (L) to flowers again and from flowers is also direct infection of young twigs possible (M), where spread to larger branches and stems can take place. Long-distance spread of bacteria in exudates on legs of birds has become very plausible for fire blight (N). After Goto (1992a).

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DISEASE CYCLE OF HALO BLIGHT (PSEUDOMONAS SYRINGAE PV. PHASEOLICOLA) OF BEAN (PHASEOLUS SPP.)

Fig. 125 Contaminated seeds (either internally or externally) are the main primary source of infections of halo blight in the field. Disease development and epidemic behaviour is, however, strongly dependant on climatic factors. This means that seed infections do not necessarily lead to substantial disease development in the field. This also holds true for infections of P. syringae pv. pisi (pea blight) in pea (Pisum sativum). After Goto (1992a).

Epidemiology

137

DISEASE CYCLE OF BROWN ROT (RALSTONIA SOLANACEARUM BIOVAR 2, RACE 3) IN TEMPERATE AND MEDITERRANEAN CLIMATES

Fig. 126 Infected (seed) tubers (A) are a primary source of infection, together with contaminated irrigation water and the weed host bittersweet, Solanum dulcamara, growing along waterways (E and DE). Through translocation and industrial/household processing (sewage!) or planting of diseased tubers further surface water or fields and plants may become contaminated or infected (B, C, D, E). Latently infected bittersweet (E) contaminates surface water (DE) that when used for irrigation (F) may lead to infection of new crops and contamination of machines (G). The infected tubers (H) that contain masses of bacteria in the vascular tissues (H1) can produce wilting plants (J) that produce (latently) infected daughter tubers and that can contaminate soil (I), especially the rhizosphere (L). Through leaching of bacteria to surface water, this water and eventually bittersweet become contaminated/infected (M) and the circle is closed. New infected crops can contaminate stores, grading belts, packing material and machines (N).

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DISEASE CYCLE OF CITRUS CANKER (XANTHOMONAS AXONOPODIS PV. CITRI) ON CITRUS SPP.

Fig. 127 In (A) infections from hold-over cankers and/or lesions from latent infections form a primary inoculum source that give rise to infections on spring shoots that can become hold-over lesions (B), that can again lead to new shoot infections later in spring (C). In later stages infections on fruits also occur (D). During later stages of the season secondary infections to other leaves, twigs and fruits may occur, often enhanced by humid climatic conditions and damage caused by leaf miners such as Phyllocnistis citrella (E). Survival during wintertime may take place in/on old leaves, soil and weeds. Windborne rain, land mowers and landscaping equipment, birds, people and young planting material are important in dispersal of the pathogen. The disease development is especially extensive on young trees; epidemics on mature trees are relatively sporadic. Highly susceptible are grapefruit (Citrus x paradisi), Mexican lime (C. aurantiifolia), sweet lime (C. limettoides), and trifoliate citrus (Poncirus trifoliata), sweet orange cultivars Navel, Hamlin, and Pineapple (C. sinensis); moderately susceptible are sweet orange cultivars except Navel, Hamlin, and Pineapple (C. sinensis), sour orange (C. aurantium), lemon (C. limon), and tangelo (C. x tangelo) and pummelo (C. maxima); susceptible are tangerine, mandarin (C. reticulata), Persian lime (C. aurantiifolia). Resistant are citron (C. medica), calamondin (x Citrofortunella) and kumquat (Fortunella spp.). After Goto (1992a).

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139

DISEASE CYCLE OF BACTERIAL LEAF BLIGHT (XANTHOMONAS ORYZAE PV. ORYZAE) IN RICE (ORYZA SATIVA) IN JAPAN

Fig. 128 Bacteria surviving in the rhizosphere of the weed host Leersia (A) will cause new infections in Leersia shoots growing near irrigation canals (B). From Leersia the bacteria are dispersed in irrigation water to lowland nurseries and transplanted in paddy fields (C) where they infect rice seedlings (D). When young plants are heavily infected the so-called kresek symptoms (severe wilting) develop (E), or otherwise the normal symptoms of leaf blight (F). Bacteria do not survive free in the soil, but they do on stubs and in straw. In the crop further (secondary) infections may occur through hydathodes and wounds especially after flooding of the rice fields and/or rainstorms. After Goto (1992a).

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Chapter IV

4. Geographical distribution of some bacterial plant pathogens Distribution of plant pathogenic bacteria is influenced by: • • • • • •

Distribution of host plants of the bacterium. Local climatic conditions. Trade activities. Presence of vectors (e.g. insects, migrating birds). Resistance of local varieties of host plants. Adaptation ability of the pathogenic bacterium to local conditions, including the ability to infect and colonize new hosts.

Geographical distribution of some plant pathogenic bacteria is presented in Figs. 129-133. Studying these distributions maps reveals the factors important for bacterial pathogen dispersal!

Fig. 129 Distribution map of Erwinia amylovora causing fire blight of apple and pear and some other rosaceous hosts. The dispersal of this bacterium is linked to occurrence of its hosts and to a temperate/subtropical and humid climatic area. Remarkably it was first observed in the USA, as early as the late 18th century in a devastating form on cultivated pear and apple. These hosts possibly became infected by populations already occurring on some indigenous plants such as hawthorn (Crataegus) and mountain ash (Sorbus) in the Hudson Valley. Local spread occurred by planting material, honeybees and wind-driven rain with epiphytotics in California. From the USA it has spread, possibly with planting material, fruits and/or migrating birds to other parts in the world. Fire blight was first found in Europe in the UK in 1958. From the UK there was rapid dispersal from 1966 over a period of c. 35 years to large parts of Europe, most recently to Italy and the Balkan countries (Jock et al., 2002). It has not yet been observed in centres of origin of apple and pear (Central Asia). Resistance has not been found in wild varieties. A very similar, but different bacterium, namely E. pyrifolii has been described and found so far only in Japan and South Korea. No races (pathotypes) of E. amylovora have been observed; the bacterium is very homogeneous in pathological, biochemical and genetic and serological characteristics. Due to its rapid spread and devastating attacks E. amylovora has been placed on quarantine lists (see Annex 3).

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Fig. 130 Distribution map of Xanthomonas oryzae pv. oryzae causing bacterial blight of rice (Oryza sativa). The dispersal of this bacterium is clearly linked to occurrence of its main host. It is endemic in India and S.E. Asia (where rice originated), also on wild rice species and on some weeds such as Leersia spp. Broadest resistance is found in (wild) rice varieties. X. o. pv. oryzae spread to parts of North and South America, Africa and Australia, possibly in seeds, only after 1965. Rice has been known in S.E. Asia since very ancient times, but was introduced in Africa only since the 16th century and America only since the 18th century. Local rice species (O. barthii, O. breviligulata) in Africa are apparently not susceptible. Many different races (pathotypes) of X. o. pv. oryzae exist, distinguished by their behaviour on different cultivars. There are broad-range pathotypes occurring over a large geographic area and others that are localized to one area such as pathotypes from Japan (island isolation). Seed infection is not common, but is, however, possible and therefore the bacterium has been placed on quarantine lists (see Annex 3).

Fig. 131 Distribution map of Xanthomonas axonopodis pv. citri causing bacterial canker of Citrus. The dispersal of this bacterium is clearly linked to occurrence of its main hosts (C. paradisi, C. aurantiifolia and Poncirus trifoliata) and to a tropical/subtropical area, especially where rainfall is heavy (>1000 mm per year). It is endemic in the source area of Citrus, (Indo-)China and prevalent in Asia: so-called Asiatic (wide-host range) strains and AsiaticW(est)(narrow-host-range) strains. From Asia it spread to other parts of the world (since the 15th-16th century). Resistance has been found especially in C. mitus (calamondin) and Fortunella (kumquat). C. reticulata (mandarin) is tolerant. Citrus canker was first introduced in the USA with trifoliate orange seedlings from Japan around 1910 and eradicated, following a very intensive campaign where thousands of trees were burned, in 1933. In 1984 it was thought that the disease was reintroduced, but this so-called bacterial spot was caused by a different bacterium (Xanthomonas campestris pv. citromelo). However, in 1986 the Asiatic citrus canker was indeed reintroduced in Florida. A closely related bacterium, X. a. pv. aurantifolii, also causing canker symptoms, has been found in South America. Due to the damage caused, the bacterium has been placed on quarantine lists (see Annex 3).

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Fig. 132 Distribution map of Clavibacter michiganensis susbsp. sepedonicus causing bacterial ring rot of potato (Solanum tuberosum). The dispersal of this bacterium is clearly linked to occurrence of its only host and to a cool climatic area that is defined by the low optimum growth temperature (21ºC) of the bacterium. It does not occur in the source area of potato (Andes mountains in South America) and resistance has not been found in wild varieties. Potato was introduced to Europe c. 1550 and cultivated since the mid-18th century (as well as in N. America and Asia). Ring rot was first observed in Germany around 1910 from where it spread most likely with seed to North America and other parts of Europe. Rapid spread in N. America occurred from 1931-1950 through seed and cutting of seed. Ring rot disappeared for a number of years in Germany but reoccurred 1980s for unknown reasons. No races (pathotypes) have been observed; the bacterium is very homogeneous in pathological, biochemical, genetic and serological characteristics. Due to the fact that infections are often latent in seed potatoes, the bacterium has been placed on quarantine lists (see Annex 3).

Fig. 133 Distribution map of Ralstonia solanacearum (Rsol) race 3, biovar 2 causing bacterial brown rot of potato (Solanum tuberosum) and bacterial wilt of tomato (Lycopersicon esculentum) and some other (solanaceous) hosts. The dispersal of race 3 is mainly linked to occurrence of its main hosts, potato and tomato, and to temperate climatic areas or cooler regions (mountainous areas) in the tropics due to its relatively low optimum growth temperature (27ºC). Rsol does occur in the source area of potato (Andes mountains in South America) and resistance has been found in wild varieties. Race 3, biovar 2 was first observed in the Mediterranean area, notably in Egypt and Portugal, perhaps introduced with potatoes shipped from S. America with allied troops during WWII. It was first observed in N.W. Europe in Sweden in 1976, with a link to potato industries that dumped untreated waste from infected potatoes from the Mediterranean area. This type of disease spread has lead to incidental outbreaks of brown rot in potato in different N.W. European countries and spread of the bacterium in surface water, especially when bittersweet (S. dulcamara) is growing along the waterways. Race 3 shows variation in its centre of origin in S. America but not in other parts of the world, indicating (clonal) spreading with potato seed or waste. Race 3, biovar 2 is very homogeneous in pathological, biochemical, genetic and serological characteristics as opposed to race 1. Due to the fact that infections are often latent in seed potatoes the bacterium has been placed on quarantine lists (see Annex 3).

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143

CHAPTER V - DAMAGE AND LOSSES CAUSED BY BACTERIAL PLANT DISEASES 1. Damage a. Reduction of assimilating surface by yellowing and necrosis This (often minimal) damage is typical for leaf spot diseases. In the field unfavourable climatic conditions (cold or warm and especially dry weather) exert their influence rapidly in the relatively thin leaves and the progression of disease stops. However when favourable temperatures and high humidity persist damage will increase. When many spots occur, leaves, fruits or young plants may be killed completely (Fig. 135). b. Death of organs or complete plants Death of organs is often caused by opportunistic pathogens, which cause rapid necrosis. Pseudomonas syringae pv. syringae for example may kill blossoms, leaves, buds and twigs. Complete killing of plants is mainly caused by primary pathogens, which cause rapid necrosis (e.g. Erwinia amylovora, Pseudomonas avellanae) or wilt diseases (e.g. Ralstonia solanacearum). Wilting diseases can be very devastating resulting in damage and high crop losses, even up to 100% (Fig. 134 and 137), e.g. when environmental conditions are conducive for Xanthomonas albilineans infections and susceptible varieties are grown, latent infections of that bacterium form into acute, epidemic disease and cause severe damage to plants and losses of more than 20% to the grower (Rott et al., 1995). Opportunistic pathogens, such as Erwinia carotovora subsp. carotovora can cause severe damage when favourable conditions prevail, such as wounded plants and high humidity and high temperature. c. Malformation and growth reduction This kind of damage is found with the diseases mentioned above, but especially with tumours caused by Agrobacterium tumefaciens. Exceptionally up to 50% growth reduction has been reported. In most cases malformation is the only (aesthetic) damage. Severity of damage caused by bacteria is dependent on environmental conditions (see Chapter IV.1). Damage can be reduced by the phenomenon of compensation by healthy neighbouring plants (Adams and Lapwood, 1983). In many cases bacterial diseases cause considerable damage due to their sudden outbreaks, lack of good bactericides and/or lack of resistant varieties. Examples are the epidemic occurrence of Xanthomonas oryzae pv. oryzae in rice in the Punjab area, India, in 1980 and of fire blight, caused by E. amylovora (Fig. 136).

2. Losses Losses caused by bacterial diseases are mainly economic, but may also be personal or aesthetic. Most direct loss for the grower is yield loss, which may be considerable. When healthy plants are removed around a diseased one, the consignment is put in quarantine or export stopped (due to quarantine regulations), there will be of course an even bigger impact. Man may be personally affected when social or psychic disturbances or suffering from hunger will be the result of destruction of crops. When trees are killed or have to be removed (e.g. hawthorns in nature reserves, due to fire blight) aesthetic losses to the landscape may occur. In Table 25 estimated losses due to bacteria in the USA are presented.

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Fig. 134

M. Schortichini

PD

IN THE GREENHOUSE ENVIRONMENT MAN OFTEN CREATES CONDITIONS FAVOURABLE FOR PROBLEMS WITH AND LOSSES DUE TO BACTERIAL DISEASES

In the greenhouse environment manipulations during cultivation (where plants often are wounded), lack of hygiene, climatic and nutritional conditions (high humidity and temperature, high N nutrition) and water management (overhead flood irrigation) cause rapid spreading of disease and extensive damage and losses. Top: Spread of bacterial canker (Clavibacter michiganensis subsp. michiganensis) in rows of tomato (Lycopersicon esculentum) by greenhouse workers. Centre: Severe crop loss due to epidemic infection of Chrysanthemum by Pseudomonas cichorii, causing stem blight, under conditions of high N fertilization, close planting and high humidity. Bottom: Similar conditions plus wounding of plants lead to a devastating disease development in courgette (Cucurbita sp.), caused by an opportunistic pathogen: Erwinia carotovora subsp. carotovora.

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Fig. 135

M. Schortichini

PD

WHEN SEEDS OR CUTTINGS ARE (LATENTLY) INFECTED MUCH DAMAGE AND HEAVY CROP LOSSES MAY OCCUR

When seeds, transplants or cuttings are externally or internally contaminated with pathogenic bacteria this may lead to heavy losses under favourable conditions for disease development. Top: Symptoms of black rot in cabbage (Brassica oleracea) seedlings. Centre: Severe infection by Xanthomonas axonopodis pv. phaseoli in bean (Phaseolus vulgaris) through infected seeds. Bottom: Severe damage and losses due to infection by Xanthomonas hortorum pv. pelargonii of Pelargonium zonale cuttings that were taken from diseased mother plants.

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Fig. 136 Erwinia amylovora causes rapid necrosis of the phloem of its hosts and is able to cause devastating infections in wild hosts such as Crataegus (top left), fruit crops such as pear (Pyrus sp., top right) and apple (Malus sp., bottom right) and ornamental hosts (such as Cotoneaster and Crataegus) in nurseries (bottom left). In cultivated hosts heavy losses may occur.

PD

PD

PD

PD

ERWINIA AMYLOVORA, CAUSING FIRE BLIGHT, RAPIDLY KILLS ITS HOSTS AND CAN GIVE RISE TO MUCH DAMAGE TO HOST PLANTS AND HEAVY CROP LOSSES FOR THE GROWER

Damage and losses

Table 25

147

Losses due to bacteria, phytoplasmas, spiroplasmas and FXLB in the USA in 1976. Kennedy & Alcorn (1980).

Pathogen or disease Agrobacterium tumefaciens1) Clavibacter michiganensis subsp. insidiosus C. m. subsp. nebraskensis C. m. subsp. sepedonicus Erwinia amylovora E. chrysanthemi Erwinia soft rots Lethal yellowing of coconut (phytoplasma) Pear decline (phytoplasma) Phony peach (Xylella fastidiosa) Pierce’s disease (Xylella fastidiosa) Pseudomonas syringae pv. glycinea P. s. pv. phaseolicola P. s. pv. syringae Ralstonia solanacearum Ratoon stunt of sugarcane (Clavibacter xyli) Spiroplasma, corn stunt Spiroplasma, Aster yellows Spiroplasma, Citrus stubborn Xanthomonas arboricola pv. juglandis X. a. pv. pruni Xanthomonas axonopodis pv. malvacearum X. a. pv. phaseoli Xanthomonas campestris pv. campestris X. translucens pv. translucens

23 17 3 1.8 5 2.3 14 3 1.6 20 3 64 2 18 9 10 0.06 0.2 1.0 2.2 2 5 5 1 1

Mainly due to export problems

M. Schortichini

1)

Losses in million US dollars

Fig. 137 Heavy crop losses may occur with plants that are grown in the field when climatic and cultural conditions are suitable for dispersal of the bacterial pathogen and disease development: severe symptoms of bacterial canker caused by Clavibacter michiganensis subsp. michiganensis in tomato (Lycopersicon esculentum) grown in the field.

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Fig. 138 Exquisite medieval miniature painting of the Limbourg brothers: The month March and the Château (castle) of Lusignan in France. The picture shows farm activities of early spring: ploughing, sowing and trimming vines. It demonstrates the interrelationship between plant protection and those active in this field (castle) that protect the crops and those producing the crops (farmers). On one hand the castle protects the farmers, but on the other hand the farmers keep the inhabitants of the castle alive! The celestial blue sky indicates that man can control many, but not all (climatic) factors. From: Book of hours ‘Les très riches Heures’ du duc de Berry (14131416).

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CHAPTER VI - PREVENTION AND CONTROL OF BACTERIAL PATHOGENS AND DISEASES 1. Principles of control of plant pathogenic bacteria and/or the diseases they cause Plant pathogenic bacteria do not form endospores. In principal, therefore, they can be easily controlled. However, only a few effective (systemic) bactericides are commercially available. Moreover certain antibiotics are not allowed to be used in plant health in many countries. Due to the (often) epidemic occurrence of bacterial diseases, the quarantine status of many of them and the substantial losses caused, control is often regulated and executed by governmental bodies. In practice it is tried to reduce damage of bacterial diseases by a combination of protective and control measures in a preventive way. Rules for efficient control of bacterial plant pathogens and diseases can be based on Robert Koch’s control system as developed at the end of the 19th century for the successful eradication of cholera. These rules may be interpreted for control of plant pathogenic bacteria as follows: - Main responsibility for controlling the disease lies with the country where infections occur. - Statistically meaningful surveys should be performed to assess presence or absence and eventual distribution of the disease. - The country involved should report as soon as possible when the disease is found. - Rapid and sure means of detection and diagnosis are vital. - Countries should try to contain and control the disease as soon as possible by: a) Holding action on infected crops or lots when appropriate b) Measures on contaminated fields and/or premises and imposing hygienic protocols (e.g. exclusion of contaminated fields for a number of years, control of volunteer plants, wild hosts and nematodes and adequate crop rotation periods). c) Tracing origins of infection (e.g. in the case of potato brown rot and ring rot diseases: clonal relationships of infected crop, trade lines, contaminated surface water). d) Checking for infections (including latent populations) before movement and trading. e) Surveys on original host and all other potential (wild) hosts. f) Establishing epidemiological risk factors and taking action upon them (e.g. for potato brown rot prohibition or discouragement of use of contaminated (surface) water or use of safe sources of surface water, if this plays a role in the disease cycle). g) Imposing safe disposal of any plant waste that may be contaminated with the pathogen. - Inspections by importing countries should be carried out. - Growers, traders, plant protection services (including policy bodies, inspectors, laboratory personnel) and public must be educated in risk analysis, avoidance and control strategies. - Production and use of healthy planting material, e.g. by indexing, and also for latent infections, developing/using resistant varieties, chemo- or thermo-therapy of basic planting material.

1)

For more information see Vidaver (1983) Hoitink and Fahy (1986); Lindow (1986); Kleinhempel et al. (1989); Cooksey (1990); Goto (1992a); Binns et al. (2000); Staskawicz (2001); Janse and Wenneker (2002); Goodman (2004); Janse (2004b); Tripathi et al., 2004; Nester et al., 2005.

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INTEGRATED CONTROL: THE ONLY WAY FOR BACTERIAL DISEASES FACTORS IMPORTANT IN INTEGRATED CONTROL OF BACTERIAL WILT, RALSTONIA SOLANACEARUM

Table 26

Factors1) important in an integrated control strategy for bacterial wilt/brown rot caused by Ralstonia solanacearum race 1 or race 3.

Factor to formulate control strategy

Race 1

Race 3

Resistance or tolerance of variety used

2

3

Cold climate

1

2

Healthy seed

3

3

R. solanacearum-free soils

7

7

Suppressive soils

2

4

Short rotation

1

4

Intercropping

2

3

Date of planting

1

3

Nematode control, resistance

4

2

Dry and or heat soil

3

2

Solarization

1

1

Rouging volunteers

2

4

Rouging wilted plants

1

2

Fumigants

3

5

Control of spread in water

3

3

Minimal till

2

1

Soil amendments

1

1

Weed host control

3

2

1)

Factor weights range from 1-7 and may be changed, according to the regional conditions. According to French a sum of 10 would usually be adequate for good control or even eradication. After French (1994).

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2. Prevention of introduction and dispersal of bacterial plant pathogens after interception by quarantine measures and legislation Long-distance movement of phytopathogenic bacteria For bacteria, the most important mechanism of global movement is international trade of plants, seeds and plant parts. With new free-trade agreements between countries in different parts of the world, the risk of introducing exotic pests is increasing. Furthermore, new, difficult to trace pathways are developing. For example, commercial companies produce basic planting material outside their (safe) regions, in regions with higher risk for contamination and under different (less safe) disease management conditions, and then introduce the products into their regions for further propagation and sale. This has led to the introduction of Ralstonia solanacearum race 3, biovar 2 with Pelargonium cuttings from Kenya into Europe and from Guatemala into the USA. Other possible pathways for the introduction of exotic phytopathogenic bacteria are the use of non-indigenous pollinating insects and biological control agents. It is suspected that Liberobacter asiaticum was introduced into Florida (USA) with parasitoids used for the control of the Asian citrus psyllid, Diaphorini citri. Certain biological control agents (including R. solanacearum, used for the control of the forest weed, Hedygium gardnerianum in Hawaii) may attack cultivated hosts (Anderson and Gardner, 1999). Approaches to prevent introduction of phytopathogenic bacteria Quarantine regulations are mainly aimed at excluding phytopathogenic bacteria from a territory. These may be laws, orders or decrees that limit the import of plants or plant products and specify the pathogens of interest. More specifically, quarantines facilitate the isolation and inspection of plants and plant products for prohibited organisms. This is only used for small-scale importations, or germplasm sent to post-entry quarantine stations for breeding or research purposes. Inspection at the point of import, is not adequate due to latent symptom development and undetectable levels of bacteria on planting material. If the host plants are not prohibited, then phytosanitary regulations are based on ensuring that the imported plants are symptomless at the point of origin, as certified by the National Plant Protection Organization (NPPO) of the exporting country. An important principle for ensuring that commodities are pathogen-free is the concept of the pest-free area, which is assigned when a disease has not been observed in a certain country or part of that country. A pest-free area can be created and officially recognized according to international standards, and commodities can be freely exported from it. A similar concept is the ‘protected zone’ that also must be free of a particular bacterium, but must be especially protected from introduction by stricter phytosanitary measures than adjoining areas. Examples of protected zones can be found in Canada and Europe for Clavibacter michiganensis subsp. sepedonicus and Erwinia amylovora, respectively. If a pest-free area cannot be established or maintained, phytosanitary regulations may allow plants or plant products to originate from a production site that has been free from the particular bacterium for a defined period of time. Quarantine regulations should also include the eradication or containment of introduced bacterial pathogens. Additionally, they should be used to ensure the production of healthy planting material by certification schemes and standard production protocols. Other phytosanitary measures applied to host plants include requirements that the previous generation of the plants, or seeds of the commodity be free of the pathogen, and that planting materials be tested, or subjected to physical or chemical eradicative treatment. Furthermore, the exchange of bacterial strains from culture collections is subject to greater regulation, mostly at the national level, but also through the World Federation for Culture Collections.

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INTEGRATED CONTROL: THE ONLY WAY FOR BACTERIAL DISEASES FACTORS IMPORTANT IN INTEGRATED CONTROL OF CITRUS CANKER

Table 27

Factors important in integrated control of citrus canker (Xanthomonas axonopodis pv. citri)

Production of X. a. pv. citri free nursery trees, indexing of budwood Choice of planting site, where strong winds do not prevail and with no citrus canker history Planting of (field) resistant cultivars Prohibition of planting highly susceptible cultivars Establishment of wind breaks in orchards Hygienic protocol for nurseries and orchards Restricted access to orchards Preventive copper sprays Disinfection of boots, implements, machines and packing material Pruning or defoliation of infected shoots during dry periods Planting of sentinel trees and monitoring/survey programs Leaf miner control Removal of plant debris and soil residue Limit exchange of personnel and machines between blocks Irrigation at times when workers are not in groves Hygienic protocol in packing houses Eradication program, destruction of infected trees and buffer zone around orchard Import restrictions and other quarantine measures Avoid dumping of waste in non-approved places, do not dump near orchards or packing stations

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Creation of regulations Sovereign states, under the International Plant Protection Convention and the Sanitary and Phytosanitary (SPS) Agreement, have the right and responsibility for preparing phytosanitary regulations to protect themselves against exotic plant pests, including bacterial diseases and pathogens. The Interim Commission on Phytosanitary Measures, established by the International Plant Protection Convention (IPPC), develops International Standards on Phytosanitary Measures. These standards establish the principles and procedures of phytosanitary measures, ensuring that countries develop their measures consistently and fairly. In addition, most countries belong to regional plant protection organizations (RPPOs, see Annex 3a-c) that coordinate and harmonize the phytosanitary actions of their member countries on a regional basis. With regard to bacterial pathogens, the European Plant Protection Organization (EPPO) advises its members which bacteria should be quarantine pests, dividing them into A1 quarantine pests (those absent from the region and against which all countries are recommended to take phytosanitary action) and A2 quarantine pests (those present in some parts of the region and of concern to only some countries) (see Annex 3). In the EPPO region it is generally thought that bacterial plant pathogens are no good candidates for bioterrorism acts and they are not part of serious biosecurity considerations. In the USA R. solanacearum biovar 2, race 3 has been placed on a bioterrorism list (Schaad et al, 2003). It is not easy to understand why this pathogen should be placed on such a list, knowing that race 1, with a much wider host range, is endemic in the southern states of the USA. The European Community (EU) issues Control Directives for some quarantine pathogens that include obligatory official testing schemes, standardized and harmonized for the whole EU region (Annex 6a and b, Anonymous, 1998).

3. Control aiming at eradication Control of bacterial diseases can be directed at a number of levels as follows: - Eradication of the pathogen (every single individual). - Eradication of the disease (every disease incidence from the production line). - Functional eradication (occasional re-occurrence and immediate action accepted). - Area-wide suppression (low level of incidence accepted). Eradication of the pathogen and disease as required for quarantine pathogens often fails through a) incomplete pathogen eradication (hidden foci), b) natural reinvasion and c) reintroduction through short- or long-distance movement of infected material from contaminated areas. What remains is functional eradication or area-wide suppression. An example of reintroduction of an eradicated bacterial disease is Asiatic citrus canker (caused by Xanthomonas axonopodis pv. citri) in Florida. It was first introduced with trifoliate orange seedlings from Japan around 1910 and eradicated in 1933, following a very intensive campaign where thousands of trees were burned. In 1984, it was thought that the disease was reintroduced, but this so-called bacterial spot was caused by a different bacterium (X. campestris pv. citromelo). However, in 1986, Asiatic citrus canker was indeed reintroduced and reappeared despite carefully planned eradication and education campaigns in subsequent years along the Gulf coast of central Florida (Schubert et al., 2001). Another example where eradication campaigns have failed in many countries is fire blight of apple, pear and other Rosaceous hosts, caused by Erwinia amylovora where (re) introduction of the pathogen by migrating birds, insects (honeybee), wind-driven bacteria in slime and undetected infections in wild hosts played an important role (Vanneste, 2000).

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ROLE OF EDUCATION AND HYGIENE IN CONTROL OF DISEASES IS OFTEN UNDERESTIMATED AND NEGLECTED

NIVAP, The Hague, NL

R. Havlick, translated

DISINFECTION OF SHOES, BOOTS AND MACHINES IN GREENHOUSES AND STORES PREVENTS DISPERSAL OF BACTERIA

Fig. 139 Education and hygiene are very important factors in the control of bacterial diseases. Top: Warning for visitors on a place of production to disinfect feet first. Bottom: Cleaning and disinfection practice on a potato farm.

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An example where eradication of a bacterial disease has been successful is from a small island called Thursday Island near Queensland, Australia. Citrus was introduced by settlers since 1877 and the fruit was and is much used by local householders for juice on fish dishes. Citrus canker (Xanthomonas axonopodis pv. citri) was found in 1984 and removed from orchards and gardens by tracing and burning trees and strong hygienic measures, during a 4-year period. If only a stump of an infected tree was neglected and allowed to form new shoots the disease reoccurred. Eradication was feasible due to the very small scale of the island and a well-executed eradication program. It is one of the few cases where eradication of an alien disease from Australia had been successful. Eradication attempts for citrus canker on Christmas Island, a slightly larger Australian island, failed (Jones, 1991). A successful eradication of bacterial brown rot (caused by Ralstonia solanacearum) was achieved in Sweden. In 1972, infections were found in fields irrigated with river water. Upon investigation, the river water and Solanum dulcamara, growing along the river with their roots and parts of the stems in the water, were found to be contaminated or (latently) infected by Ralstonia solanacearum. The source of contamination/infection appeared to be two potato processing industries, that used potatoes coming from known brown rot contaminated areas, dumping unprocessed waste and waste water into the river. After measures were taken (such as eradication of bittersweet, prohibition of irrigation with surface water, taking contaminated fields out of potato production for a number of years, disinfection of premises and machines), the disease was eradicated over a 4-years period.

4. Prevention and control at farm or nursery level: the integrated approach Apart from governmental actions to avoid, prevent or control diseases the following actions can be taken by producers and traders or their organizations as single actions or in combination (integrated approach, also see Tables 26 and 27): a) Removal of plant debris. Bacteria can survive very well in plant debris (Fig. 141). b) Removal of volunteer plants and wild hosts, especially important for broad host range pathogens like R. solanacearum race 1. c) Disinfection or sterilization of soil, potting material, transport material, machinery (including grading machines, see Elphinstone and Pérombelon, 1986), cutting knives, greenhouses, etc (Figs. 139 and 140). In the case of heat sterilization, temperatures of 80oC should be reached for at least 60 min everywhere in the layers to be sterilized (Fig. 148). Quaternary ammonium compounds, formalin, 70% ethanol and chlorine compounds all have been found to have a good bactericidal action. The quaternary ammonium compounds are very sensitive to organic matter; other compounds may be corrosive (Tables 33 and 34). d) Crop rotation. Its duration is dependent on bacterial strains and host plant control in intermittent periods. Ralstonia solanacearum for example could be controlled by a crop rotation of 3-5 years in tobacco in North Carolina, USA, 7-10 years in tobacco in Sumatra, Indonesia, while a 2-year rotation with banana in South America was sufficient. e) Cultivation of plants in containers, eventually on concrete floors. f) Use of plant material tested for freedom of bacterial pathogens. g) Use of resistant, tolerant or less susceptible varieties.

Chapter VI

HYGIENE MUST NOT ONLY BE PRACTISED BY THE PRODUCER AND HIS WORKERS, BUT ALSO BY THE INSPECTOR AND THE TESTING LABORATORY

PD

Fig. 140 Top left and right Disinfection equipment in a greenhouse for workers that sample cuttings and a disinfection sink and mat that have to be used (disinfection of hands and feet, before entering the greenhouse). Centre Hygiene is practised when taking samples for (quarantine) diseases. The inspector wears a disposable overall, shoes and gloves. This in order to avoid dispersal of the pathogen from one lot to the other or from one production place to the other.

PD

156

Bottom When handling samples in the laboratory hygiene remains very important in order to avoid any possibility of crosscontamination between samples.

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h) Removal of diseased plants and plants parts after the growing season (Fig. 142). i) Other cultural measures such as removal of second bloom, good drainage, avoiding close planting and overhead irrigation and in greenhouses the lowering of humidity and (soil) temperature. j) Application of pesticide sprays (Chapter VI.9), eventually in combination with a disease forecasting system (page 125) or biological control agent (Chapter VI.8).

AWARENESS OF GROWERS, TRADERS, INSPECTORS AND LABORATORY PERSONNEL OF (HYGIENE) RISKS WILL ASSIST IN AVOIDING CONTAMINATION WITH PATHOGENS Table 28 Variable

List of hygiene variables important in control and prevention of bacterial diseases. After Janse and Wenneker (2002). Variable description

Wild host/volunteer control

Whether wild hosts/volunteers are mechanically or chemically controlled

Clean premises

Whether greenhouse/storage facilities are cleaned and disinfected before new plants are brought in

Clean machines

Whether machines (incl. conveyer belts for grading) and tools are cleaned and disinfected after each production cycle or when used in other premises

Trucks

Whether trucks are cleaned/disinfected before taking new loads or when used to remove infected material

Manure use

Whether manure is used from risk sources like potato processing industry, contaminated areas, cattle fed with risk crop

Water source

Surface water, deep soil water, tap water

Water system

Overhead irrigation, furrow, drip, tidal

Water treatment

Disinfection or not

Storage system

Cooled or not, ventilation or not

Greenhouse climatic system

Humidity, temperature, misting frequency

(Infected) waste disposal

Dump, burning, burial

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M. Schortichini

HYGIENE: REMOVAL OF HOST DEBRIS, IMPORTANT FOR REMOVAL OF INOCULUM

Fig. 141 Removal of host debris is an important hygiene factor in removing inoculum and controlling bacterial diseases.

PD

SAFE TRANSPORT OF INFECTED/CONTAMINATED WASTE IN CLOSED AND COVERED CONTAINERS FOLLOWED BY SAFE DISPOSAL PREVENTS FURTHER DISPERSAL

Fig. 142 Safe waste transport in closed and covered containers followed by safe disposal are important factors in control of further spreading of pathogens and diseases.

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SUCCESS OF SAMPLING AND TESTING IS LIMITED BY THE PROBLEM OF SAMPLING ERROR Probability of detection (%) as a function of sample size in lots of infinite size (random disease distribution).

Table 29

Disease incidence (%) Sample size

1.5

0.5

0.1

0.01

200 400 1000 2000

95 99.9 100 100

63 98 99 99.9

18 32 62 86

1.8 3.9 9.5 18

This statistical table shows that even taking relatively large samples (e.g. 2000 tubers out of 25 tons, N.B.: statistically a 25 ton lot is an infinite sized lot) still yields only a low probability of finding low disease incidences (18% for 0.01% disease incidence).

a

Table 30

Factors important in success of testing for latent infections.

Factor

G & Pa

Bb

Cc

Total freedom

+++

+/±

-

Sampling error

-

++

+++

Test sensitivity

+

+

+

Traceability

+++

++ b

-/± c

Germplasm and pre-basic material; Basic material; Consumable product.

From this Table it is clear that chances for total freedom from a pathogen and traceability becomes progressively less during multiplication from the small numbers in germplasm material to the production of vast numbers of a consumable product. Sampling error increases progressively.

Fig. 143 Risk evaluation in the potato production column. After Struik and Wiersema (1999).

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Fig. 144 Citrus variety improvement program through indexing as applied in Spain. After Navarro (1986).

Fig. 145 Meristem culture for the production of disease-free basic planting material of cassava (Manihot esculentum). From a selected mother plant (A) a stem cutting (B) is taken and a new plant (C) raised and kept at 35ºC to express symptoms of diseases, if present. From the plant a bud is selected that includes a growing tip or meristem (D). The meristem is aseptically removed (E) and placed on agar (F). From the meristem a new plantlet is raised (G, H). This new plantlet is indexed for diseases (I) and when found to be free of diseases further multiplied by taking a stem cutting (J). From this stem cutting a new plantlet (K) is raised that can be further multiplied (L). After Frison (1994).

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5. The role of education and hygiene Education of growers plays an important role in distribution, occurrence, establishment and/or eradication of bacterial plant diseases. Producers often have only little knowledge of disease epidemiology and economic consequences (and therefore often efficiently maintain and disperse pathogens in production systems). Plant breeders have more knowledge (although they often introduce pathogens in new areas or new variants in areas where the pathogen already occurs). In fact, this aspect is underestimated, neglected and often not admitted. Hygiene during the early and later stages of multiplication of basic planting material (and of plant material in general) is a very important factor in maintenance of pathogen freedom. This is usually better understood and practised by producers of basic planting material, and those dealing with veterinarian and human disease problems than by bulk producers, especially those active in arable farming. Some variables useful as a basis for hygiene in greenhouse and arable farming are presented in Table 28 and some examples given in Figs. 139-142. The use of surface water for irrigation or pesticide sprays, contaminated with Ralstonia solanacearum for example, clearly presents a risk of contamination of seed potato crops or tomato seedlings. In a classical case, two geographically separated plots were planted with different potato seed varieties for variety evaluation. One plot became infected with R. solanacearum due to irrigation from a contaminated river, whereas the other plot (not irrigated with the same water) remained completely free of the disease. In developing countries in the tropics, where soils often are heavily contaminated with Ralstonia solanacearum, the farmer may even reintroduce the disease by his feet or shoes and contaminated implements. Here again hygiene and education are main factors in disease control.

6. The role of healthy basic material and indexing/testing in control strategies Germplasm collected from nature or produced by plant breeders and further multiplied and dispersed by them forms an important source for dispersal of pathogens. Even though the awareness of plant breeders has increased in recent years regarding the risk of pathogen dispersal in plant material, much still has to be done. This is demonstrated by the results of testing pome fruit germplasm in quarantine programs of the Plant Germplasm Quarantine Office, Fruit Laboratory, USDA, over a period of 12 years (1986-1997). In these programs c. 54% of the 550 accessions were found to be infected with one or more viruses or phytoplasmas. There was not much change in this percentage over the 12-year period. When selecting mother plants for nuclear stock production, e.g. through meristem culture (Fig. 145), these plants as well as first-generation progeny should be subjected to as many tests for as many different pathogens as possible (indexing). In addition, they should be kept under quarantine conditions as long as possible. There should be special conditions and hygiene rules for places of production producing basic material. These are generally laid down in certification schemes. A good example for such a scheme is presented in Fig. 144 for health improvement in Citrus (through indexing and shoot-tip grafting) and checking of imported citrus germplasm (Navarro, 1986). Achievement of pathogen freedom through indexing by testing can take place at different stages in the production column and serve different purposes: germplasm and pre-basic material, basic material and consumption material. These stages differ concerning their possibilities for removing the disease, tracing back infected material and influence of sampling error (Tables 29 and 30 and Fig. 143).

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EVALUATION OF GENETIC MATERIAL FOR RESISTANCE AGAINST BACTERIAL DISEASE

RESISTANCE TYPE

RESISTANCE AGAINST

RESISTANCE DURING DIFFERENT

DIFFERENT PATHOTYPES

GROWTH STAGES

(MOLECULAR) INVESTIGATION OF GENETIC BASIS OF RESISTANCE

IMPROVED RESISTANCE SOURCES

DIFFERENTIATING VARIETIES

HYBRIDIZATION BLOCK

VIRULENCE OF PATHOTYPES

NEW RESISTANT VARIETY

Fig. 146 Principles of breeding for resistance.

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Furthermore the effectiveness of indexing (i.e. sampling) harvested product is determined by a) statistical probability based on lot and sample size and b) in the case of latent or epiphytic presence of the pathogen, it may also be based on test sensitivity. In ‘infinite’ lots (true seeds, tubers) the sampling factor is by far the most important. Both statistical probability and sampling factor determine that a zero occurrence can never be completely guaranteed. Table 29 shows that usually, with infection rates within the range of 0.1-1.5%, disease incidences can be found with a 95% probability, where the amount of sampling is still practicable. But other factors also determine effectiveness, such as: 1. When and where the samples are taken (e.g. sampling of tubers from the field is less effective than tubers from storage; in the case of ring rot and brown rot, due to less chance for progression of the pathogen into daughter tubers, random sampling is more difficult in big stores than when potatoes are stored in crates). 2. Maintenance of accurate trace-back records by the trade. 3. How the product (and its by-products and waste) flow through the system (e.g. when biologically produced tomato seeds are not disinfected with a HCl treatment, they present potentially more risk for transmission of the seed-borne Clavibacter michiganensis subsp. michiganensis). A curative treatment is sometimes possible by heat treatment (thermotherapy) or chemical treatment of planting material. Thermotherapy may be performed by a) hot water treatment, usually 50-54°C for 5-30 min, b) aerated steam at 50°C for 1 h or c) dry heat at 70°C for 3-7 days. Thermotherapy has been applied to a number of bacterial diseases in different plant parts with reasonable success for true seed, e.g. cabbage seed, bulbs; e.g. Hyacinthus (4-6 weeks 30oC, 2 weeks 38ºC, 3 days 44ºC); rhizomes, e.g. ginger and plantlets or cuttings, e.g. sugarcane and grape (Mahmoodzadeh et al., 2003). When thermotherapy has been performed and DNA-based or serological methods are used for detection, the stability of DNA or antigens from dead cells of the pathogen should be assessed as well as the time of testing. Detection should preferably be based on methods detecting living cells. Unfortunately heat damage may occur resulting in lower germination of seed, or malformation of bulbs. Composting infected material is in fact a thermotherapy, too. When proper temperatures (over 60ºC) are reached for several days in all the material, this is an effective way of killing the pathogen (Hoitink and Fahy, 1986). Chemical control proves to be effective in some cases in freeing basic material from bacterial plant pathogens. Phytotoxicity and difficulties with penetration into internal tissues of plants are a problem. Public health dangers have excluded a number of products, e.g. mercury compounds (once much used to disinfect potato tubers), and certain antibiotics. The following substances have been used with relative success: a) NaCLO or CaClO (less effective, pH important: undissociated HOCl at lower pH) and chlorine dioxide (ClO2); b) antibiotics (high risk of phytotoxicity; c) organic acids (lactic acid, acetic acid, soak for 5-10 min to control e.g. Pseudomonas syringae pv. lachrymans in cucumber seed and Acidovorax avenae subsp. citrulli in watermelon seed and d) 0.1 M hydrochloric acid for 1 h, e.g. very important in the disinfection of tomato seeds from C. michiganensis subsp. michiganensis. Eradication of Xanthomonas campestris pv. zinniae from Zinnia seeds was obtained by soaking seeds for 30 min in 10,500 ppm sodium hypochlorite. Soaking in a streptomycin solution was phytotoxic and hot water treatment for 30 min at 53ºC had a negative effect on germination.

7. Breeding for resistance Probably the most durable form of plant protection is breeding for resistance. Especially in the case of bacterial plant diseases, where (systemic) pesticides are rare or absent, resistance breeding is the only option apart from hygiene and other phytosanitary measures. Resistance is based on a pathogen host interaction that is characterized by genetic variability of both host and pathogen

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BREEDING FOR RESISTANCE IS THE BEST WAY TO CONTROL BACTERIAL DISEASES

Table 31

List of examples where resistance to plant pathogenic bacteria was successfully incorporated into commercial crops and indication of the occurrence of cultivar-specific races in the pathogen (indicated with *).

Bacterium

Host plant

Clavibacter michiganensis subsp. nebraskensis

Zea mays

Pseudomonas syringae pv. glycinea*

Glycine max

P.s. pv. lachrymans

Cucumis spp.

P. s. pv. mors-prunorum*

Prunus spp.

P. s. pv. phaseolicola*

Phaseolus spp.

P.s. pv. pisi* (also see Table 16)

Pisum sativum

P.s. pv. tabaci*

Nicotiana spp.

Ralstonia solanacearum*

Arachis spp. (peanut), Capsicum spp, Musa spp. (banana), Nicotiana spp., S. melongena (eggplant), S. tuberosum 1)

Xanthomonas axonopodis pv. malvacearum* X. a. pv. manihotis

Gossypium hirsutum (cotton) Manihot esculentum

X. a. pv. phaseoli*

Phaseolus spp.

X. campestris pv. campestris

Brassica oleracea*2)

X. oryzae pv. oryzae*

Oryza sativa

X. vesicatoria*

Lycopersicon esculentum

1)

Only tolerance ; 2) Limited success only.

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and by variable reaction according to environmental conditions. This makes evaluation of resistance difficult. The following conditions are very important to obtain good results: a) Resistance of different plant parts can be totally different and the tissues to be inoculated should be carefully selected. b) The method of inoculation (e.g. injection or rubbing using carborundum powder) can determe the detection of resistance. c) In all inoculum experiments a standardized inoculum, preferably containing one or a mixture several virulent strains, must be used. When more than one strain is used, they should originate from different geographic origins, if possible. If a toxic product of the bacterium is used in initial in vitro screenings, it should always be followed by greenhouse and field experiments. d) Experimental conditions (especially humidity, temperature and light) should be kept as constant as possible and therefore first experiments are usually in vitro or in greenhouses. Some resistance as in hosts of race 1 of R. solanacearum is temperature dependent (resistance broken above 32ºC). e) Test plants should be in the same developmental stage and pathogen free. f) Field experiments can be of more value when natural infections are nearby. g) Rating of disease must accurately reflect differences in resistance among plants. h) Controls in the form of highly susceptible and highly resistant cultivars should always be used; furthermore intermediate checks for disease pressure and reisolations from symptoms should be performed. The different steps in the process of resistance breeding are given in Fig. 146. First the genetic susceptibility spectrum has to be carefully determined. For most cultivated plants the diversity in susceptibility is very low and for resistance breeding therefore wild varieties or species have to be incorporated into the breeding program, demanding a lot of effort and a long time period. For example it took more than 14 years to develop by breeding a moderate resistance against R. solanacearum from three wild potato species (Solanum chacoense, S. sparsipilum and S. multidissectum). After that period there was still excessive wildness and considerable glycoalkaloid content (French et al., 1997). Breeding methods can follow a) selection of resistant plants, already present in cultivars or wild relatives; b) combination using a crossing program to introduce resistance genes in cultivars that already possess other valuable genes; c) hybrid and (molecular) mutation breeding, where mutations are selected and/or created by the breeder. Resistance is either based on one gene (gene-for-gene relationship or vertical resistance, also see Table 32) of both the pathogen and host (it is pathogen race-specific) or is based on many genes (horizontal resistance, usually not total resistance). In plant pathogenic bacteria the basis of resistance is often not known and is more horizontal than vertical. Moreover in many cases resistant plants are shown to be tolerant to the bacterial pathogen. Tolerance is usually not desired as tolerant plants may harbour large populations of the pathogen that can still be spread to other non-resistant varieties and species. Due to the fact that traditional breeding for resistance is very time-consuming and partly based on trial and error, it has been tried in recent years to enhance the process by introducing genetic engineering. Resistance and avirulence genes can be cloned and studied and eventually introduced into a desired host. In this way multiple disease resistance genes could be introduced (pyramiding) and a wider range of sources for resistant genes would be available. In this respect the combination X. oryzae pv. oryzae and rice (bacterial blight) has been much studied. Much is already known about resistance in rice due to the excellent work of the International Rice Research Institute in the Philippines. Bacterial blight of rice has been controlled through the use of single-gene resistance introduced by traditional breeding methods, but it has proven to be

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BIOLOGICAL CONTROL SUCCESS STORY FOR AGROBACTERIUM RADIOBACTER K84 AGAINST A. TUMEFACIENS (CROWN GALL) STRAINS THAT CATABOLIZE NOPALINE

Fig. 147 Use of Agrobacterium radiobacter strain K84 for biological control of crown gall (Agrobacterium tumefaciens, nopaline-producing strains); also see text.

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rather unstable. Instability of resistance to X. oryzae pv. oryzae is due to the appearance of new strains in nature that are virulent to known resistant varieties. Genetic manipulation of the resistance genes may allow the design of more stable resistance. In interactions between X. oryzae pv. oryzae and rice, resistance is governed by an interaction between single, dominant resistance genes (R genes) in rice and corresponding pathogen genes called avirulence (avr) genes (Mew, 1987; Leach and White, 1995). The products of avr genes control factors that elicit a plant resistance response. Races of X. oryzae pv. oryzae are defined by the presence (or expression) of a unique combination of avr genes in the pathogen. Race 2, for example, should contain avrXa10, avrXa5, and avrXa7. Another strategy to enhance resistance in the plant is introducing (by genetic manipulation) antimicrobial proteins from other (micro-)organisms into plants. This resistance may develop at the site of treatment, but also in tissues distant from the initial infection sites (inducible resistance or systemic acquired resistance or SAR). One group of such proteins are lytic peptides with an amphipathic α-helical structure that damage the bacterial cell membrane. Cecropins are such lytic peptides from the haemolymph of Hyalophora cecropia, (giant silk moth). Transgenic tobacco plants expressing cecropins have increased resistance to Pseudomonas syringae pv. tabaci, the cause of tobacco wildfire. Bacterial blackleg of potato caused by Erwinia carotovora subsp. atroseptica can be reduced by the synthetic lytic peptide analogs, Shiva-1 and SB-37, when introduced into potato. Transgenic apple expressing an SB-37 lytic peptide analog or apple and pear expressing attacins (antibacterial proteins produced by Hyalophora cecropia pupae) showed increased resistance to E. amylovora in field tests (Norelli et al., 1998). Chemical substances that activate SAR have also been found, such as 2,6-dichloroisonicotinic acid (INA), potassium salts, amino butyric acid and especially benzo (1,2,3) thiadazole-7-carbothioic acid-S-methyl ester (acibenzolar-S-methyl, ASM or BTH). ASM is active in different plant species such as bean, cauliflower, cucumber, tobacco, apple and pear and decreased disease severity substantially in the case of bacterial canker in tomato (Clavibacter michiganensis subsp. michiganensis) and X. oryzae pv. oryzae in rice (Babu et al., 2003).

8. Biological control Biological control is based on antagonism between organisms and can be direct (antibiosis, competition, parasitism), or indirect (induced resistance by application of the (micro-) organism or products thereof). In some cases bacterial diseases have been controlled with reasonable success using biological control. The total microbial population can be used, by activating it (soil cultivation, aeration or use of green manuring), which has been used in control of potato scab. More attention has been paid to special organisms that can be grown on artificial media and can be applied as a pesticide on the plant or in the soil (Table 32). The best example is the use of Agrobacterium radiobacter strain K84 or its genetically engineered form K1026 for control of the crown gall bacterium Agrobacterium tumefaciens (Farrand, 1990). This soil-inhabiting saprophytic bacterium is very closely related to A. tumefaciens, but does not possess a tumour-inducing (Ti) plasmid. A. radiobacter is a good root colonizer (better than A. tumefaciens) and produces a bacteriocin, Agrocin 84, which is toxic for the crown gall bacterium A. tumefaciens. The bacteriocin is an adenine-based nucleotide and not a protein as are most other bacteriocins. Genes for the production of the bacteriocin are located on a plasmid, named pAgK84 (Fig 147). The bacteriocin is only active against A. tumefaciens strains that produce the opine compounds nopaline and agrocinopine. This explains some failures in biocontrol in some countries with some hosts. In

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Bacteria that have been used with reasonable success in biocontrol of Table 32 Ralstonia solanacearum in tomato, tobacco, aubergine and banana. After Trigalet et al. (1994) and others.

Bacterium Actinomycetes Bacillus polymyxa Bacillus subtilus Bacillus spp. Burkholderia glumae Erwinia spp. Pseudomonas aeruginosa Pseudomonas fluorescens Ralstonia solanacearum, spontaneous avirulent mutants (EPS-negative) R. solanacearum, artificial mutants, hrp-, EPS+ mutants Stenotrophomonas maltophilia Streptomyces mutabilis

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Fig. 148 Steam sterilization at 80ºC under plastic is very effective in eliminating the non-sporeforming plant pathogenic bacteria. Care should be taken that the heat reaches sufficiently deep soil layers over a sufficiently long period (usually 1 h to penetrate up to 60 cm). If the procedure is insufficient disease explosion may occur the following season due to absence of most antagonists in the soil.

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the latter case bacteriocin-insensitive strains producing octopine and agropine are present. In the USA and Australia up to 90% control was achieved with peach and cherry by dipping roots in a suspension of this saprophyte. Nopaline-producing strains of A. tumefaciens cause plant cells to produce agrocinopine, which is used by the bacterium as a carbon-source. The Ti plasmid of the pathogen codes for a specific agrocinopine permease that enables uptake of agrocinopine. Agrocin 84 is taken up through this permease accidentally in the A. tumefaciens cell and, as a nucleotide analogue, blocks DNA synthesis and cell growth in the pathogen (Fig. 147). Strains of A. tumefaciens may become resistant to the bacteriocin apparently due to genetic exchange with A. radiobacter. A. radiobacter contains a large plasmid pNOC that can transfer itself and the small pAgK84 to other A. radiobacter cells, but also to A. tumefaciens (Fig. 147). Therefore genetically engineered A. radiobacter strains have been constructed, by deleting the transfer genes from the large plasmid so that it can no longer transfer bacteriocin-resistance (strain K1026). Agrobacterium strains effective against A. vitis have also been found (Creasap et al., 2005). Another example of biological control is the use of Erwinia herbicola (Pantoea agglomerans). Certain saprophytic strains of this bacterium can compete with pathogenic bacteria and other bacteria which are ice-nucleus active and which play a role in frost damage. Control of pathogens was not very successful; frost damage, however, could be much reduced, using this bacterium. Fluorescent saprophytic Pseudomonas spp. have also been used as antagonistic organisms, e.g. in the control of P. tolaasi in the mushroom Agaricus bisporus, with quite some success in control of R. solanacearum in potato and of plant pathogenic fungi. A P. savastanoi strain deficient in the production of indole acetic acid was moderately successful in protection against olive knot in olive (Olea europaea). For E. amylovora a strain of P. fluorescens (A506) proved to be very effective and became commercially available. Furthermore strains of Bacillus subtilus, E. herbicola (Pantoea agglomerans) and Rahnella aquatilis were also found to be promising. Several factors might encourage growers to use strain A506: in addition to controlling fire blight, A506 also gives some control of frost injury and limits russeting on pears. Due to its natural resistance to streptomycin it is the only biocontrol microbe that can be used for orchards with streptomycin-resistant strains of the pathogen (Laux et al., 2003; Vanneste, 2000). In an exceptional case, when antibiotic resistance prevents the use of antibiotics and durable resistance is not present in the host, bacteriophages have been used for control, e.g. in the control of X. vesicatoria in tomato (Balogh et al., 2003).

9. Chemical control All the preventive measures mentioned above cannot prevent the occurrence of calamities. In some cases these can be controlled by bactericides (Table 33). In The Netherlands streptomycin sulphate (an antibiotic produced by Streptomyces griseus), kasugamycin (the antibiotic kasumin produced by Streptomyces kasugaensis) and copper compounds are permitted for control of fire blight and for control of bacterial diseases. For control of diseases in ornamental greenhouse plants only streptomycin is permitted. In the United States, streptomycin is registered for use on twelve (fruit, vegetable and ornamental) plant species. Both antibiotics are applied primarily for the control of bacterial diseases, oxytetracyclin also for control of phytoplasmas. Tree fruits account for the majority of antibiotic use on plants in the USA. In 1995 antibiotics were applied to less than 20% of apple, 35-40% of pear, and 4% of peach acreage. Copper compounds are not systemic, they have to be applied frequently (giving a risk of the

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Table 33

List of chemical compounds used in recent years as a single application or in combination with other (biological) compounds for the control of bacterial plant pathogens and disinfection.

Bactericidal compounds for spray control and disinfection Copper compounds

Disinfectants

Ammoniacal copper sulfate

Acetic acid 1 M

Copper oxide

Benzalkonium chloride

Copper oxyquinolate

Ethanol 70 or 80%

Copper hydroxide

Isopropanol 70%

Copper oxychloride

Propionic acid 1 M

(Tri)basic copper sulphate

Quaternary ammonium compounds

Copper sulphate + lime

Calcium hypochloride

Copper oxychloride + maneb, mancozeb or chorothalonil

Sodium hypochloride Chlorine dioxide

Antibiotics

Stabilized chlorine compounds

Kasugamycin

Hydrogen peroxide with peracetic acid

Oxytetracyclin

Ozone

Streptomycin

UV light Phenolic and cresolic compounds

Other compounds

Formaldehyde

Flumequin

Potassium permanganate

Fosetyl-aluminium

Hydroxychinolinsulphate

7-Chloro-1-ethyl-6-fluoro-1,4-dihydro-4exo-3-quinoline carboxylic acid Oxolinic acid

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FREQUENT SPRAY WITH ANTIBIOTICS OR COPPER MAY LEAD TO DEVELOPMENT OF BACTERICIDERESISTANT STRAINS OF THE PATHOGEN

Fig. 149 Cycle of antibiotic resistance development under selection pressure of the antibiotic. The example is for the likely development of E. amylovora streptomycin-resistant strains by plasmid transfer from saprophytic bacteria to the pathogen and subsequent selection of resistant cells when this antibiotic is frequently used in control sprays in orchards. In orchards, populations of saprophytic bacteria belonging to the genera Acinetobacter, Flavobacterium, Erwinia herbicola (=Pantoea agglomerans) and Pseudomonas occur that possess streptomycin-resistance genes on a moving genetic element (transposon) located in its plasmid. The movable plasmid can be transferred by chance through conjugation (also see Fig. 22) from the saprophyte (E. herbicola in the example) to non-resistant E. amylovora cells. Under the selection pressure of streptomycin sprays susceptible E. amylovora cells are killed, but resistant ones can multiply and will soon outnumber the susceptible cells and cause disease under favourable conditions.

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Table 34

Effect of several disinfectants on Erwinia carotovora subsp. atroseptica (Eca) and Clavibacter michiganensis subsp. sepedonicus (Cms). After Letal (1977).

Compound

Metal

Wood

Jute

Eca

Cms

Eca

Cms

Eca

Cms

10% NaOCL

-1)

-

+

+

-

-

0.1% Mercury chloride

-

-

-

-

-

-

Quaternary ammonium compound

-

-

+++

++

+++

-

10% Dettol

-

-

+

-

-

-

2% Formaldehyde

-

-

-

+

+

-

5% Formaldehyde

+++

+

-

-

-

-

H20 + soap

-

-

+++

+++

+++

++

0.13% Zephiranchloride

-

-

++

+

+

+

Hibitane

-

-

+++

+++

+++

-

- = no growth; +/++/+++ = growth 1) after application for 10 min

Table 35 Pathogen

Streptomycin resistance as observed for bacterial plant pathogens. Plant(s) affected Location(s)

Erwinia amylovora

apple, pear

Israel, New Zealand, USA

Pseudomonas cichorii Pseudomonas syringae pv. syringae Xanthomonas campestris pv. vesicatoria

celery apple, pear, ornamental and landscape trees

USA

tomato, pepper

USA Argentina, Brazil, Taiwan, Tonga, USA

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development of copper-resistant populations) and they have the risk of phytotoxicity. They are often used for leaf spotting organisms. Compounds most frequently used are copper chloride basic, CuCl2.3Cu(OH)2; basic copper sulphate, CuSO4.3Cu(OH)2 and copper hydroxide, Cu(OH)2. Strains of Pseudomonas syringae and X. axonopodis pv. vesicatoria have been found to develop resistance against copper. These bacteria accumulate blue Cu2+ ions in the periplasm and outer membrane of the cell (Cooksey, 1996; Voloudakis et al., 2005). Streptomycin is taken up by the root and is transported through the vascular system. It can penetrate the leaf to a certain extent. Risk of phytotoxicity is present, but relatively low. Kasugamycin has a preventive effect, but a therapeutic or curative effect is also claimed, as it is completely systemic and it is easily translocated to target sites. It has a very low phytotoxicity and can be used in combination with other pesticides. Antibiotics have the disadvantage that the target bacterium may develop resistance against the compound, as is the case for e.g. resistance against streptomycin found in Erwinia amylovora, P. cichorii and P. syringae pv. papulans in the USA and X. vesicatoria in S. America (Fig. 149 and Table 35, McManus & Jones, 1994; Vaneste, 2000; McManus et al., 2002). Antibiotic use on crops and ornamental plants is usually highly regulated by Environmental and Health Agencies and pesticide laws. As with other pesticide instructions of products, these instructions should be strictly followed concerning type of clothing, boots, gloves, and respirators.

10. Sanitation and disinfection Good sanitation is extremely important in the case of contagious bacterial diseases. Plant pathogenic bacteria can survive on many different materials, sometimes for many years. These materials can be sanitized with disinfectants. Success of disinfection is dependant on the concentration of the compound, duration of the application, nature of the material to be disinfected and especially the amount of organic material present. Organic material inactivates many compounds very quickly (Tables 33 and 34). Disinfection of (surface) irrigation water, also when recycling is applied in greenhouses, is possible using (combinations of) filtration, UV irradiation, chlorine dioxide and/or hydrogen peroxide with peracetic acid (Runia, 1995; Runia & Amsing, 2001). Hydrogen peroxide (H2O2) formulations with peracetic acid or a similar catalyst are active at relatively low concentrations (<0.01% H2O2) and are relatively insensitive to organic matter and can be used for disinfection of surface (irrigation) water (Niepold, 1999, unpublished results, author). Diseased crop residues can be removed and burned in a safe place, or disposed of in a safe waste disposal. In many countries waste can only be left in licensed disposals and landfills (see e.g. http://www.defra.gov.uk/planth/publicat/waste/ ).

VII. I Examples of bacterial diseases – Bulbaceous plants

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A. Noordijk, PD

174

Fig. 150 Top:

Bottom:

Symptoms caused by Curtobacterium flaccumfaciens pv. oortii on garden tulip (Tulipa gesneriana), leaf symptoms: silver grey stripes and cracks along the main vein (called hell fire) and yellow pustules on the outer white scales of the bulbs. Yellow discoloration of the vascular tissue in stems and bulbs. Colour drawing. Pustules on outer scale (left) and yellow discoloration of vascular and surrounding tissue of outer scale (right). Natural infection.

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- EXAMPLES OF BACTERIAL DISEASES OF CULTIVATED AND WILD PLANTS1)

A Bacteria pathogenic to monocotyledonous plants 1. Bulbaceous plants 1.1

Curtobacterium flaccumfaciens pv. oortii (Saaltink and Maas Geesteranus 1969) Collins and Jones 19832) Syn.3) Corynebacterium oortii

Yellow pustule of tulip (bulb symptoms) or hell fire (leaf symptoms) Pustule jaune de la tulipe (feu d’enfer)4) Gelbpocken der Tulpe (Höllisches Feuer) 4)

Main hosts: Garden tulip (Tulipa gesneriana) Symptoms and transmission: On the bulbs tiny slightly swollen white/grey spots occur on the outermost white scales that later become raised and turn yellow (hence the name yellow pustule). On the leaves silver grey spots up to 5 mm are the first symptoms. In later stages, (usually in March-April, after some night frosts) upper and lower epidermis may (badly) crack and become loosened from the other tissue, usually along the veins (hence the name hell fire). The vascular tissue, both in leaves and bulbs, shows yellow discoloration. Heavily infected plants are stunted and the flower buds wither easily or these plants do not appear. C. f. pv. oortii is a systemic vascular pathogen and a wound parasite. Cold weather and wounds caused by rubbing of leaves, hail and cultivation practices in spring favour disease development. Transmission is by infected bulbs and between plants in the field by wind-blown rain and sand and by contaminated implements used to cut the flowers (to prevent virus transmission by aphids). Geographical distribution and importance Denmark, Germany, Japan, The Netherlands, UK. The disease is of no economic importance. Control Direct control of the disease is not possible. Preventive spray or dip in captan containing pesticides yields some protection. Removal of infected plants and bulbs and avoidance of working in the fields during humid weather conditions (especially when removing the flowers) reduces the risk of infection. Late planting (c. 30 November) to avoid critical night frost in spring may be beneficial. 1)

2)

3) 4)

Examples of some well-known and lesser-known bacterial pathogens and diseases in diverse cultivated and wild hosts will be presented, purely on the basis of selection by the author. More information and description of other diseases and bacterial pathogens can be found in Smith (1905b, 1911, 1914); Elliot (1951); Stapp (1956), Starr (1983); Bradbury (1986); Collmer et al. (1987); Kleinhempel et al. (1989); Smith et al. (1992); http://www.eppo.org/ and http://www.cabicompendium.org/cpc/home.asp. Authors and year of publication in which the bacterium was described or renamed. These publications are generally not presented in the literature list, but they can be found in (on line) catalogues of official culture collections, also see Chapter II.3e and page 78. syn. = synonym. Only more recent synonyms are mentioned, for others see literature under 1) Common name of the disease in French and/or German, when known.

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Fig. 151 Top:

Bottom:

Pinkish-brown discoloration of (inner) scales of a bulb of Hyacinthus orientalis, caused by Erwinia rhapontici in transverse section of the bulb. Natural infection. As described above, showing extent of discoloration on inner (left) and more outer (right) scales. Natural infection.

Examples of bacterial diseases – Bulbaceous plants

1.2

177

Erwinia rhapontici (Millard 1924) Burkholder 1948 Syn. Pectobacterium rhapontici

Internal browning of hyacinth Main hosts: For bulbous plants hyacinth (Hyacinthus orientalis) and onion (Allium cepa) are the main hosts, but E. rhapontici has also been reported from A. sativum (garlic) and Hippeastrum (amaryllis). Amaranthus hybridus (smooth pigweed) was reported as a host from Mexico and the bacterium found to be seed transmitted. E. rhapontici has also been reported to be the cause of foot and crown rot of Rheum rhaponticum and R. hybridum (rhubarb) and pink seed (pale pinkish brown-to-bright pink discoloration throughout the seed coat) of Triticum aestivum (wheat), Pisum sativum (pea) and Phaseolus vulgaris (bean) in the USA. Pink seed has also been described from Lens culinaris (lentil) and Cicer arietinum (chickpea) in Canada (Huang et al., 2002). Furthermore Cyclamen persicum (cyclamen), Dianthus (carnation), Armoracia rusticana (horseradish) and Morus (mulberry) have been reported as hosts. Symptoms and transmission: Pinkish brown discoloration of inner scales of Hyacinthus (Fig. 151) and onion (turning later into brown to black soft rot), crown rot of rhubarb and pink discoloration (as if treated with pesticides) of seeds of wheat, pea, bean and lentil. The pathogen is spread by bulbs, rhubarb crowns used for propagation and infected seeds. Plant to plant infection may occur by insects spreading the bacterium and infection with the nematode Anguillulina dipsaci enhances the occurrence of crown rot of rhubarb. This nematode probably also transmits the organism. Wounding is necessary during procedures connected with cultivation and vegetative propagation. Irrigation in connection with wounding of pods appears to enhance the occurrence of pink seed of legumes. Geographical distribution and importance Belgium, Canada, former Czechoslovakia, France, Iran, Israel, Italy, Japan, Korea, Lithuania, Malaysia, The Netherlands, Norway, Poland, UK, Ukraine, USA. E. rhapontici is an opportunistic pathogen, only causing problems under adverse conditions for the plant and remains incidental in bulbaceous plants. The seed infections give problems of quality, not so much of yield loss. Control In rhubarb E. rhapontici can be readily transmitted via transplanting infected crowns. In establishing new plantings only disease-free crowns should be selected. Documented movement has occurred when infected crowns were used to establish new plantings. New crowns must not be replanted in areas where the disease has previously been observed. Evidence exists that rootand foliage-feeding insects can move the bacteria from infected to uninfected plants. Therefore good insect management will also reduce localized spread. Healthy seeds and use of healthy crowns or bulbs and early spraying with insecticides to reduce insect populations on the hosts are the only way for prevention or control.

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Fig. 152 Top left:

Bulb of Scilla tubergeniana, showing typical symptoms of yellow disease, caused by Xanthomonas hyacinthi: yellow discoloration in the vascular tissues (and some of the surrounding parenchyma) of the scales and of the bottom. Natural infection. Top right: Bulb of Eucomis autumnalis with typical symptoms of yellow disease, description similar to that of top left. Some secondary rotting is already present. Natural infection. Bottom left: Leaf of Scilla tubergeniana with glassy leaf spots and streaks, following artificial spraying with a suspension of Xanthomonas hyacinthi and subsequent wound/stomata and hydathode infection. Bottom right: Bulbs of Puschkinia scilloides with typical symptoms of yellow disease, description similar to that of top left. Natural infection.

Examples of bacterial diseases – Bulbaceous plants

1.3

179

Xanthomonas hyacinthi (ex Wakker 1883) Vauterin, Hoste, Kersters and Swings 1995 Syn. Xanthomonas campestris pv. hyacinthi

Yellow disease of hyacinth Jaunisse bactérienne de la jacinthe Gelbfäule de Hyazinthe

Main hosts: Hyacinth (Hyacinthus orientalis). Natural infections of X. hyacinthi have also been reported from Scilla tubergeniana (scilla, Fig. 152 top and bottom left), Eucomis autumnalis (pineapple lily, Fig. 152 top right) and Puschkinia scilloides (striped squill, Fig. 152, bottom right; Janse and Miller, 1983; van der Tuin et al., 1995c). Symptoms and transmission: When the bulb is already infected at planting yellow discoloration of the vascular tissue and surrounding parenchyma can be found in the scales and bottom plate (Fig. 152, also see Fig. 26a-c on page 34). Sometimes yellow spots are found on the outer scales, following mechanical damage. From heavily diseased bulbs no plants develop or those plants suddenly wilt and die. Bulbs rot completely and have an unpleasant smell. In others dark streaks following the veins in the leaves develop from the leaf base. Primary infection in the field (usually not before May) gives rise to leaf spots and dark streaks along the veins at the margin (hydathode infection) or other parts of the leaf (Figs 26 and 152). Leaf tips may turn black and wither (also in secondary infections). No vectors have been described, but mechanical transmission by man is easily possible (Kamerman, 1975). Geographical distribution and importance Originally described from The Netherlands in 1883 by Wakker. Australia, Finland, France, Hungary, Ireland, Italy, Japan, The Netherlands, Poland, Romania, Russian federation, Sweden, UK, USA and former Yugoslavia.In susceptible cultivars heavy losses may occur under intensive cultivation. In all other cases the disease appears to be of little importance. Control In early springtime volunteers should be removed. Visitors should not be allowed to enter the fields. Further field inspections should trace diseased plants. The leaves of these plants and those present in the vicinity should be removed and destroyed by HCl or covered with sand. Bulbs and surrounding soil can be treated with diquat or 5% formalin. Surrounding plants can be treated with kasugamycin (if permitted). Alkyl dimethyl benzyl ammonium chloride (if permitted) can be applied when leaf symptoms occur, but is not completely effective. Bulbs from diseased spots should be harvested last and separately and destroyed or heat-treated. After harvest, disinfection of machines and implements should take place and bulbs should be dried rapidly. For bulbs a heat treatment has been found to be quite effective. Bulbs are stored first for a period of 4 weeks at 30°C, thereafter for 2 weeks at 38°C, with a final treatment for 3 days at 44°C. Subsequently bulbs are stored at 28-30°C (Kruyer and Vreeburg, 1981). To avoid negative effects of the heat treatment sufficient ventilation is essential. Before planting bulbs should be carefully inspected. Susceptibility of cultivars is different and resistance breeding has been moderately successful. A classification system has been developed to produce yellow disease-free hyacinths (OEPP/EPPO, 1998).

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Fig. 153 Rectangular leaf spots, initially dark green and water-soaked, later brown and necrotic, sometimes with a white centre or silvery film of bacterial slime, caused by Xanthomonas campestris pv. gummisudans on Gladiolus convilli ‘Alba’. Natural infection.

Examples of bacterial diseases – Plants with bulbous roots (corms)

181

2. Plants with bulbous roots (corms) Xanthomonas campestris pv. gummisudans (McCulloch 1924) Dye 1978 Syn. Xanthomonas gummisudans

Leaf spot and scorch of Gladiolus Main hosts: Gladiolus (Gladiolus x hortulanus). Symptoms and transmission: Dark green, water-soaked leaf spots that are more or less rectangular are the first symptoms of the disease (Figs. 153 and 154). These leaf spots later turn brown and necrotic. Bacterial ooze is present on the lesions under wet conditions. When there are many leaf spots, which can coalesce, plant and corm growth is retarded. Bacteria enter the plant through stomata. Development of the disease is strongly dependent on free water (irrigation, wet weather). Geographical distribution and importance Reported by McCulloch from the USA for the first time in 1924, where it caused severe damage in the first years it was observed. Thereafter only sporadic reports were published, where no serious damage was mentioned. Australia, Canada, Finland, The Netherlands, South Africa, USA. Control Chemical control is not possible. If possible foliage should be kept dry. Susceptibility and resistance of varieties varies.

PD

2.1

Fig. 154 Early infection by Xanthomonas campestris pv. gummisudans on Gladiolus convilli ‘Alba’. Rectangular dark green, watersoaked spots. Natural infection.

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182

Fig. 155 Early symptoms, in the form of small water-soaked spots along the veins, on maize (Zea mays) of leaf freckles and wilt, caused by Clavibacter michiganensis subsp. nebraskense.

Examples of bacterial diseases – Gramineous plants

183

3. Gramineous plants 3.1

Clavibacter michiganensis subsp. nebraskense (Vidaver and Mandel 1974) Davis, Gillaspie, Vidaver and Harris 1984 Syn. Corynebacterium nebraskense

(Nebraska) leaf freckles and wilt or Goss’s bacterial wilt and blight of corn Main hosts: Maize (Zea mays). Saccharum officinarum (sugarcane), Sorghum bicolor (common sorghum), S. sudanense (sudan grass) and Zea mexicana (teosinte) have also been reported as natural hosts. Echinochloa crus-galli (barngrass), Triticale and Triticum aestivum (wheat) have been reported as secondary hosts. Symptoms and transmission: First symptoms are water-soaked leaf spots (on seedlings or older plants) that develop into longitudinal streaks parallel to the main veins. Sometimes drops of bacterial slime are present.In later stages of infection plants wilt, show leaf blight and may be stunted. Leaf streaks turn grey to light green or yellow to red stripes with wavy or irregular margins, which follow leaf veins (confusion with leaf symptoms of Erwinia stewartii and cold damage is possible, see Fig. 156a). Discrete, water-soaked spots (freckles) along the veins are characteristic of the disease. These spots are dark-green to blackish in appearance and look like freckles (hence the name) when infected leaves turn brown. When streaks coalesce leaf scorch results that may be confused with symptoms of drought. Systemically infected plants may show yellowish discoloured vascular bundles and a dry or water-soaked to slimy-brown rot of the roots and lower stalk may occur. From these infected stalks and roots, leaves can become reinfected. Plants at any stage of development can become infected, wilt and die. The main source of inoculum appears to be maize crop residues that have been left over winter. Seed infection (internal and external) and transmission occurs, but appears to be minor as compared to residue transmission (Schuster, 1975). Geographical distribution and importance Canada, USA. C. m. subsp. nebraskense was first described from Nebraska, USA, in 1969 and the disease has gradually spread across the USA corn belt. Heavy losses have been reported for individual fields but over larger areas they appear to be minor (Wysong et al., 1981). Control Control measures are crop rotation, destruction of maize debris and general hygiene, as well as testing of seed and planting of seed lots that tested free from the pathogen. Planting of resistant varieties is sometimes possible. High resistance in dent maize hybrids and inbreeds has been found, but sweet corn varieties appear to be less resistant (Schuster, 1975; Wysong et al., 1981; Smidt and Vidaver, 1986).

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Fig. 156a

Nebraska Inst. Agr. Natural Resources, South Central Res. & Ext. Center, Clay Center, NE, USA

Typical yellowish leaf streaks of Stewart’s disease, on maize (Zea mays), caused by Pantoea stewartii subsp. stewartii.

Fig. 156b Corn flea beetle (Chaetocnema pulicaria) and its damage (scaling the epidermis) on maize (Zea mays).

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Pantoea stewartii subsp. stewartii (Smith 1898) Mergaert, Verdonck and Kersters 1993 Syn. Erwinia stewartii

Stewart’s disease, bacterial wilt Maladie de Stewart, flétrissement bactérien Stewart’s krankheit, Bakteriellen Welkenkrankheit der Mais

Main hosts: Maize (Zea mays), especially sweet corn. Also fodder grasses such as Tripsacum dactyloides (eastern gamma grass) and Zea mexicana (= Z. mays spp. mexicana, teosinte, a wild relative of maize) have been reported as natural hosts. Some other grasses have been mentioned as secondary hosts, such as Agrostis gigantea (bench couch grass), Dactylis glomerata (orchardgrass), Panicum spp. (panic grass), Poa pratensis (junegrass), Setaria lutescens (foxtail grass) and Sorghum sudanense (sudan grass). Symptoms and transmission: Short to long pale-green to white-yellow streaks on the leaves are formed and sweet corn hybrids may already show rapid wilting in an early stage of the disease (Fig. 156a). Streaks turn brown in later stages of the disease. The entire vascular tissue may be infected and bacteria can be found in roots, stalks, leaves, tassels, cobs, husks and kernels. Bacteria may exude as droplets of slime from the husks. Bacteria penetrate the seed, but not the embryo. The disease may be confused by other leaf blights such as caused by the bacteria Acidovorax avenae subsp. avenae, Burkholderia andropogonis and Clavibacter michiganensis subsp. nebraskensis or fungi like Setosphaeria turcica and Cochiobolus heterostrophus. The pathogen survives in living host plants and seed and may be also spread by seed. Most important dispersal (in the field), however, is by an efficient insect vector, the corn flea beetle Chaetocnema pulicaria (Fig. 156b). P. s. pv. stewartii overwinters in the alimentary tract of C. pulicaria, which emerges from hibernation and feeds on young maize plants (Pepper, 1967). Overwintering beetles cause the first infections, but also the second (summer) generation is active in transmission. Other (minor) vectors have been described in North America. The vectors are absent from Europe. Geographical distribution and importance EPPO A2 quarantine pest Smith (1898) described the bacterium adequately for the first time in the USA. Bolivia, Brazil, Canada, Costa Rica, Guyana, Puerto Rico, Peru, USA. Isolated findings or interceptions in some European countries (Austria, Greece, Poland, Romania, Russia). In Europe Stewart’s disease has and will have limited importance due to absence of the vector. Heavy losses have been incidentally reported from the USA in susceptible sweet corn hybrids (Patacky and Eastburn, 1993; Pataky et al., 2000) and incidentally in Italy (Mazzucchi, 1984). Control Healthy seeds and use of low susceptibility or resistant varieties and early spraying with insecticides to reduce vector populations are the most important ways to prevent or control disease caused by P. stewartii subsp. stewartii. Stewart’s wilt can be predicted on the basis of the average daily temperature in December, January and February, which relates to survival of C. pulicaria. If the average daily temperature during these months is above freezing, flea beetles survive and Stewart’s wilt is likely to be severe on susceptible hybrids. At average daily temperatures of lower than -3°C, most flea beetles do not survive and it is unlikely that Stewart’s wilt will be severe (Esker and Nutter, 2002). There is sufficient resistance present in maize hybrids (Michener et al., 2003).

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Fig. 157 Top:

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Typical field symptoms of bacterial stripe blight in oat (Avena sativa), caused by Pseudomonas coronofaciens. Leaf stripes with a narrow yellow halo and necrotic leaf tips. Bottom: Symptoms of bacterial stripe blight in individual oat plants, as above.

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Pseudomonas coronofaciens (Elliot 1920) Schaad and Cunfer 1979 Syn. Pseudomonas syringae pv. coronofaciens (formerly connected to halo blight of oat) Pseudomonas syringae pv. striafaciens (formerly connected to bacterial stripe blight of oat and barley)

Halo blight of oat and bacterial stripe blight of oat and barley Maladie bactérienne de l’avoine et Taches foliaires jaune-brune de l’avoine Bacterielle Blattdürre des Hafers und andere Gramineen-Arten und Bakterielle Streifenkrankheit des Hafers und der Gerste

Main hosts: Oat (Avena sativa) and barley (Hordeum vulgare). Furthermore Secale cereale (rye), Triticum aestivum (wheat), Avena byzantina (red oat, Algerian oat), Agropyron repens (quackgrass), Bromus spp. (brome grass) and Phleum pratense (timothy) have been reported as natural hosts. Symptoms and transmission: Formerly it was thought that the two diseases mentioned above were caused by two different pathogens (mentioned under synonyms), based on slight differences in pathogenicity and host range. Schaad and Cunfer (1979) proved that the same organism causes both diseases. Halo blight symptoms are characteristic small, pale green, oval leaf spots with slightly sunken centres. Around the spot a yellow to red-brown halo usually develops that can be more than 1 cm in diameter or elongates along the entire leaf length. On chaff small spots with a yellow halo may also occur and on glumes yellow translucent spots between the veins can be observed. Bacterial stripe symptoms are small water-soaked leaf spots, often in rows (stomatal infection). When these spots coalesce they form narrow water-soaked stripes, sometimes surrounded by a narrow yellow halo. Under humid conditions bacterial slime can sometimes be observed. Spots and stripes turn red to brown-black in later stages (Fig. 157). P. coronofaciens can be transmitted by seed and can survive in crop debris for several years (Kleinhempel et al., 1989). In the field bacteria are spread by wind-driven rain. The role of insects and surface water is unknown. Geographical distribution and importance Described for the first time by Elliot (1920) from the USA. Widespread in most oat- and other grain-producing countries in the world. Although devastating infections were described from the USA in the 1920s, the disease is now sporadic and generally causes no economic losses, except for some sporadic outbreaks. Cold, wet weather conditions or aberrant drainage and irrigation conditions favour these outbreaks. Control Crop rotation, plowing under of crop debris, and the use of certified seed are the only possible and usually also the only necessary means for control. Copper-based pesticides are sometimes used in the USA for preventive spraying or seed treatment.

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Fig. 158 Top:

Typical symptoms (rapid yellowing, wilting and dying) of Kresek disease in rice (Oryza sativa) transplants, caused by Xanthomonas oryzae pv. oryzae in a wet rice field. Bottom: Advanced stages of bacterial blight of rice caused by X.o. pv. oryzae. Natural infection.

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Xanthomonas oryzae pv. oryzae (ex Ishiyama 1922) Swings, Van den Mooter, Vauterin, Hoste, Gillis, Mew and Kersters 1990 Syn. Xanthomonas oryzae; X. campestris pv. oryzae

Bacterial blight of rice, Kresek disease Maladie bactérienne des feuilles du riz Bakterielle Weissfleckenkrankheit, bakterieller Blattbrand

Main hosts: Rice (Oryza sativa). Also other Gramineae such as Brachiaria mutica (para grass), Cenchrus ciliaris (buffel grass), Cynodon dactylon (bermudagrass), Cyperus difformis (rice sedge), C. rotundus (purple nut sedge), Echinochloa crus-galli (barnyardgrass), Leersia spp. (cutgrass, obligate wetland weed), Leptochloa spp. (sprangletop), wild Oryza spp., Panicum maximum (guineagrass), Paspalum scrobiculatum (kodomillet), Zizania spp. (wild rice) and Zoysia spp. (creeping grass) have been described as natural hosts. Symptoms and transmission: Leaf blight: On the leaf blade pale green to yellowish, water-soaked stripes develop, often near to the top of the leaf. Hydathode infection occurs. The stripes later increase in length and width and often show a wavy margin. Sometimes milky to yellowish bacterial slime can be observed on young stripes under humid conditions (early in the morning). In later stages the stripes turn yellow to white (Fig. 158 bottom). Severely infected leaves and plants tend to dry and die quickly. Sometimes stripes become grey due to infection with secondary fungi. In severe infections panicles may be sterile and unfilled. Symptoms may be confused with those caused by the related X. o. pv. oryzicola. The lesions of this pathogen remain more linear and not wavy and are more a problem in combination with insect damage. Seed transmission is possible, but appears of minor importance, as is survival in and transmission by soil. X. o. pv. oryzae populations rapidly decline in soil under dry, hot conditions. In the growing period water plays an important role in distribution of X. o. pv. oryzae, especially when other (wild) hosts such as Leersia spp. are present (also see Fig. 128). Kresek disease (in seedlings): so-called kresek symptoms are usually observed 1-3 weeks after transplanting, especially when leaf tips of transplants have been cut before transplanting with non-disinfected tools. Green water-soaked stripes at the leaf tips are the first symptoms, soon followed by severe wilting, yellowing and death of the entire plant (Fig. 158 top). Geographical distribution and importance EPPO A1 quarantine pest First described from Japan by Ishiyama in 1922. Widespread in S.E. Asia. Furthermore reported from Australia, Bolivia, Colombia, Burkina Faso, Cameroon, Costa Rica, El Salvador, Ecuador, Gabon, Honduras, Mali, Mexico, Niger, Panama, S.E. Russia, Senegal, Togo, USA and Venezuela. Not yet found in Europe. Heavy losses (up to 50%) have been reported from dwarf high-yielding cultivars (Ray and Sengupta, 1970). Control Healthy seeds and use of low susceptibility or resistant varieties, hygiene during transplanting, proper fertilization and water management are main ways to prevent or control bacterial blight. Extensive resistance breeding and characterization of races of X. o. pv. oryzae are performed with good success by the International Rice Research Institute, Manila, Philippines. Resistance is sometimes overcome by the pathogen by development of new races (Nelson et al., 1994).

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Fig. 159 Top:

Typical symptoms (water-soaked to yellow-brown streaks, wilting and complete necrosis of leaves) of bacterial blight in English ryegrass (Lolium perenne), caused by Xanthomonas translucens pv. graminis in a grass field for seed production. Bottom left: Detailed view of the infection described above. Bottom right: Droplets of slime exuding from stripes of an oat (Secale cereale) leaf. Natural infection.

Examples of bacterial diseases – Gramineous plants

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Xanthomonas translucens (Jones, Johnson and Reddy 1917) Dowson 1939 Syn. Xanthomonas campestris pv. translucens

Formerly a number of xanthomonads pathogenic for Gramineae were classified as pathovars of Xanthomonas campestris. Vauterin et al. (1992, 1995) showed that these pathovars in fact belong to one species, already described and known as a pathogen of wheat and rye, Xanthomonas translucens, and differ mainly in host range.

Black chaff, leaf streak, bacterial stripe or bacterial blight of cereals and grasses Maladie bactérienne des céreales Schwarzspelzigkeit oder Bakterielle Streifenkrankheit des Weizens

Main hosts: pv. arrhenateri Arrhenaterum elatius (false oatgrass) pv. cerealis Agropyron and Bromus spp. (wheatgrass and brome grass) pv. graminis Lolium, Dactylis, Festuca (ryegrass, orchardgrass, fescue) pv. hordei Hordeum vulgare (barley) pv. phlei Phleum pratense (timothy) pv. phleipratensis Phleum pratense (timothy) pv. poae Poa trivialis (rough bluegrass) pv. secalis Secale cereale (rye) pv. translucens Secale cereale, Triticum x Secale, Triticum (rye, triticale and wheat) pv. undulosa Triticum (wheat) Symptoms and transmission: On young leaves generally water-soaked small leaf spots that develop later into longitudinal yellow to brown streaks. Leaves may wilt (Fig. 159). When the weather is wet small droplets of yellowish bacterial slime can be observed on the lesions. On the stem brown to black stripes may be formed. On the chaff of many hosts water-soaked spots are formed that later develop into brown to black spots. Ears may show brown discoloration and distortion. Seeds may be black and shrivelled. All the X. translucens pathogenic varieties can be transmitted by contact (mowing, harvesting, plant-to-plant), insects (aphids) especially when they come into contact with bacterial slime, splash water (rain, irrigation) and seeds. Geographical distribution and importance X. t. pv. translucens: EPPO A2 quarantine pest X. t. pv. translucens was described for the first time in 1917 by Jones et al. from the USA. Cereal pathogens are widespread in North America, Russia and surrounding states. X. t. pv. translucens has been described from Argentina, Asia (widespread), Australia, Bolivia, Brazil, Ethiopia, Israel, Kenya, Libya, Madagascar, Morocco, Paraguay, Peru, Romania, S. Russia, South Africa, Spain, Syria, Tanzania, Tunisia, Turkey, Ukraine, Uruguay and Zambia. X. translucens can occasionally cause serious crop losses of up to 15%. Grass pathogens have been found in Western Europe and New Zealand, especially in grass cultivated for seed production and crop losses can be up to 40%. X. t. pv. translucens is ice-nucleating active and can be especially damaging when helped by frost damage (Sands and Fourrest, 1989). Control Healthy seeds through seed certification programs (and eventually by decontamination with acidic cupric acetate, but this is not 100% effective) and use of low susceptibility or resistant varieties are the only way to prevent or control diseases caused by X. translucens. Chemical control in the field is not possible (Duveiller, 1994).

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Fig. 160 Typical reddish-brown leaf streaks with a pale brown centre on sorghum (Sorghum bicolor), caused by Xanthomonas vasicola pv. holcicola. Natural infection.

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Xanthomonas vasicola pv. holcicola (Elliot 1930) Vauterin, Hoste, Kersters and Swings 1995 Syn. Xanthomonas holcicola; X. campestris pv. holcicola

Bacterial leaf streak Main hosts: Sorghum or Indian millet (Sorghum bicolor = S. vulgare = Andropogon sorghum) and its varieties caffrorum (= S. caffrorum), durra (= S. durra) and technicus (= S. technicum). S. almum (Columbus grass or five-year sorghum), S. halepense (Arabian millet or Aleppo grass), S. sudanense (Sudangrass), Panicum milliaceum (broomcorn millet) and Zea mays (maize) have also been reported as natural hosts. Symptoms and transmission: Small water-soaked leaf spots are the first symptom of the disease. These spots later enlarge and elongate, become reddish-brown with a pale brown centre and may cover a large part of the leaf (Fig. 160). In a final, severe, stage leaves wither and drop early. Yellowish bacterial slime is exuded from the spots under humid conditions and dries as a white papery film in dry weather. Symptoms can be confused with those caused by Burkholderia andropogonis (bacterial leaf stripe). Stripes caused by the latter bacterium, however, are more uniformly reddish-brown. Symptoms may also be confused with those caused by Pseudomonas syringae pv. syringae (bacterial eye spot or leaf spot). Here spots remain round to ellipsoid and bacterial slime is not observed. The bacterium is seed transmitted. Geographical distribution and importance First described by Elliot (1930) from the USA. Argentina, Australia, Ethiopia, Gambia, India, Israel, Mexico, New Zealand, Niger, Philippines, South Africa, Thailand, Romania, S. Russia, Ukraine, USA. Never observed in Europe. The disease has only limited importance, is important only occasionally in springtime under warm weather conditions and becomes less serious during hot and dry summer months and there is considerable variation in resistance. Control Healthy seeds and use of low susceptibility or resistant varieties are the main ways to prevent or control diseases caused by X. vasicola pv. holcicola. Furthermore rotations with non-grasses or grain crops in a 1:3 scheme, removal of crop residue, controlling weeds and (in the case of fodder grasses) sowing of mixtures are useful in reducing risk of disease. In some cases burning of old grass in spring may reduce the initial inoculum.

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Fig. 161 Top:

Brown necrotic leaves and wilting as (severe) symptoms of Xanthomonas axonopodis pv. vasculorum in betel nut palm (Areca catechu) used as an ornamental. Natural infection. Bottom: Detailed view of early symptoms of X. a. pv. vasculorum on betel nut palm: water-soaked reddish discoloration of leaf tips and yellow stripes along the leaf blade. Natural infection. PD

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Examples of bacterial diseases – Palm trees

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4. Palm trees 4.1

Xanthomonas axonopodis pv. vasculorum (ex Cobb 1893) Vauterin, Hoste, Kersters and Swings 1995 Syn. Xanthomonas vasculorum; X. campestris pv. vasculorum

Gummosis or Cobb’s disease of sugarcane Gommose de la canne à sucre (on sugarcane)

Main hosts: Betel nut palm (Areca catechu), Dictyosperma album (princess palm), D. rubrum (hurricane palm), Roystonia regia (royal palm) in the Family Palmae; sugarcane (Saccharum officinarum). Thysanolaena maxima (= T. agrostis, tiger grass or broom bamboo), Tripsacum laxum (Guatemala grass) and Zea mays. Some strains causing a more or less similar disease on Zea mays (maize) and Tripsacum laxum (Guatemala grass) appeared to differ in several aspects from X. a. pv. vasculorum strains and they belong possibly to a group of strains reclassified as X. vasicola pv. vasculorum (Stead, 1989; Péros et al., 1994; Vauterin et al., 1995). Dookun et al. (2000), have described even more diversity in this species. Symptoms and transmission: On the leaves water-soaked to chlorotic to red stripes occur, starting from the tips (Fig. 161 bottom). The vascular bundles show a reddish discoloration and bacterial slime oozes out of this tissue and bacterial pockets in the parenchyma. In sugarcane the tips of canes may die and at the basis of the necrotic tip bacterial pockets, filled with slime are also found. In later stages leaves yellow and/or become completely brown and necrotic. When canes become infected reddish discoloration and bacterial slime in the vascular bundles can be found. Plants stunt, wilt and eventually die. Plants become infected through wounded roots, but also wind-driven rain and insects (flies) play a role in transmission, as well as cuttings. Geographical distribution and importance EPPO A2 quarantine pest Dränert first reported the disease from Brazil in 1869, but Cobb in Australia described the causal bacterium only in 1893. Antigua and Barbuda, Australia (not in recent years), Argentina, Belize, Brazil, Colombia, Dominica, Dominican Republic, French Guyana, Fiji (but not in recent years), Ghana, New Guinea, Jamaica, Martinique, Mascareigne Islands, Madeira, Malagasy Republic, Malawi, Mauritius, Mexico, Mozambique, Nevis, Panama, Papua New Guinea, Puerto Rico, Reunion, South Africa, St. Kitts and Nevis, St. Lucia, St Vincent and the Grenadines, Swaziland, Zimbabwe. In palm trees X. v. pv. vasculorum occurs only rarely. In Australia, Mauritius and Reunion gumming disease of sugarcane caused sporadic epidemics (Hughes, 1961), but appears to be of low economic importance nowadays. Control Use of healthy planting material (cuttings), hygiene and disinfection of cutting knifes, and use of low susceptibility or resistant varieties (Ricaud and Autrey, 1989) and early spraying with insecticides to reduce vector populations are the only way to prevent or control diseases caused X. v. pv. vasculorum.

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Fig. 162 Top:

Typical, large black spots, surrounded by a light green or yellow halo, caused by Acidovorax avenae susbsp. cattleyae on mature Cattleya orchid. Natural infection. Bottom: Dark brown spots, surrounded by a yellow halo and complete necrosis of leaves, caused by Acidovorax avenae subsp. cattleyae on a young Vuylstekaera orchid. Natural infection.

Examples of bacterial diseases – Orchids

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B Bacteria pathogenic to dicotyledonous plants 5. Orchids 5.1

Acidovorax avenae subsp. cattleyae (Pavarino 1911) Willems, Goor, Thielemans, Gillis, Kerster and De Ley 1992 Syn. Pseudomonas cattleyae. On the basis of DNA:DNA hybridization, PAGE and carbon utilization studies Willems et al. (1992) placed several species formerly classified in the genus Pseudomonas in the new genus Acidovorax. In Acidovorax avenae three subspecies were recognized: subsp. avenae (on oat), subsp. citrulli (on watermelon) and subsp. cattleyae (on orchids).

Bacterial leaf spot and bud rot or brown spot of orchids Main hosts: Cattleya spp., Phalaenopsis and Paphiopedilum (Venus’ slipper) spp. and hybrids. Also Catasetum spp., Cypripedium spp. (Lady’s slipper), Dendrobium spp., Doriaenopsis sp., Epidendrum spp., Epiphronitis veitchii, Ionopsis utricularioides, Miltonia sp., Oncidium spp. Ornithocephalus bicornis, Renanthera sp., Rodricidium, Rodriguezia, Rhyhchostylis spp. Sophronitus carnus, Trichocentrum sp., Vanda spp., Vanilla sp. and Vuylstekaera sp. have been reported as natural hosts. Symptoms and transmission: Small water-soaked spots are the first symptoms caused by A. a. pv. cattleyae. These spots enlarge rapidly under humid conditions and turn brown to black, often surrounded by a yellow halo. In advanced stages of the infection, spots are sunken and invaded by secondary organisms (Fig. 162). Spots may coalesce and whole leaves and plants are killed especially when the growing point becomes infected. Spots can be found on seedlings (where the infection can be devastating), but also on older plants (especially in Cattleya). Infection occurs through wounds and stomata. Overhead irrigation and manipulations during cultivation can cause rapid dispersal of the pathogen in greenhouses. Geographical distribution and importance The disease was first described by Pavarino in Italy in 1911, the causal organism in 1946 by Ark and Thomas in the USA. Australia (Stovold et al., 2001), Italy, The Netherlands, Philippines, Portugal, Taiwan (Huang, 1990), USA, probably Venezuela (Trujillo and Hernández, 1999). Especially in seedlings the disease can cause heavy losses. Control A combination of high temperature and high humidity should be avoided if possible. In an early stage attempts can be made to cut out the infected spots, after which the cut surface should be dusted with an antibiotic (if permitted). When the infection is widespread in the greenhouse spraying with a solution of natriphene or soaking of whole plants in orthophenylphenol, natriphene or Physan (quartenary ammonium compound) for c. 1 h is often effective in reducing the disease. Avoidance of overhead irrigation, splash water, disinfection of tools and isolation of infected plants are also helpful in control. Sprays with copper compounds or benzalkonium chloride (if permitted) can be applied as prevention.

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Fig. 163 Top:

Typical brown rot, surrounded by a yellow halo in the heart of the plant caused by Erwinia cypripedii on mature Cypripedium maudiae orchid. Natural infection. Bottom: Same infection as described above, on Paphiopedilum orchid. Natural infection.

Examples of bacterial diseases – Orchids

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Erwinia cypripedii (Hori 1911) Bergey, Harrison, Breed, Hammer and Huntoon 1923 Syn. Pectobacterium cypripedii

Brown rot of orchids Main hosts: Cypripedium and Paphiopedilum orchid spp. and their respective hybrids. Aerides japonicum, Phaleonopsis spp. and Carica papayae (pawpaw) have also been reported as natural hosts. Symptoms and transmission: On orchids E. cypripedii initially causes water-soaked round to oval leaf spots that enlarge, turn dark brown and become slightly sunken. When the leaf base and growing point are infected, plants rot totally and die (Fig. 163). In pawpaws water-soaked spots turn black and when lesions coalesce a black rot develops on leaves and stems. On the fruits black spots may be observed (Leu et al., 1980). It is not clear whether this disease on pawpaw is similar to that described by Gardan et al. (2004). In their study Gardan et al. noted similarity of their strains to E. cypripedii. Those authors attributed the disease to a newly described species: Erwinia papayae. The disease and organism may be spread by overhead irrigation, manipulations during cultivation and with diseased transplants. Geographical distribution and importance First described from Japan by Hori in 1911. Australia, Japan, The Netherlands, South Africa, Taiwan, USA. Control Good ventilation and keeping roots aerated during cultivation and rapid drying of plants after watering are preventive measures to avoid rot caused by E. cypripedii. Compost should be dry before use and should not be too acid. Avoidance of overhead irrigation, splash water, disinfection of tools and isolation of infected plants are also helpful in control. Infected plants should be removed and destroyed. When the infection is widespread in the greenhouse spraying with a solution of natriphene or soaking of whole plants in orthophenylphenol, natriphene or Physan (quartenary ammonium compound) for c. 1 h is often effective in reducing the disease.

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Agriculture Canada

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Fig. 164 Top left:

Symptoms of ring rot in potato (Solanum tuberosum): mottling, intercostal yellowing, brown necrosis along leaf margins and slight wilting. Natural infection. Top right: Same infection as above, with detail of curling leaf margins and intercostal yellowing. Centre left: Typical tuber symptoms of ring rot. Yellowing of vascular bundles with exuding tissue debris and bacterial slime, separation of inner and outer cortex of vascular tissue and blackening of tissues due to secondary invaders. Centre right: Tissue debris can be pressed out of vascular bundles in the case of ring rot. In infections of brown rot (Ralstonia solanacearum) slime exudes spontaneously from vascular tissue that is still firm. Bottom left: Eggplant (Solanum melongena) ‘Black Beauty’ inoculated with a potato extract from heel ends of tubers with latent infection of ring rot, showing glassy spots (red arrows) as early symptoms of the infection with Clavibacter michiganensis subsp. sepedonicus. Bottom centre: As bottom left, but later stage of the infection with intercostal yellowing and wilting. Bottom right: Symptoms of infection with Verticillium dahliae that may be very similar to those of C. m. subsp. sepedonicus.

Examples of bacterial diseases – Arable and cash crops

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6. Arable and cash crops 6.1

Clavibacter michiganesis subsp. sepedonicus (Spiekermann and Kotthoff 1914) Davis, Gillaspie, Vidaver and Harris 1984 Syn. Corynebacterium sepedonicus

Bacterial ring rot Bactériose annulaire, flétrissement bactérien Bakterienringfäule

Main hosts: Potato (Solanum tuberosum). After artificial inoculation Lycopersicon esculentum (tomato) and S. melongena (eggplant) also become infected. Eggplant is used as a test plant in official testing schemes (see European Communities, 1987). A finding of latent infection in Beta vulgaris var. esculenta (sugar beet) and sugar beet seed (Bugbee et al., 1987; Bugbee and Gudmestad, 1988) has never been substantiated. Symptoms and transmission: First symptoms are slow wilting, initially limited to the margins of leaves low on the stem. Later leaves show chlorotic, yellow to orange, intercostal areas (Fig. 164 top). Infected leaflets, leaves and even stems may become necrotic and eventually die. Symptoms can be easily missed or mistaken for senescence symptoms since they are usually observed only late in the season. In tubers the first symptoms are a slight glassiness of the tissue without softening round the vascular system, particularly near the heel end. Later (parts of) the vascular ring show(s) a yellowish coloration and when the tuber is gently squeezed, pillars of cheese-like material emerge, containing millions of bacteria (Fig. 164 centre). When vascular tissue turns brown symptoms may resemble those of Ralstonia solanacearum (Fig. 207 top right). When larger parts of the vascular ring are attacked the outer cortex may become separated from the inner cortex. Then reddish-brown cracks can also appear on the surface of the tuber. In advanced stages of infection secondary pathogens may cause other symptoms of (soft) rot that may obscure the true symptoms of ring rot (Fig. 164 centre left). In exceptional cases the vascular ring does not turn yellow, but black. C. m. subsp. sepedonicus is transmitted mainly by (latently) infected seed and cutting knifes or machines; it is not true seed transmitted. Geographical distribution and importance EPPO A2 quarantine pest Described for the first time by Spieckermann and Kotthoff in 1914 from Germany, the disease was not found here between the 1940s and 1980s but reappeared. In France the disease occurred but was eradicated. Austria, Belgium, Canada, China, Czech Republic, Denmark, Estonia, Finland, Greece (Crete), Germany, Japan, Kazakhstan, Korea DPR, Korea Republic, Latvia, The Netherlands, Norway, Poland, Russia, Sweden, Taiwan, Ukraine, USA, Uzbekistan. The disease was particularly damaging in temperate areas (growth temperature optimum of C. m. subsp. sepedonicus is 21ºC, see Fig. 132) when seed was cut and picker-type planting machines used, with damage of up to 50% crop loss. Usually the disease is much less damaging (Sletten, 1985; Manzer et al., 1987). Resistance is only tolerance in potato varieties. Control Healthy seeds through seed certification, avoidance of cutting seed or proper disinfection of cutting knifes/machines and strict hygiene, with particular attention to disinfection of packaging, storage and transport facilities and materials (grading machines) are efficient in reducing ring rot and minimizing its effects.

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Fig. 165 Top left: Top right: Bottom:

Severely yellowing and stunted potato plant (Solanum tuberosum), due to infection of Erwinia carotovora subsp. atroseptica, which was already present in the mother tuber. Natural infection. Characteristic symptoms of blackleg in potato, a black discoloration of the stem base and internal black rotting of the pith and brown discoloration of the vascular bundles. These stems have a bad smell of rotting fish. Similar infection as described above. Field symptoms of blackleg in a potato crop: yellowing, growth reduction, wilting and curling of leaflets. Natural infection.

Examples of bacterial diseases – Arable and cash crops

Erwinia carotovora subsp. atroseptica (Van Hall 1902) Dye 1969 Syn. Erwinia atroseptica; Pectobacterium atrosepticum

Potato blackleg Jambe noir de la pomme de terre Schwarzbeinigkeit der Kartoffel

Main hosts: Potato (Solanum tuberosum). Other hosts like Allium cepa (onion), Asparagus officinalis (asparagus), Brassica oleracea var. botrytis (cauliflower), Brassica oleracea var. capitata (cabbage), Cichorium intybus and Daucus carota (carrot) have also been mentioned as hosts, but it is not sure whether these hosts are attacked by strains belonging to other Erwinia carotovora subspecies such as subsp. betavasculorum, odorifera or wasabiae or by intermediate strains. A slightly different strain causing blackleg of potato in Brazil has been described as E. c. subsp. brasiliensis (De Boer, 2003; Duarte et al., 2003). Symptoms and transmission: Shortly after emergence or later plants show yellowing and wilting of leaves and stunting of some or all stems. Leaflets often show curled margins (Fig. 165). Wilting symptoms are strongest at midday. In later stages whole stems and plants wilt completely and collapse. When stems are pulled out of the ground they show a typical black discoloration and have a typical bad smell of rotting fish. The vascular tissue may be brown. Tubers show a brown to black rot that is glassy, but not soft, like the soft rot caused by E. c. subsp. carotovora. The infection usually starts at the heel end and can also be observed on the outside of the tuber as sunken purple to black zones around the heel end (Fig. 166). E. c. subsp. atroseptica is mainly transmitted by seed, but in the field wind-driven rain, insects and soil water also play a role. The bacterium is active at lower (15-20ºC) temperatures than E. chrysanthemi, causing stem rot, and is dependent on wounds. It can survive on the surface, in lenticels and vascular and storage tissues of the stored tuber, in volunteer plants and also, although less easily, in the rhizosphere of weeds and in plant debris. External contamination can occur during harvest (Naumann et al., 1978). Geographical distribution and importance E. c. subsp. atroseptica is reported from all potato-growing areas on all continents. Substantial losses occur especially in cool climates and can be 10-25%, and also during storage or, especially when in combination with soft rot, caused by E. c. subsp. carotovora (Pérombelon and Kelman, 1980).

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Control Healthy seeds through seed certification, strict hygiene, careful cultivation (planting in moderately moist and warmed-up soil, adequate N and Ca nutrition, not cutting seed), good (dry) harvest and storage (quick drying and good ventilation) conditions and use of low susceptibility or resistant varieties are ways to prevent or control blackleg.

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Fig. 166 Left: Right:

Symptoms of blackleg, caused by Erwinia carotovora subsp. atroseptica on potato tubers. Purple to black necrotic, sunken spots around the heel end. Internal symptoms of blackleg: rotting tissue that is still firm. Natural infection.

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Fig. 167 Symptoms of common scab of potato (Solanum tuberosum), caused by Streptomyces scabiei or related species (a and b) and very similar symptoms of so-called powdery scab caused by the plasmodiophorid (usually included in the fungi) Spongospora subterranea (c) (see text). In the scabby blemishes of Spongospora brown spore balls can be found and the blemishes are often situated like a girdle around the tuber. Colour drawing. Bottom: Netted scab, caused by Streptomyces reticuliscabiei: very superficial scabby lesions on the potato skin. Natural infection. PD

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Streptomyces scabiei (ex Thaxter 1892) Lambert and Loria 1989 Syn. Streptomyces scabies. Related species S. europaeiscabiei and S. stelliscabiei also cause common scab of potato in Europe, S. turgidiscabies, S. luridiscabiei, S. puniciscabiei and S. niveiscabiei were found to cause common scab in Japan and Korea (Park et al., 2003) as well as other Streptomyces spp., namely S. acidiscabiei and S. caviscabiei in N. America. A related disease, called netted scab of potato, is caused by S. reticuliscabiei (Bouchek-Mechiche et al., 2000; Goyer et al., 1996). So-called russet scab, observed in N. America, has sometimes been associated with S. aureofaciens (Faucher et al., 1993).

Common scab Gale commun de la pomme de terre Gewöhnlicher Kartoffelschorf

Main hosts: Potato (Solanum tuberosum). Arachis hypochea (groundnut, in rotation with potato, de Klerk et al., 1997), Beta vulgaris (beet), B. vulgaris var. rubra (red beet), Brassica rapa (turnip rape), Daucus carota (carrot) and Pastinaca sativa (parsnip) have also been reported as hosts for S. scabiei. Symptoms and transmission: Relative to the susceptibility of the potato variety and reactions of the potato after infection (degree of phellem and phellogen formation), the following types of scab can be recognized: superficial scab, characterized by irregular lesions with scabby margins; deep scab, as for superficial scab, but due to aggressiveness of the pathogen deep scabby cavities are formed; elevated scab, due to strong defence reactions of the host, wart-like scabby pustules are formed. Sometimes these different symptoms are present on the same tuber. Symptoms are easily confused with those caused by the plasmodiophorid (usually included in the fungi) Spongospora subterranea; however, in the scabby blemishes caused by this pathogen spore balls of the fungus can be found. Moreover the blemishes are often situated like a girdle around the tuber (Fig. 167 top a-c). Streptomyces spp. are soil inhabitants and are spread by soil particles and planting material, including potato seed. They infect young tubers through lenticels. Depending on the situation in the soil a scabby seed potato crop can produce a scabfree crop in the next season and a scab-free crop can give a heavily scabbed crop the next year. Geographical distribution and importance S. scabiei was described for the first time by Thaxter from the USA in 1891 as Oospora scabies and is ubiquitous. It is also found in unfarmed land and potato scab is widespread in all potatogrowing areas, especially in light soils. Damage caused by scab is mainly aesthetical. Only when potatoes are heavily scabbed and badly stored secondary soft rot organisms may cause serious losses. Control Common scab can be fairly well controlled by irrigation; just after tubers have set and when weather conditions are warm and dry, irrigation should be continued. S. reticuliscabiei is not sensitive to this treatment. Rotation with green manure crops (e.g. legumes, in order to stimulate antagonism, see Wiggins and Kinkel, 2005), use of physiological acid fertilizers and avoidance of excess calcium can control scab to a certain extent. There is much difference in susceptibility to scab in potato varieties.

Lyle J. Buss, Univ. of Florida, Ent. & Nem. Dept., Gainsville, FL, USA

Phil Sloderbeck, Kansas State Univ. Southwest Res. & Extension Center, Garden City,

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Fig. 168 Top left: Top right: Centre left: Centre right: Bottom:

Typical leaf spots on the lower side of a leaf of cotton (Gossypium hirsutum), caused by Xanthomonas axonopodis pv. malvacearum. Natural infection. Same infection as above, with detail of angular leaf spots and longitudinal necrosis along the main veins. Mating cotton stainer (Dysdercus suturellus), a bug that transmits X. a. pv. malvacearum into the cotton bolls. The insect contains bacteria internally and externally. Cotton fleahopper (Pseudatomoscelis seratius), which is also able to transmit X. a. pv. malvacearum in the USA. Bolls of cotton showing diverse stages of spot development after natural infection with X. a. pv. malvacearum.

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Xanthomonas axonopodis pv. malvacearum (Smith 1901) Vauterin, Hoste, Kersters and Swings 1995 Syn. Xanthomonas malvacearum

Angular leaf spot Taches angulaires du cotonnier ‘Eckige Blattflecken’-Krankheit der Baumwolle

Main hosts: Gossypium hirsutum (upland cotton) and G. barbadense (sea island or pima cotton). G. herbaceum, G. populifolium (wild cotton), Hibiscus vitifolius (weed in Indian cotton fields) and Hibiscus rosa-sinensis (Chase, 1986), and outside the family of Malvaceae Lochnera pusilla (weed in Indian cotton fields), Ceiba pentandra (kapok tree) and Jatropha curcas (purging nut) have been reported as natural hosts. Symptoms and transmission: Small green, water-soaked spots are the first symptoms on the underside of the leaves. These spots become angular and turn brown to black and are visible on the upper surface. Elongated spots can also occur along the main veins (Fig. 168 top left and right). Lesions dry and darken with age and leaves may be shed prematurely resulting in extensive defoliation. Spots can also occur on cotyledons (seedling blight) and on stems and bolls (Fig. 168 bottom). When lesions girdle main branches these may break, causing losses of fruits (called blackarm). When the bacteria penetrate the boll cortex they can cause internal bollrot and seed infection. Bacteria can also invade the boll and cause bollrot after introduction by sucking insects such as the cotton stainer (Dysdercus spp., Fig. 168 centre left, Verma et al., 1981) in Africa, and the tarnished bug (Lygus vosseleri) and the cotton fleahopper (Pseudatomoscelis seratius, Fig. 168 centre right) in the USA. Bacteria overwinter on seed and plant debris. Penetration is through stomata or wounds. Bacteria are spread by infected seed and in the field from crop residues and infected plants by water, and wet conditions are necessary for disease development. Winddriven rain, hail and sandblasting significantly increase disease severity. Geographical distribution and importance Widespread in all cotton-growing regions of the world. In Europe reported from Bulgaria, Greece, Italy, Moldavia, Poland, Romania, Russia, Spain, Ukraine and former Yugoslavia. The disease was a major problem, with high crop losses, after introduction into Africa in the first and the USA and Australia in the second half of the last century. After the development of resistant varieties the disease has become a more minor problem. Control In G. hirsutum the highest level of resistance is available. There are 10 major genes for blight resistance (B1-B10) that were and are used to develop resistant varieties (Hillocks and Chinodya, 1988). Integrated control using the following measures is successful in controlling angular leaf spot in cotton: use of high-quality, disease-free, acid-delinted seed and resistant varieties if available, crop rotation, regular monitoring in the field, removal or ploughing-in of plant debris as much as possible, avoidance of cultivation and movement of equipment through fields when foliage is wet and avoidance of sprinkler irrigation using furrow irrigation instead.

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Fig. 169 Top:

Hazelnut trees (Corylus avellana) in autumn. The tree in the foreground is healthy. The tree in the background is infected by Pseudomonas avellanae. The tree wilted and leaves became brown and necrotic, but remained attached to the tree. White strips were used to locate isolation places in an experiment. Artificial infection. Bottom: Same infection as above, but here only a part of the tree wilted and died. Natural infection.

Examples of bacterial diseases – Fruit and nut trees, fruits

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Pseudomonas avellanae Janse, Rossi, Angelucci, Scortichini, Derks, Akkermans, De Vrijer and Psallidas 1997 Syn. Pseudomonas syringae pv. avellanae

Canker or decline of hazelnut Chancre du noisetier Haselnusskrebs

Main hosts: Hazelnut (Corylus avellana). Symptoms and transmission: The first symptoms are rapid wilting of twigs, branches and trees during spring and/or summer. Leaves usually remain attached to the twigs after withering (Fig. 169). The bacterium infects mainly in autumn through leaf scars and, from there, the pathogen can move systemically within the plant, with the potential to reach the roots and cause cankers on the trunk and branches. In severe infections trees are completely killed in one season. Acidic soils and spring frost probably are important for predisposing trees. Not much is known about the mode of transmission.Very similar symptoms, but with a less aggressive disease development are caused by strains that are not P. avellanae but closely related to Pseudomonas syringae pv. syringae (Scortichini et al., 2002) and recently described as P. syringae pv. coryli (Scortichini et al., 2005). Geographical distribution and importance First reported in Greece in 1976 (Psallidas and Panagopoulos, 1979) and the causal organism first described from Greece by Psallidas in 1984. The disease has been observed in Greece and Italy. P. avellanae has caused substantial losses in N. Greece and especially also in central Italy (Scortichini et al., 2001). Control Preventive sprays with copper oxychloride during leaf fall and budbreak are the only way to try to control this disease. Trials with acibenzolar-S-methyl to induce systemic-acquired resistance gave a promising 25-30% disease reduction, compared to untreated trees (Scortichini et al., 2002).

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Fig. 170 Top:

Leaf of kiwifruit (Actinidia deliciosa) with typical brown angular leaf spots, sometimes surrounded by a narrow yellow halo, caused by Pseudomonas syringae pv. actinidiae. Natural infection. Bottom: Internal glassy to brown necrotic discoloration of the bark and dark brown necrotic spots in the wood, due to infection by P. s. pv. actinidiae. Natural infection.

Examples of bacterial diseases – Fruit and nut trees, fruits

Pseudomonas syringae pv. actinidiae Takikawa, Serizawa, Ichikawa, Tsuyumu and Goto 1989 Syn. Pseudomonas syringae pv. avellanae

Bacterial canker and leaf spot of kiwifruit Main hosts: Kiwifruit (Actinidia deliciosa) and Chinese gooseberry (Actinidia chinensis). The wild species A. arguta and A. kolomikta (Ushiyama et al., 1992) have also been reported as natural hosts. Symptoms and transmission: Early infections in springtime are glassiness of canes sometimes with bacterial slime oozing from lenticels and small water-soaked leaf spots that become angular and brown and necrotic and are often surrounded by a narrow yellow halo; on the lower leaf surface they are sometimes covered with slime (Fig. 170 top). Flower buds may turn brown and wither. In mid-winter cankers develop on trunks and vines. Also here slime may be found and when the bark is removed a reddish-brown discoloration with a glassy margin can be observed (Fig. 170 bottom and Fig. 171). Infected limbs may be girdled and suckers formed in the healthy tissue below the canker (Serizawa, 1994). Remarkably some strains of the pathogen in Japan produce phaseolotoxin as does P. syringae pv. phaseolicola, a pathogen of bean (Phaseolus vulgaris). Very similar disease symptoms can be caused by P. syringae pv. syringae and P. viridivlava or related Pseudomonas species (Hu et al., 1999; Young et al., 1997). The pathogen is spread by planting material, wind-driven rain and (pruning) tools in the nursery or orchard. Geographical distribution and importance First described by Takikawa et al. (1989) from Japan. Japan, Italy (Scortichini, 1994), Korea and New Zealand. Disease develops most rapidly at temperatures between 10 and 20ºC at high humidity and stops when weather conditions become more warm and dry. The bacterium is not transmitted by seed. Control Healthy planting material and preventive sprays with copper compounds or antibiotics (if allowed) in areas where the disease occurs are the only ways to control bacterial canker of kiwifruit. Copper and streptomycin resistance has been observed (Nakajima, 1995; Vanneste and Voyle, 2003).

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Fig. 171 More advanced canker symptoms in a small branch of kiwifruit (Actinidia deliciosa), as shown in Fig. 170 bottom, caused by Pseudomonas syringae pv. actinidiae. Natural infection.

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Fig. 172 Leaf of mulberry (Morus latifolia) showing brown necrotic spots on the lamina and on the larger veins as well as distortion, caused by Pseudomonas syringae pv. mori. Natural infection. Bottom: Infection as above, but only vein necrosis and distortion. Natural infection.

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Pseudomonas syringae pv. mori (Boyer and Lambert 1893) Young, Dye and Wilkie 1978 Syn. Pseudomonas mori

Mulberry blight or mulberry leaf spot Bacteriose du mûrier Maulbeerbrand

Main hosts: White mulberry (Morus alba). M. alba var. tartarica, M. bombycis (Japanese mulberry), M. kagayamae, M. latifolia, M. multicaulis, M. nigra (black mulberry) and M. rubra (red mulberry) have been reported as natural hosts of P. s. pv. mori. Symptoms and transmission: On the leaves water-soaked spots that easily coalesce are the first symptoms. These spots later turn brown to black necrotic, sometimes surrounded by yellowing tissue. On the larger veins elongated brown slightly sunken necrotic spots can be observed and also bacterial slime drops. Leaves often become distorted (Fig. 172 top and bottom). On young and second-year shoots brown sunken streaks can also develop from which cream-yellowish bacterial slime often oozes. From lenticels this slime sometimes exudes as bacterial strands. When lesions enlarge cracks appear and when they girdle shoots they shrivel and die. The pathogen attacks mainly the parenchyma of leaf and bark tissues (Smith, 1920). Trees rarely die, but may become stunted and unprofitable. The bacterium is not seed transmitted but can overwinter in leaf debris in the soil. Geographical distribution and importance First (incompletely) described by Boyer and Lambert from France in 1893. Australia, Brazil, Bulgaria, Canada, former Czechoslovakia, China, Dominican Republic, France, Germany, Hong Kong, Hungary, Iran, India, Italy, Japan, Korea DPR, Republic of Korea, The Netherlands (van der Tuin et al., 1995a), New Zealand, Pakistan, Romania, South Africa, Taiwan, Tanganyika, Tanzania, Turkey, Uganda, UK, former USSR and Zimbabwe. Control Use of healthy planting material, good cultural practices (water management, no overhead irrigation, and pruning of dead shoots in autumn) and eventually preventive copper compound sprays are the only ways to control this disease.

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Fig. 173 Canker on sweet cherry (Prunus avium) with exuding wound gum, caused by Pseudomonas syringae pv. morsprunorum. Natural infection.

Examples of bacterial diseases – Fruit and nut trees, fruits

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Pseudomonas syringae pv. morsprunorum (Wormald 1931) Young, Dye and Wilkie 1978 Syn. Pseudomonas mors-prunorum

Bacterial canker of stone fruits, blossom blast, gummosis, leaf spot of stone fruits Chancre bactérien du cerisier Bakterienbrand des Steinobstes

Main hosts: Sweet cherry (Prunus avium). P. amygdalus (almond), P. armeniaca (apricot), P. cerasifera (cherry plum), P. cerasoides (wild Himalayan cherry), P. cerasus (sour cherry), P. domestica (European plum), P. institia (damson), P. persica (peach), P. pissardi (Pissardi plum, purple leaf plum), P. salicina (Japanese plum) and P. triloba (flowering almond) have also been reported as natural hosts. Symptoms and transmission: Main symptoms are slightly sunken, brown to black cankers on shoots, branches and limbs that often show strong formation of wound gum during late spring and summer (Fig. 173). Leaves may yellow, and may show curling margins and wilting. When stomatal infection takes place, water-soaked spots are formed on the leaves, which turn brown to black in a later stage, often surrounded by a narrow yellow halo. The centre of the spot may drop out (shothole symptom). Spots can also develop on fruits. Buds may be killed from the inside and also blossom blight may occur. Symptoms are very similar to those caused by P. s. pv. syringae. The pathogen infects mainly through leaf scars and wounds and is spread from cankers and other diseased tissues and as an epiphyte by splashing rain. Bacteria can survive in cankers or in dormant buds (and can therefore be spread by planting material also). During the growing season epiphytic populations are present on all plant parts. Frost enhances the infection, as in the case of infections by P. s. pv. syringae. Hail and storms enhance formation of stomatal infection and leaf spots. Races with some host specificity have been determined using bacteriophages (Crosse, 1966; Sundin et al., 1988; Hatting et al., 1989). Geographical distribution and importance Australia, Canada, former Czechoslovakia, Denmark, Finland, France, Germany, Greece, India, Ireland, Italy, Japan, Lebanon, The Netherlands, Norway, Poland, Romania, South Africa, Sweden, Switzerland, UK, Ukraine, USA, former Yugoslavia. P.s. pv. morsprunorum produces coronatine toxin, but not syringomycin (Liang et al., 1994). Cool wet weather stimulates disease development. When cankers girdle branches or stems substantial damage to trees may occur. In most countries the disease is a minor problem. In British Columbia, Canada, and the USA substantial damage has been reported, especially in young orchards. Control Preventive spraying with copper compounds (copper resistance has not yet been observed) or antibiotics (if allowed) in critical times in spring and autumn, adequate fertilization and pruning and burning of diseased parts (in summer under dry conditions) are main factors in the control of bacterial canker of stone fruits (Sayler and Kirkpatrick, 2003). Furthermore cultural practices such as proper water management may be effective in prevention of disease. Differences in susceptibility are present in the different cultivars of host species and in certain rootstocks (Garrett, 1986).

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Fig. 174 Top:

Leaf of red currant (Ribes rubrum) showing tiny water-soaked spots at the lower leaf side, caused by Pseudomonas syringae pv. rubri. Natural infection. Bottom: Infection as above, advanced stages of infection with necrosis of larger leaf parts. Natural infection.

Examples of bacterial diseases – Fruit and nut trees, fruits

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Pseudomonas syringae pv. ribicola (Bohn and Maloit 1946) Young, Dye and Wilkie 1978 Syn. Pseudomonas ribicola

Bacterial leaf spot Taches angulaires

Main hosts: Golden currant (Ribes aureum), Red currant (Ribes rubrum). Symptoms and transmission: Inconspicuous, round to angular water-soaked leaf spots are the first symptoms (Fig. 174 top). These spots later turn reddish-brown and necrotic with a slightly sunken centre. The tissues that immediately surround the spots may turn light brown and necrotic. When spots coalesce larger parts of the leaf turn necrotic (Fig. 174 bottom). Young leaves may be distorted. Spots may also occur, especially after hailstorms, on petioles and small shoots, as well as on the fruits that ripen prematurely. In severe cases a strong defoliation also takes place (Bohn and Maloit, 1946; van der Tuin et al., 1995b). Not much is known about the mode of transmission. The symptoms are very similar to those caused by P. s. pv. syringae on other hosts and biochemically the bacterium is closely related, but its O polysaccharide (OPS) moiety of the lipopolysaccharide (LPS) separates it clearly from other pvs of P. syringae in a study using monoclonal antibodies against OPS (Ovod et al., 2000). Geographical distribution and importance Bohn and Maloit (1945) first reported the disease and the bacterium was first described by Bohn and Maloit (1946) from the USA. The Netherlands and USA. Control Use of healthy planting material, good cultural practices (water management, no overhead irrigation, and pruning dead shoots in autumn) and eventually preventive copper compound sprays are the only ways to control this disease.

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Fig. 175 Top left:

Advanced symptoms of bacterial knot disease of ash (Fraxinus excelsior) in (A). Excrescences of dark, necrotic proliferated bark tissue with bacterial cavities and cork layers. In (a) blister-like swellings caused by mining of larvae of the ash twig miner, Prays fraxinella, from which infections of P. s. pv. fraxini may start as shown in (B). In (C) scab symptoms caused by the ash bark beetle (Leperisinus varius), which may be confused with those caused by P. s. pv. fraxini. Top right, top: Early symptom of ash knot disease, starting from the borehole of the ash twig miner, Prays fraxinella. Yellowish slime exudes from the swelling. Natural infection. Top right, centre: Ash twig miner, Prays fraxinella. Top right, bottom: Ash bark beetle, Leperisinus varius. Bottom left: Large excrescenses on an ash tree, caused by P.s. pv. fraxini. Natural infection. Bottom centre: Regular patches of scab on an ash tree, caused by the ash bark beetle. Bottom right: Bacterial cankers on ash, with proliferating bark still present. Natural infection.

Examples of bacterial diseases – Fruit and nut trees, fruits

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Pseudomonas savastanoi pv. fraxini Janse 1982 Syn. Pseudomonas savastanoi; P. syringae subsp. savastanoi; P. savastanoi pv. savastanoi

Bacterial knot disease or bacterial canker of ash tree Chancre du frêne Bakterienkrebs der Esche

Main hosts: Common ash tree (Fraxinus excelsior) and some of its cultivated varieties. Symptoms and transmission: First symptoms are red-brown, lens-shaped blisters on the bark of younger branches or the trunk, often at wounds caused by the twig miner Prays fraxinella or lenticels (Fig. 175 top left and top right, top). In later stages the swellings burst and brown-black proliferated dead bark tissue with vertical and lateral cracks develops (Fig. 175 top left A). Finally large excrescences develop, consisting of swollen bark tissue that contains bacterial cavities surrounded by cork layers (formed in defence by the tree) and necrotic, black bark parenchyma (Fig. 175 bottom left). When secondary parasites, notably fungi, aggravate the syndrome and cambium is destroyed, this leads to formation of open wounds (Fig. 175 bottom right). The proliferated, thickened diseased bark is still present at the margins. The bacterial cankers may be confused with older stages of cankers caused by the fungus Nectria galligena because sporodochia-like cushions of hyphae may resemble bacterial swellings. N. galligena cankers are always accompanied by dieback of branches and leaf drop. Bacterial excrescences may also be confused with damage caused by boring of adult ash bark beetles, Leperisinus varius (Fig. 175 top right, bottom). The beetles return every year to the same places so that scab-like patches develop that are regularly dispersed, more regular than is the case with the bacterial infection (Fig. 175 bottom centre) on the trunk and older limbs. On younger branches callus swellings that developed in reaction to the boring of the beetles may be confused with bacterial excrescences. The bacterium is a wound parasite and it often infects through wounds made by the twig leaf miner Prays fraxinella (Fig. 175, top right, centre). Splashing rain and branches that rub against each other can spread P. s. subsp. fraxini. The bacterium can survive in the excrescences on a tree for years and it has been found as an epiphyte on ash leaves in springtime (Riggenbach, 1956; Janse, 1981, 1982). Geographical distribution and importance Noack described the disease as a bacterial disease for the first time in 1893 from Germany. Brown (1932) in the USA described the bacterium, studying material from Austria. Austria, Belgium, former Czechoslovakia, France, Russia, Switzerland, UK, former Yugoslavia. Growth of young trees may be severely restricted and eventually they may die, but usually the disease is bad from an aesthetic viewpoint only. Larger trees will only show restricted growth of branches and deformation of the wood, especially when the open canker form is prevalent. Control Chemical control is not possible. The only ways of control are removing and burning of infected branches when still possible. Otherwise infected trees should be removed and burned. In isolated cases, where no industrial use of the trees is intended control could be considered as not necessary. There is some resistance present in some varieties of ash tree.

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Fig. 176 Top left:

Typical irregular, smooth galls on olive (Olea europaea), caused by Pseudomonas savastanoi pv. savastanoi. Galls formed after artificial inoculation. Top right, top: Olive fly (Bactrocera = Dacus oleae), which plays a role in transmission of P. s. pv. savastanoi as determined by Petri (1910) in Italy. Very few reports after his detailed study have confirmed this relationship subsequently. Top right, bottom: Galls on inflorescences of Nerium oleander caused by P. s. pv. nerii. The high frequency of flower and inflorescence infection (where even pistils become infected) implies an insect vector, but this has not been substantiated till now. Bottom left: Infection as in top right, but in an earlier stage. Swollen buds and distorted flowers. Bottom right: Flower of Nerium oleander, just before opening, showing infected, blackened and shrivelled pistil from which a pure culture of P. s. pv. nerii was isolated.

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Pseudomonas savastanoi pv. savastanoi (ex Smith 1908) Gardan et al. 1992 and P. savastanoi pv. nerii (Janse 1982) Syn. Pseudomonas savastanoi; P. syringae subsp. savastanoi pv. oleae; P. syringae pv. savastanoi Syn. Pseudomonas nerii; P. syringae subsp. savastanoi pv. nerii; P. syringae pv. savastanoi

Olive knot, Oleander knot Bactériose d’olivier, Bactériose du laurier-rose Tuberkelkrankheit des Ölbaums, Tuberkelkrankheit des Oleanders

Main hosts: Olive tree (Olea europaea). Japanese privet (Ligustrum japonicum), jasmine (Jasminum spp.), Phillyrea, Spanish broom (Retama sphaerocarpa, Alvarez et al., 1998), all in the family of Oleaceae, have also been reported as hosts. The related P. s. pv. nerii causes very similar galls on Nerium oleander (Apocynaceae). Another variety of P. savastanoi was recently described as the cause of galls on Forsythia intermedia (golden bells, Besenyei and Hevesi, 2003). Symptoms and transmission: Irregular, smooth to warty galls or knots are formed mainly on shoots and branches (Fig. 176 top left and top right, bottom), but can also be found on roots, trunks, leaves, leaf petioles and fruit stems. Initially galls are soft and yellowish, but after some time they become hard and brown with necrotic areas. Symptoms are often related to wounds made by leaf drop (leaf scars), frost, insects, or as in olive, by man, e.g. during harvest of olives when branches are beaten with a stick, or as a result of careless pruning. P. s. pv. nerii can also infect inflorescences and flowers in Nerium oleander (Fig. 176 bottom left and right). In the latter host the bacterium can also spread internally via laticiferous ducts and produces a yellow halo around galls present on leaves. The inflorescences, fruits and leaves of Nerium often become distorted. Bacteria can survive in the gall tissue and can be spread from galls (from which they may exude as slime drops) by rain splash and insects (accidentally, like mining insects, but a more specific relationship exists for pv. oleae and the olive fly (Bactrocera = Dacus oleae Fig. 176 top right, top), where the bacterium was found in the intestinal tract and salivary glands of the fly (Petri, 1910), and from pruning tools and contaminated nursery stock. The bacterium can occur in an epiphytic phase, especially in spring and autumn (Ercolani, 1978). Geographical distribution and importance Savastano (1889) in Italy described the bacterium for the first time as Bacillus oleae tuberculosis. E.F. Smith in the USA (1908), who named it Bacterium savastanoi later, completed his description. Algeria, Argentina, Australia, Austria, Brazil, Colombia, Cyprus, Greece, France, Iran, Iraq, Israel, Libya, Mexico, Morocco, The Netherlands, New Zealand, Peru, Poland, South Africa, Spain, Sweden, Switzerland, Tanzania, Tunisia, Turkey, UK, Uruguay, USA, former USSR, former Yugoslavia. When knots are girdling shoots or branches they may die. Furthermore there is reduced growth and the disease has a negative effect on fruit yield and quality and flavour of the olive oil. Control Chemical control is not possible. The best ways of control are use of healthy planting material, avoiding wounds (careful harvesting), removal and burning of infected branches when still possible, and disinfection of pruning tools. Otherwise infected trees should be removed and burned. Preventive sprays with copper compound or antibiotics (if allowed) against epiphytic populations can be performed. There is some resistance present in varieties of olive tree (Varvaro and Surico, 1978).

T.R. Gottwald US Hort. Res. Lab. Orlando FL

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Fig. 177 Top left:

Typical, early symptoms of citrus canker: slightly raised, brown leaf spots (that become pustules in a later stage, see top right) on the lower side of a leaf of kaffir lime or jeruk perut (Citrus hystrix), caused by Xanthomonas axonopodis pv. citri. Natural infection. Top right: Grapefruit (Citrus paradisi) leaf showing galleries of the Asian citrus leaf miner (Phyllocnistis citrella) that, in the lower part, became ports of entry for X. a. pv. citri, that caused the typical wart-like pustules. Natural infection. Centre: Fruit of orange (Citrus sinensis) with typical wart-like pustules, superficially resembling California red scale (Aonidiella aurantii), caused by X. a. pv. citri. Natural infection. Bottom left: Five-day-old culture of X. a. pv. citri on YPGA agar, colonies c. 3 mm in diameter. Bottom right: Asian citrus miner (Phyllocnistis citrella). For leaf mine, caused by larvae, see top right.

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Xanthomonas axonopodis pv. citri (Hasse 1915) Vauterin, Hoste, Kersters and Swings 1985 Syn. Xanthomonas citri; X. citri group A (Asiatic strain); X. campestris pv. citri oriental canker strain. Strains causing relatively mild disease in Citrus in Mexico and S. America are named X. citri group B (cancrosis B), C (Mexican lime cancrosis) and D (Citrus bacteriosis) or X. a. pv. aurantifolii. Group E strains, causing citrus bacterial spot, are also named X. campestris pv. citrumelo. (Gabriel, 1989; Stall and Civerolo, 1991). Only the A strain or X. a. pv. citri is described here.

Citrus canker, citrus bacterial canker Chancre bactérien des agrumes Citrus-Krebs

Main hosts: Citrus spp., hybrids and cultivars. Grapefruit (Citrus paradisi) and lime (C. aurantiifolia) are highly susceptible. Sour orange (C. aurantium), lemon (C. limon) and orange (C. sinensis) are moderately susceptible. Furthermore Fortunella japonica (kumquat). F. margarita, Poncirus trifoliata, Severinia buxifolia and Swinglea glutinosa have been reported as natural hosts. Symptoms and transmission: Small spots (called cankers in this case, Fig. 177 top left) are the first symptoms on leaves, shoots, twigs and fruits and these spots become raised pustules or blister-like eruptions. On the leaves these pustules are first visible on the upper leaf surface. Later the lesions become up to 10 mm in size and brown and necrotic, with a depressed centre and sometimes surrounded by a yellow halo. On fruits (Fig. 177 centre) these lesions can be mistaken for scale insects (e.g. the California red scale, Aonidiella aurantii). The bacterium is a wound parasite and the citrus leaf miner (Phyllocnistis citrella, Fig. 177 top and bottom right) contributes to the spread and disease severity. Citrus canker is especially epidemic and damaging on seedlings and young trees, especially after storms (hurricanes) with rain under warm weather conditions. Fullgrown trees show much less disease and damage (Goto, 1992b). The bacterium can survive in (in a latent form) and on diseased shoots and discoloured bark tissue of the trunk. Geographical distribution and importance EPPO A1 quarantine pest The disease was first described by Stevens in 1914 and the bacterium by Hasse in 1915 from the USA. Originating from and widespread in Asia. Australia (eradicated), Argentina, Belau, Brazil, Caroline Islands, Cocos Islands, Comoros, Congo Democratic Republic, Ivory Coast, Fiji, Gabon, Madagascar, Mauritius, Mozambique (eradicated), Netherlands Antilles, New Zealand (eradicated), Micronesia, Palau, Papua New Guinea, Paraguay, Réunion, Seychelles, South Africa (eradicated), Uruguay, USA. Heavy losses have been reported in epidemics due to leaf drop and fruit loss due to premature fruit drop and fruits with spots that cannot be marketed or start to rot and have to be destroyed. Furthermore quarantine measures such as burning of trees and destruction of fruits may add to these losses (Goto, 1992b). Control Use of healthy planting material, and use of other measures as mentioned in Chapter VI, Table 27 in an integrated way have been applied in the control of Citrus canker with some success. Weather forecasting has been tried in Japan (Goto, 1992b). Resistance has been found especially in C. mitus (calamondin) and Fortunella (kumquat). C. reticulata (mandarin) is tolerant.

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Fig. 178 Top left:

Leaf of strawberry (Fragaria x ananassa) showing inconspicuous water-soaked leaf spots and an area where spots coalesced and the tissue became brown and necrotic with a yellow zone, typical for infection of Xanthomonas fragariae. Natural infection. Top right: Tiny brown spots with yellow halo, often along veins and reddish-purple to brown discoloured veins and sectorial leaf parts with yellowing on upper (upper photo) and lower side (lower photo) of a strawberry (Fragaria x ananassa) leaf, caused by X. arboricola pv. fragariae. This bacterium evokes symptoms rather similar to those of X. fragariae, but is not closely related in its genetic composition (also see Figs. 24 and 58). Natural infection. Bottom: Typical angular, small, water-soaked leaf spots on the lower side of a strawberry (Fragaria x ananassa) leaf, caused by X. fragariae. Natural infection.

Examples of bacterial diseases – Fruit and nut trees, fruits

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Xanthomonas fragariae Kennedy and King 1962 Angular leaf spot Taches angulaires Blattfleckenkrankheit

Main hosts: Cultivated strawberry and its varieties (Fragaria x ananassa which was originally a cross between F. chiloensis, beach strawberry from Chile, and F. virginiana, wild strawberry from N. America). Varieties emerging from crosses between F. x ananassa and F. vesca, the European wild strawberry, are also natural hosts. Symptoms and transmission: On the leaves very small up to 4 mm angular, shiny, water-soaked spots are first visible on the lower surface of the leaf and when the infected leaf is held against the light these spots are yellowish-translucent (Fig. 178 top left and bottom). Leaf spots enlarge and also become visible on the upper side of the leaf as reddish-necrotic spots. When spots coalesce larger leaf parts become reddish-brown and necrotic, sometimes surrounded by a yellow zone. These symptoms may be confused with those of another bacterium, X. arboricola pv. fragariae (Fig. 178 top right, Janse et al., 2001) and of fungi such as Diplocarpon earliana (leaf scorch), Gnomonia comari (leaf blotch) and Phomopsis obscurans. On the spots bacterial slime is often present, which dries as a silvery scale when the weather conditions are dry. Occasionally, under natural conditions, infection follows the major veins, resulting in water-soaked to black streaks along the veins. In heavy infections systemic infection of the crown may also occur. The disease often appears in late summer till late autumn with relatively cool (c. 20ºC) weather conditions, even cooler night-time temperatures and high humidity. When these conditions re-occur the infection has an alternating character. Spots may also occur on calyx, runners and crowns. These crown infections play an important role in the survival of the bacterium and infections to new leaves in the next season and long-distance spread as runners with (latently) infected or contaminated crowns (Stefani et al., 1989). Fruits and roots are not infected. The bacterium can overwinter in infected dead leaves and was also detected with PCR in tissue culture production. X. fragariae is easily transmitted by splash water (wind-driven rain and overhead irrigation) and man and his tools during cultivation (Kennedy and King, 1962a and b). Geographical distribution and importance EPPO A2 quarantine pest First described by Kennedy and King from the USA in 1962. Argentina, Australia (eradicated), Austria, Belgium, Brazil, Canada, Chile (eradicated), Ecuador, Germany, France, Greece, Italy, The Netherlands, New Zealand (eradicated), Paraguay, Portugal, Romania, Spain, Switzerland, Ukraine, Uruguay, USA, Venezuela. Substantial losses have been reported from the USA and occasionally from Europe (Maas et al., 1995; Mazzuchi et al., 1973; Roberts et al., 1997). Control Preventive measures are most important, such as the use of healthy planting material, avoiding working in wet fields and overhead irrigation, working last in contaminated fields, disinfection of materials, removal and burning of plant debris, rotation of a minimum of 2 years. Susceptibility of varieties is variable and resistance has been found in different species and lines. Fragaria vesca is immune (Maas et al., 2000). Copper and antibiotic sprays (if allowed) can be applied, but are only moderately effective and may give rise to phytotoxicity and health risks for treated fruits.

Chapter VII

Jack Kelly Clark, University of California Cooperative Extension, CA, USA.

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Fig. 179 Top:

Japanese plum tree (Prunus salicina), showing necrotic, folded leafs (leaf scald), caused by Xylella fastidiosa. Natural infection. Bottom left: Chlorotic spots (variegiated chlorosis) on upper leaf side and brown necrotic spots on lower leaf side of orange (Citrus sinensis), caused by Xylella fastidiosa. Natural infection. Bottom right: The red-headed sharpshooter, Xyphon (formerly Carneocephala) fulgida is an important vector of Xylella fastidiosa in N. America. It feeds and lays eggs mainly on Bermuda grass (Cynodon dactylon).

Examples of bacterial diseases – Fruit and nut trees, fruits

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7.10 Xylella fastidiosa Wells, Raju, Hung, Weisburg, Mandelco-Paul and Brenner 1987

Pierce’s disease, California vine disease (grapevine); phony disease (peach); variegated chlorosis (citrus) Maladie de Pierce (grapevine); chlorose variégée (citrus)

Main hosts: Grapevine (Vitis vinifera, V. labrusca, V. riparia), peach (Prunus persica) and orange (Citrus sinensis). On C. limon, C. reshni, C. volkameriana rootstocks: citrus variegated chlorosis). Medicago sativa (alfalfa: dwarf), Prunus dulcis (almond: leaf scorch), P. armeniaca (apricot), P. salicina, P. domestica (plum: leaf scald, Fig. 179 top), P. angustifolia, Acer rubrum, Morus rubra, Platanus occidentalis, Quercus rubra, Ulmus americana and Vinca minor have also been reported as natural hosts. Many crops and wild plants carry Pierce’s disease strains without symptoms (e.g. wild grasses, sedges and trees), systemically or non-systemically infected (Hill and Purcell, 1995). Strains from different hosts may differ slightly in characteristics and host range (Almeida and Purcell, 2003). Symptoms and transmission: On grapevine: first symptoms are sudden drying of large parts of a green leaf. These parts become brown and the surrounding tissues become yellow to red (Fig. 103). In later stages more yellowing occurs and leaves shrivel and drop. On stems irregular brown and green tissue can be found and trees may be reduced in growth, stunted and have a low and short production. Main (persistent) vectors are Xyphon (= Carneocephala fulgida, Fig. 179 bottom right), Draeculacephala minerva, Graphocephala atropunctata and Oncometopia nigricans (Fig. 113) in N. America, Cicadella viridis and Philaenus spumarius (meadow spittle bug) in S. E. Europe (Redak et al., 2004). Not seedborne. On peach: first symptoms are young shoots that have earlier and more and darker green leaves than normal and these shoots have a stunted appearance. Moreover they have earlier flowers. Both leaves and flowers remain on the shoots longer than normal. Lateral branches grow horizontally or droop. Fruit production is severely impaired and fruits are small. Main (persistent) vectors are Homalodisca coagulata, H. insolita, Oncometopia orbona, Graphocephala versuta and Cuerna costalis. Not seedborne. On citrus: chlorosis of leaves in parts of a tree or all over the tree (Fig. 179 bottom left). Symptoms are very similar to zinc deficiency symptoms. In later stages brown necrotic spots develop on the lower side of the leaf, corresponding to the chlorotic areas on the upper side. Fruits remain small, have higher sugar content and have a harder rind than normal. Transmission is by planting material and probably insect vectors (Hartung et al., 1994). Geographical distribution and importance EPPO A1 quarantine pest Described for the first time by Wells et al. (1987) from the USA. Argentina, Brazil, Costa Rica, Mexico, Paraguay, Slovenia, USA, Venezuela. Plum leaf scald is widespread in S. America. In the USA Pierce’s disease is a devastating disease, killing plants, especially in California wine production areas. In peach the main damage is in fruit (size) reduction and not killing plants. In citrus it is a devastating disease in Brazil (Purcell and Hopkins, 1996). Control Control consists of burning of infected trees, production and use of healthy planting material and certification schemes, insect and wild host control. Normal dormant season pruning of grapevines and citrus can eliminate many incipient infections of X. fastidiosa. When the infection is widespread pruning is no longer effective.

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M.M. Lopez via M. Scortichini

Gabrijel Seljak

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Fig. 180 Top:

Bacterial blight symptoms caused by Xylophylus ampelinus on grapevine (Vitis vinifera) shoots: wilt, droop and drying up in spring. Natural infection. Bottom: Reddish-brown discoloration, cracks and canker-like lesions on grapevine shoots due to advanced infection with Xylophilus ampelinus. Natural infection.

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7.11 Xylophilus ampelinus (Panagopoulos 1969) Willems, Gillis, Kersters, Van den

Broecke and De Ley 1987 Syn. Xanthomonas ampelina. The ‘maladie d’Oléron’ of grapevine, described in France in 1895 (Ravaz, 1895) was first thought to be caused by Erwinia vitivora, but has been shown to be due to X. ampelinus (Prunier et al., 1970).

Bacterial blight of vine Nécrose bactérienne de la vigne

Main hosts: Grapevine (Vitis vinifera). Symptoms and transmission: Earliest symptoms are observed in spring and development continues up to June. Reddishbrown to black streaks develop on lower nodes of 12-30 cm long shoots (Fig. 180 top, and 181). Later cracks and cankers develop up to the pith of the shoot (Fig. 180 bottom). No callus tissue is formed and shoots wilt and eventually die. Adventitious buds may develop that also wither. Stalks of grape bunches may show similar symptoms. Leaves show stomatal and hydathodal infection as angular, reddish-brown spots or as infections starting from the petiole into veins and they become brown, necrotic and dry (Fig. 181). Flowers may become black and necrotic and die. Roots may also be affected and show brown internal discoloration. X. ampelinus is transmitted by pruning tools and (irrigation) water. Infections occur in wet and warm conditions. The bacterium can occur as an epiphyte and survives in wood of producing plants (in vascular tissue) or planting material and can be spread with cuttings (Grall and Manceau, 2003). Geographical distribution and importance EPPO A2 quarantine pest First described from Crete, Greece (Panagopoulos, 1969). France, Greece, Italy, Slovenia, Turkey (eradicated), Moldova, Spain, South Africa. Severe damage was reported from South Africa (Matthee et al., 1970) and France in the past, but the disease appears to be of minor importance nowadays.

Gabrijel Seljak

Control Healthy planting material, hygiene and good viticultural practice (e.g. pruning in dry weather, disinfection of pruning tools, avoidance of overhead irrigation, indexing of mother plants) are the only way to prevent or control disease caused by Xylophilus ampelinus.

Fig 181. Deep black necrotic streaks on shoots of grapevine cultivar ‘Rebula’ with a dry leaf due to infection with Xylophilus ampelinus. Natural infection.

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U. Mazzucchi, Univ. Bologna, Italy

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Fig. 182 Grey-green stems with brownish longitudinal cracks and cankers on carnation (Dianthus caryophyllus), caused by Burkholderia caryophylli. Natural infection.

Examples of bacterial diseases – Ornamental plants

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8. Ornamental plants 8.1

Burkholderia caryophylli (Burkholder 1942) Yabuuchi, Kosako, Oyaizu, Yano, Hotta, Hahimoto, Ezaki and Arakawa 1993 Syn. Pseudomonas caryophylli

Bacterial wilt or stem crack of carnation Chancre bactérien de l’oeillet Welkekrankheit oder Wurzelfäule der Nelken

Main hosts: Carnation (Dianthus caryophyllus, Caryophyllaceae). Limonum sinuatum (statice, Plumbaginaceae) has been reported as a host from the USA (Florida) and Japan (Jones and Engelhard, 1984; Nishiyama et al., 1988). Russell prairie gentian (Eustoma grandiflorum) was reported as a host from Japan. Strains from the latter host were pathogenic to carnation and Russell prairie gentian (Furuya et al., 2000). Symptoms and transmission: Plants become grey-green and then yellow; in later stages they wilt and die. The vascular bundles show yellow to brown discoloration and roots are destroyed. Young plants may show an acute form of the disease, where first symptoms are distortion of the leaves, wilting and rotting of the roots. In older plants a more chronic form often occurs that may take up to 2 years, where plants show only some growth depression and 1-5 cm long cracks in the stems (Fig. 182). In the final stages the vascular tissue in stem and roots is destroyed, showing a yellow exudate and plants also die. Symptoms may be confused with those of Fusarium oxysporum f. sp. dianthi (wilting) and Fusarium roseum (root rot). Bacteria can be dispersed by (latently) infected planting material and through infected soil or substrate and root-toroot contact. Disease development is strongly enhanced by higher (up to 35ºC) temperatures. Ecto- and endoparasitic nematodes may also enhance disease (Stewart and Schindler, 1956). Geographical distribution and importance EPPO A2 quarantine pest The disease was first described from the USA by Jones (1941) and the causal organism in 1942 by Burkholder. Argentina, Brazil, China, Denmark, France, Germany, Hungary, India, Israel, Italy, Japan, The Netherlands (eradicated), Norway, Poland (disappeared), Sweden (eradicated), Switzerland (disappeared), UK (eradicated), Uruguay, Yugoslavia, USA. The disease has been important in the USA and was present in Europe to some extent in the 1950s, but disappeared from most countries, most probably through use of different carnation cultivars and modern cultivation practices on substrate. The disease appears mostly to be of minor importance now. Control Hellmers (1958) who studied the disease in much detail developed a method for planting material that can detect diseased plants within 3-4 days. In this method lower internodia of cuttings are selected and disinfected. Thereafter these internodia are placed in nutrient broth, containing 1.5% glucose and incubated at 28-30ºC. Cuttings from internodia which show bacterial growth are discarded. Clean mother plants and hygienic measures when producing cuttings are very important in the control of this disease. Resistance breeding has been successful in Japan and resistance was found in polyploidal D. henteri and D. acicularis. In EPPO a certification scheme for production of carnation planting material was developed (OEPP/EPPO, 1991).

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Fig. 183 Top:

Early symptoms of black spot of larkspur (Delphinium sp.): water-soaked to black, irregular leaf spots, caused by Pseudomonas syringae pv. delphinii. Natural infection. Bottom: Advanced stages of spot formation (non-water-soaked, tar-like spots) on larkspur (Delphinium ‘Belladonna’ hybrid), caused by Pseudomonas syringae pv. delphinii. Natural infection.

Examples of bacterial diseases – Ornamental plants

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Pseudomonas syringae pv. delphinii (Smith 1904) Young, Dye and Wilkie 1978 Syn. Pseudomonas delphinii

Black spot, black blotch or bacterial leaf spot of Delphinium Taches noires foliaires du Delphinium Schwarzfleckigkeit des Rittersporns

Main hosts: Larkspur or staggerweed (Delphinium spp.). Symptoms and transmission: Irregular black tar-like spots (up to 2 cm) become visible on the upper and lower leaf side. They may or may be not water-soaked (Fig. 183 top and bottom). When spots are not water-soaked (usually the older spots) they may be confused with those of fungi, especially as concentric rings may be formed. Leaves may become distorted. Spots can also be formed on flowers, petioles and stems. Bacteria infect through stomata and hydathodes (Smith, 1911) and may survive in the soil in plant debris. From soil they can be spread to plants by splash water (Stapp, 1956). Geographical distribution and importance First described by Smith (1904, 1905b) from the USA. Australia, Brazil, Canada, former Czechoslovakia, Denmark, France, Germany, Italy, Kenya, The Netherlands, New Zealand, UK, USA. Heavy losses have been reported from the USA, where in some areas on the east coast cultivation of Delphinium had to be given up (Bryan, 1928). In Europe the disease occurs only sporadically and causes losses only occasionally under cool, wet growing conditions. Control Healthy seeds or planting material and use of low susceptibility or resistant varieties are the only way to prevent or control diseases caused by P. syringae pv. delphinii. Infected plant material should be removed and destroyed as much as possible in combination with crop rotation. Overhead irrigation should be avoided. Copper-based pesticides (if allowed) may be used in a preventive way during cultivation.

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Fig. 184 Top:

Foliar symptoms (small water-soaked spots and larger brown and necrotic spots = tip burn, at the leaf margin surrounded by a broad yellow zone) of Xanthomonas axonopodis pv. dieffenbachiae on Anthurium andraeanum. Natural infection. Bottom: Symptoms of systemic infection (water-soaked leaf spots near the main veins and yellow bacterial slime protruding from the petiole) of Xanthomonas axonopodis pv. dieffenbachiae on Anthurium andraeanum. Natural infection.

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Xanthomonas axonopodis pv. dieffenbachiae (McCulloch and Pirone 1939) Vauterin, Hoste, Kersters and Swings 1995 Syn. X. campestris pv. dieffenbachiae. There are at least three groups of strains affecting Araceae: 1) strains from Anthurium, more virulent on Anthurium than other strains and with a broader host range, 2) strains from Syngonium, also virulent on Anthurium, with a narrow host range; 3) strains from other Araceae, including strains from Syngonium weakly virulent on Anthurium and with a narrower host range. For certain strains of Syngonium the name pv. syngonii has been proposed (Chase et al., 1992).

Bacterial blight of aroids, bacterial blight or tip burn of Anthurium Dépérissement de l’anthurium Bakterienbrand der Flamingopflanze

Main hosts: Flamingo flower or Anthurium (Anthurium amnicola, A. andraeanum, A. cristalinum, A. scherzerianum, Araceae). Furthermore Aglaonema commutatum, A. robeliniii, A. ‘pseudobracteatum’, Caladium hortulanum, Colocasia esculenta, Dieffenbachia picta=D. maculata, Epipremnum aureum, P. oxycardium, Philodendron scandens, P. selloum, Raphidophora, Scindapsis, Spathiphyllum, Syngonium podophyllum, and Xanthosoma caracu, X. lindenii and X. sagittifolium, all Araceae, have been reported as natural hosts. Symptoms and transmission: On Aglaonema and Anthurium, the disease has two stages. Foliar symptoms are found on the leaves and spathe, starting close to the leaf margin on the underside of the leaf as small watersoaked spots, eventually with some yellowing around the spots. Infection is usually through hydathodes and/or stomata. Under dry conditions the early spots may appear dry and dark brown. In later stages, leaf spots become brown and necrotic, and coalesce, resulting in large, irregular necrotic areas with a bright yellow border (Fig. 184 top). Symptoms of systemic invasion of the pathogen start with yellowing of the older leaves and petioles. Systemically infected leaves or flowers easily break off and may show dark brown streaks at their base. Yellow bacterial slime may occur on infected petioles (Fig. 184 bottom). When petioles are cut, yellow-brown vascular bundles are visible. Eventually the entire plant is killed. Systemic infection may also produce water-soaked leaf spots, when bacteria invade the leaf parenchyma from the infected vascular bundles (Fig. 184 bottom). Dry necrotic spots may be confused with those of other diseases, injury or nutritional deficiencies. Bacteria spread by splash water, (latently) infected plants, tools and clothes, plant debris in soil and nematodes. The bacterium can survive in tissue culture material (Norman and Alvarez, 1994, 1996) and roots. Geographical distribution and importance EPPO A2 quarantine pest Reported for the first time on Dieffenbachia maculata in the USA by McCulloch and Pirone in 1939 and described as Bacterium dieffenbachiae. Australia, Barbados, Bermuda, Brazil, Canada, Costa Rica, Dominica, French Polynesia, Guadeloupe, Italy, Jamaica, Martinique, The Netherlands, Philippines, Portugal, Puerto Rico, Reunion, Saint Vincent and the Grenadines, South Africa, Taiwan, Trinidad and Tobago, Turkey, USA, Venezuela. Heavy losses have been reported from Hawaii and other Caribbean islands. Control Sanitation and exclusion are the main cultural measures to be taken (Lipp et al., 1992). In particular, overhead irrigation and overcrowding must be avoided and infected leaves removed. Streptomycin or oxytetracycline have been used for preventive control (Sato, 1983) as well as cupric hydroxide (Knauss et al., 1972). For resistance breeding in Anthurium andraeanum see Kamemoto et al. (1990) and Fukui et al. (1998) and in Dieffenbachia see Norman et al. (1997).

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Fig. 185 Top:

Foliar symptoms due to stomata and hydathode infection (circular to angular reddish-brown, water-soaked spots) by Xanthomonas hortorum pv. hederae on Hedera helix. Natural infection. Bottom: Similar symptoms as described above on Hedera helix caused by X. h. pv. hederae. Natural infection.

Examples of bacterial diseases – Ornamental plants

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Xanthomonas hortorum pv. hederae (Arnaud 1920) Vauterin, Hoste, Kersters and Swings 1995 Syn. X. hederae; X. campestris pv. hederae

Bacterial leaf spot of ivy Maladie bactérienne du lierre Efeukrebs

Main hosts: Ivy (Hedera helix, Araliaceae); Brassaia actinophylla (Australian umbrella tree), Fatsia japonica (Japanese Aralia), Polyscias spp. (Ming Aralia) and Schefflera arboricola (Hawaiian Elf), all Araliaceae, have also been reported as natural hosts in the USA (Norman et al., 1999). Symptoms and transmission: First symptoms are small, circular, dark green, water-soaked (oily) lesions on the leaves. Spots may enlarge into 1-2 cm circular to angular spots with green-brown, water-soaked margins and a pinkish to reddish brown or black centre, often with a yellow halo (Fig. 185 top and bottom). The upper side of older leaf margins of lesions are reddish in colour, but the underside of the leaf margins are still water-soaked. On the spots droplets of orange-red bacterial slime may be observed under moist conditions. Under dry conditions the central tissues in the spots become dry and cracked. Heavily infected leaves yellow and drop. Under warm, wet conditions, brown to black canker-like lesions may develop on the stems and petioles. These lesions may girdle the stem or petiole, causing plants to dwarf and upper plant parts to wither and die. Twig tips may turn black and die back into the old wood. Symptoms are especially found on lower (shaded) plant parts, close to (wet) areas on the ground. The bacterium infects via wounds, stomata and hydathodes. Spreading is by splash water (rain and overhead irrigation), contaminated planting material and contact during manipulations, when plants are wet. X. h. pv. hederae can survive in the soil in plant debris. X. h. pv. hederae strains from Hedera helix appear to be very homogeneous in biochemical and genetic characteristics, but differ to some extent from those of other hosts (Norman et al., 1999). Geographical distribution and importance Described as a bacterial disease (but without isolation and characterization) in 1894 by Lindau in Germany. In 1920 Arnaud reported the disease from France, still without description of the bacterium. Burkholder and Guterman (1932) and later White and McCulloch (1934) characterized the causal bacterium in the USA. Canada, Denmark, Germany, Hawaii, Japan (Suzuki et al., 2002), The Netherlands, New Zealand, UK and USA. The disease appears only of minor importance. From the UK substantial losses have been reported in nurseries under wet, cool climatic conditions. Control For (preventive) control the following practices can be advised: selection and planting of only vigorous, disease-free plants in new or sterilized beds, removal and burning of diseased plants or plant parts, preferably before new growth in spring, and avoidance of close planting, avoidance of excessive (overhead) irrigation, high humidity and high temperatures. Frequent preventive sprays with copper compounds (if allowed) may reduce disease.

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Fig. 186 Top:

Symptoms of systemic invasion by Xanthomonas hortorum pv. pelargonii on Pelargonium hortorum. Greying of plant, yellowing, wilting and brown necrosis of whole leaves. Natural infection. Bottom: Symptoms of systemic invasion by Xanthomonas hortorum pv. pelargonii on Pelargonium peltatum (ivy geranium). Yellowing of leaves, wilting absent. Natural infection.

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Xanthomonas hortorum pv. pelargonii (Brown 1923) Vauterin, Hoste, Kersters and Swings 1995 Syn. Xanthomonas pelargonii; X. campestris pv. pelargonii

Bacterial leaf spot, blight, stem rot or wilt of Pelargonium Bactériose du Pelargonium Bakterielle Pelargoniumwelke

Main hosts: Pelargonium (Pelargonium x hortorum, P. zonale) and their hybrids. P. graveolens (scented geranium), P. peltatum (ivy geranium and its hybrids), Geranium spp. (cranesbill geranium, incl. G. sanguineum), Pelargonium x domesticum (regal geranium). P. acerfolium, P. tomentosum, P. ‘Torento’, and P. scarboroviae are hosts, but show some tolerance. Symptoms and transmission: After stomatal infection small (1-5 mm) dark green, water-soaked spots appear on the lower leaf surface. These spots become reddish and are sometimes surrounded with a yellow halo. Infected leaves turn yellow, and later brown then necrotic (at first often in wedge-shaped sectors) and shrivel. In later stages, or when bacteria have infected the plant through the roots, plants show symptoms of systemic infection in the vascular tissues. These symptoms are initially greying of leaves, and later yellowing (Fig. 186 top), wilting and blight, often accompanied by black streaks on the stem near leaf axils. In the final stages leaves become brown and necrotic, shrivel (Fig. 186 bottom), and stems show yellow-brown discoloured vascular tissue and may rot; whole plants may die. Symptoms may be confused with those of Ralstonia solanacearum, Pythium debaryanum or Botrytis cinerea. On ivy geraniums symptoms are less conspicuous and wilting is usually absent. In regal geraniums systemic infection is absent. (Latently) infected stock plants are the main source of infection and cuttings from these plants have systemic infection. The bacterium can be spread in the greenhouse by tools, hands, splash water, plant contact and soil and occasionally by whitefly (Trialeurodes vaporarium) and aphids (Bugbee and Anderson, 1963). X. h. pv. pelargonii may survive for 3-7 months in plant debris; it survives less well in soil. The bacterium is not known to be seed-transmitted. Geographical distribution and importance First described by Brown in 1923 from the USA. Argentina, Australia, Austria, Belgium, Brazil, Canada, Denmark, Egypt, France, Germany, Greece, Hungary, India, Iran, Italy, Japan, Morocco, The Netherlands, Portugal, Romania, South Africa, Sweden, Switzerland, UK, USA, former USSR and former Yugoslavia. Extensive financial losses have been reported from the USA. Control Healthy mother plants, certification and indexing, strict hygiene, removing and burning of infected plants, avoidance of overhead irrigation and good ventilation are important tools in prevention of infections by X. h. pv. pelargonii. When taking cuttings it is better to break than to use cutting knives. Baskets with geraniums should not be hung above benches. Copper or streptomycin and insecticide sprays may reduce spreading of the bacterium.

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Fig. 187 Top left:

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Fig. 188 Winter symptoms of fire blight on hawthorn (Crataegus monogyna). Slightly sunken reddish to dark brown patches on the bark, reddish discoloration visible when bark is removed.

Symptoms of fireblight (Erwinia amylovora) on pear (Pyrus communis): dead black branches with black leaves. Natural infection. Top right: Dark, glassy discoloration and exudation of slime on a pear (Pyrus communis) fruit infected with E. amylovora. Natural infection. Bottom left: Typical crook on an apple (Malus sp.) twig infected with E. amylovora and reddish slime exudates (yellow arrow). Natural infection. Bottom centre: Crook formation, blossom blight (flower clusters remain attached to the twig) and leaf blight of Cotoneaster salicifolius var. floccosus (rock spray) infected with E. amylovora. Natural infection. Bottom right: Crook formation on a Viburnum sp. caused by Pseudomonas syringae pv. syringae. Similar crooks are formed by P. s. pv. syringae on hosts of E. amylovora! Natural infection.

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9. Stone fruits 9.1

Erwinia amylovora (Burrill 1882) Winslow, Broadhurst, Buchanan, Krumwiede, Rogers and Smith 1920 Fire blight Feu bactérien Feuerbrand

Main hosts: Members of the Pomoideae: Amelanchier spp. (serviceberry), Chaenomeles spp. (Japanese quince), Cotoneaster spp. (rock spray), Crataegus spp. (hawthorn), Cydonia oblonga (quince), Eriobotrya japonica (loquat), Malus spp. (apple), Mespilus germanica (medlar), Photinia spp. (including P. = Stranvaesia davidiana), Pyracantha spp. (firethorn), Pyrus spp. (pear), Rosa rugosa, Sorbus spp. (mountain ash). Rubus spp. (blackberry, Rosoideae) in N. America and Prunus salicina (Japanese plum, Amygdaloideae). A related bacterium, Erwinia pyrifoliae, causes a similar disease on Asian pear, Pyrus pyrifolia, in Korea and Japan (Kim et al., 2001). Symptoms (Fig. 187 and 188) and transmission: a) Blossom blight after primary blossom infection (also see Fig. 124) shows wilt and death of flower clusters that become black and stick to the plant; b) Shoot blight where shoots wither and die, often with bending in the form of a crook (Fig. 187). Droplets of cream-coloured to orange sticky slime or slime strands may be observed; c) leaf blight shows brown to black necrotic patches starting from the leaf margin or the midrib. Also here slime may exude (see photo on page 33); d) Fruit blight in the form of dark glassy patches or total blackening and shrivelling of fruits that remain attached to the spur; e) branch, limb and trunk blight, where diffuse, large brown to orange coloured patches that may be water-soaked can be found that may girdle a branch or tree and kill it. Cankers are slightly sunken and darker in colour than surrounding healthy tissues (Fig. 188). The disease may be confused with dieback caused by Pseudomonas syringae subsp. syringae (Fig. 187 bottom right). The pathogen is efficiently spread over short distances by insects (honeybees), furthermore by wind (-driven rain), tools and most probably also by (migrating) birds and (latently) infected propagation material. Holdover cankers provide inoculum in spring and bacteria enter through wounds, stomata and nectaries. Geographical distribution and importance EPPO A2 quarantine pest Armenia, most countries of Europe, except Latvia and Ukraine, furthermore Guatemala, Iran, Israel, Jordan, Lebanon, N. America, including Mexico and Bermuda, New Zealand, Turkey. Epidemics are sporadic, but devastating, especially after first introduction into a country. According to climatic conditions and sensitivity of hosts the disease is of minor or major importance (Lecomte and Paulin, 1989; Mani et al., 1996). A severe epidemic occurred in Michigan in apple in 2000 with total losses estimated over $42 million (Longstroth, 2001). Control Integrated control, using weather forecast data (see Chapter IV.3, page 125), preventive chemical sprays with antibiotics such as streptomycin or kasugamycin, copper compounds or other compounds such as flumequine or fosetyl-Al, sanitation, pruning, eradication, nutrition and resistant or tolerant varieties, are the only ways to control fire blight. Resistance has been discovered in several hosts. Biological control has been used with variable results, mainly using strains of Pantoea agglomerans (Vaneste, 2000). In some areas in Europe, where the disease does not yet occur, protected zones have been delineated (Petter and de Guenin, 1993).

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Fig. 189 Top:

Brown necrotic spots, sometimes with a water-soaked halo, on a leaf of Persian walnut (Juglans regia), caused by Xanthomonas arboricola pv. juglandis. Natural infection. Centre: Black, slightly sunken spots, with a water-soaked halo, on fruits of Persian walnut (J. regia), caused by X. a. pv. juglandis. Natural infection. Bottom left: Yellow, slimy colonies of X. a. pv. juglandis after 4 days’ growth on yeast-peptone-glucose agar. Bottom right: Colony of the large walnut aphid Chromaphis juglandicola that can play a role in transmission of X. a. pv. juglandis.

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Xanthomonas arboricola pv. juglandis (Pierce 1901) Vauterin, Hoste, Kersters and Swings 1995 Syn. X. juglandis; X. campestris pv. juglandis

Black spot of walnut or walnut bacterial blight Maladie des tâches noires du noyer, bactériose du noyer Bakterieller Walnussbrand

Main hosts: Persian walnut (Juglans regia) and Eastern black walnut (J. nigra). Furthermore J. ailantifolia (Japanese walnut), J. ailantifolia var. cordiformis, J. californica (Southern California black walnut), J. cinerea (butternut) and J. hindsii (Northern California black walnut). Symptoms and transmission: On leaves (lamina and veins) and petioles water soaked spots appear, that later coalesce into large brown-black necrotic lesions (Fig. 189 top). Tips of twigs become black and subsequently the whole twig will die. Water-soaked black spots can also develop on the male and female catkins and cause severe damage. The pollen of such catkins is contaminated with the bacteria and with this pollen the pathogen can be spread. Fruits show water-soaked spots that become black and slightly sunken (Fig. 189 centre). The infected black tissue can reach from the epicarp into the nut and destroy it. The bacterium invades primarily the parenchymal tissues and wood older than 1 year is not attacked. Trees are not killed. Symptoms on leaves and fruits may be confused with those caused by the fungus Marssonina juglandis, but in the case of this fungal attack acervulus fruiting bodies can always be found. The disease develops usually in spring under wet weather conditions from bacteria that survive in buds and catkins and to a lesser extent in holdover cankers on twigs. X. a. pv. juglandis may be dispersed by the large walnut aphid Chromaphis juglandicola (Fig. 189 bottom right) and the black walnut blister mite or walnut leaf gall mite Eriophyes erineus (= Aceria tristriatus) (Rudolph, 1943). Geographical distribution and importance First described by Pierce in 1901 from California. Argentina, Australia, Azebaijan, Bermuda, Canada, Chile, China, widespread in Europe (not reported from Finland, Norway and Sweden), Georgia, India, Iraq, Iran, Israel, Lebanon, Mexico, New Zealand, South Africa, Uruguay, USA, Uzbekistan and Zimbabwe. Heavy losses have been reported from the USA, up to 30%, exceptionally 100% crop loss (Rudolph, 1933). In other parts of the world the disease is apparently less damaging, probably due to variation in resistance. Californian cultivars and a Hungarian selection A 117 show high susceptibility, French cultivars medium susceptibility; the Italian cultivar Sorrento is tolerant (Tamponi and Donati, 1990). Control Infected parts should be pruned a good way back from visible damage and burnt. Soil pH should be kept above 6, wetting of foliage, especially during bloom, with spray irrigation must be avoided, nitrogen fertilization should be balanced and by pruning an open structure for aeration should be created. Copper sprays (e.g. Bordeaux mixture) during the flowering stage can reduce disease severity and incidence, especially when combined with maneb (+ zinc) in case sensitivity to copper is decreased. A walnut blight forecast system that may reduce chemical sprays has been developed in the USA (http://www.fieldwise.com). Copper resistance has been observed in California, France and Italy (Teviotdale and Schroth, 1998; Scortichini et al., 2001).

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Fig. 190 Top left: Top right: Bottom:

Small water-soaked spots on fruit and cankers on twigs of peach (Prunus persica) caused by Xanthomonas arboricola pv. pruni. Natural infection. Black leaf spots with a light green halo and small, black, slightly sunken summer cankers on twigs of peach (P. persica) caused by X. a. pv. pruni. Natural infection. Small water-soaked spots, sometimes with a light brown centre, as the first symptom of bacterial canker of stone fruit (X. a. pv. pruni). Natural infection.

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Xanthomonas arboricola pv. pruni (Smith 1903) Vauterin, Hoste, Kersters and Swings 1995 Syn. X. pruni; X. campestris pv. pruni

Bacterial leaf spot, shot-hole, black spot Tache bactérienne, bactériose Bakterielle Blattfleckenkrankheit des Steinobstes

Main hosts: Peach (Prunus persica), Japanese bush cherry (P. japonica), plum (P. domestica and Japanese plum) and apricot (P. armeniaca). Furthermore P. amygdalis (almond), P. avium (sweet cherry), P. cerasus (sour cherry), P. davidiana (David’s peach), P. mume (Japanese apricot) and P. laurocerasus (cherry laurel). Symptoms and transmission: Peach: Leaf spots occur first as light-green water-soaked spots on the lower surface (Fig. 190 bottom). When the disease reaches the upper surface angular, purple to black spots appear, often surrounded by a yellow halo. Yellowish bacterial slime may ooze from the spots. The diseased areas drop out (shot-hole symptom). Spots are most frequent near the leaf tip, due to accumulation of bacteria in drops of rain or dew. Severely infected leaves turn yellow and drop off. Twigs that overwinter may show small cankers that appear as water-soaked blisters that later turn purple to black. In later stages these cankers become sunken and cracked (Fig. 190 top left). On fruits, small circular, slightly sunken, water-soaked brown spots can be observed, sometimes surrounded by a light green halo (Fig. 190 top right). Gum can exude from the spots, and symptoms may look very similar to insect damage. In severe cases, when fruits became infected in an early phase, fruits are malformed and show cracks. Plum and apricot: Leaf symptoms are very similar to those on peach, with pronounced shothole effect. Cankers, when formed, are perennial, in contrast to peach. They continue to develop in 2- to 3-year-old twigs and become much bigger and more devastating than in peach. On fruits some cultivars show only small pit-like spots, others large, sunken black necrotic spots. Cherry: Leaf symptoms are similar to those on peach, but not of importance. Fruit infection resulting in malformed fruit is more prevalent. Bacteria may be found throughout the fruit. The bacterium overwinters in summer cankers or twig tips from where bacteria may be spread by rain, wind and insects to other parts of the tree and infect through leaf scars, lenticels, stomata and (insect) wounds (Dunegan, 1932; Hayward and Waterston, 1965; Moffett, 1973). The disease may be confused with symptoms caused by Pseudomonas syringae pv. morsprunorum or P. s. pv. syringae. Temperatures of 15-28ºC, heavy rain and wind in spring favour epidemic infection. There seems to be no strain differentiation between hosts (Scortichini et al., 1996). Geographical distribution and importance EPPO A2 quarantine pest First described by E.F. Smith in 1903 from the USA. Argentina, Australia, Austria, Bermuda, Brazil, Bulgaria, Canada, China, France, India, Italy, Japan, Korea, Lebanon, Mexico, Moldova, Morocco, New Zealand, Pakistan, Romania, Russia, Saudi Arabia, Slovenia, Tajikistan, Ukraine, Uruguay, USA, South Africa and Zimbabwe. Epidemics have also been reported in recent years from southern USA and Italy (Dunegan, 1932; Battilani et al., 1999). Control Budwood from disease-free trees, chemical control using copper or antibiotics (when allowed) and use of resistant cultivars are the way to control bacterial canker of stone fruit (Young, 1987; Ritchie, 1995).

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Fig. 191 Top left:

Large white, necrotic spots caused by the toxin of Clavibacter michiganensis subsp. michiganensis on tomato (Lycopersicon esculentum). In these spots bacteria are not present, they can only be found in the vascular tissue lower in the plant. The low molecular weight toxin has diffused throughout the plant and caused necrosis (also see Fig. 68). Natural infection. Top right: Severe wilting in a final stage of infection of tomato (L. esculentum) by C. m. subsp. michiganensis. Natural infection. Bottom left: Yellow-brown discoloration in the vascular tissue of a tomato stem caused by C. m. subsp. michiganensis. When the tissue at a node is touched with a knife it is soft, as opposed to tissue infected by Verticillium and Fusarium fungi (see bottom right). Natural infection. Bottom centre: Light brown discoloration in the vascular tissue of a tomato stem caused by a fungus, Verticillium albo-atrum. When the tissue at a node is touched with a knife it is hard, as opposed to tissue infected by C. m. subsp. michiganensis (see bottom left). Natural infection. Bottom right: So-called bird’s eye symptom on tomato fruit: light brown spots with a white halo after stomatal infection, following overhead irrigation, caused by C. m. subsp. michiganensis, otherwise mainly a vascular pathogen. Natural infection.

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10. Vegetables 10.1 Clavibacter michiganensis subsp. michiganensis (Smith 1910) Davis, Gillaspie,

Vidaver and Harris 1984 Syn. Corynebacterium michiganense; C. michiganese subsp. michiganense

Bacterial canker of tomato Chancre bactérien de la tomate Bakterielle Tomatenwelke

Main hosts: Tomato (Lycopersicon esculentum) and sweet pepper (Capsicum annuum) and wild Solanum douglasii, S. nigrum and S. triflorum (Bradbury, 1986). Symptoms and transmission: In the normal case of vascular infection unilateral wilting of leaflets and leaves can be observed. Especially in young plants wilting can develop quite rapidly. In the end severe wilting occurs and fruits may show a characteristic marbling and may fail to develop and plants are killed (Fig. 191 top right). Vascular tissue shows a yellow-brown discoloration and tissues at nodes are soft when touched with a knife. This is contrary to tissues infected by fungi such as Verticillium and Fusarium, which remain firm (Fig. 191 bottom left and centre). Cankers can be formed on stems and petioles during the later stages of pathogenesis, but are often absent. Older plants, with vascular infection still only in the base of the stem, may show large white necrotic spots, due to rapid diffusion of toxin. In these spots bacteria are absent (see Fig. 191 top left). So called ‘bird’s-eye spots’, consisting of small, tan lesions surrounded by white halos, may develop on tomato fruits after overhead irrigation or heavy rain and stomatal infection. After hydathode infection or through broken trichomes (hairs), yellow-to-brown regions of marginal necrosis, so-called ‘firing’ symptoms, develop on leaflets of diseased plants. Bacteria penetrate the seed through the vascular system and give rise to (latently) infected seedlings. Spread of the disease is much facilitated by infected seed and manipulations during the growing period (also see Fig. 123). Geographical distribution and importance EPPO A2 quarantine pest First reported by E.F. Smith (1910) from Michigan, USA. Argentina, Armenia, Australia, Austria, Azerbaijan, Belarus, Belgium, Belize, Brazil, Bulgaria, Canada, Chile, China, Colombia, Cyprus, Czech Republic, Dominica, Dominican Republic, Egypt, Ecuador, France, Greece, Grenada, Guadeloupe, Hungary, India, Indonesia, Iran, Israel, Italy, Japan, Kenya, Lebanon, Martinique, Mexico, Morocco, The Netherlands, New Zealand, Panama, Peru, Poland, Romania, Russia, Slovenia, Spain, South Africa, Switzerland, Tanzania, Togo, Tonga, Tunisia, Turkey, Uganda, Ukraine, Uruguay, USA, Yugoslavia, Zambia and Zimbabwe. Yield losses of 20-70% have been observed in France and the USA (Rat et al., 1991). Control Healthy seeds, use of HCl extraction of seeds (Dhanvantari, 1989), strict hygiene (including destruction of infected plants and disinfection of tools) and avoidance of overhead irrigation are important measures to control or avoid bacterial canker (Gleason et al., 1993). Some cultivar resistance has been developed and copper or antibiotic sprays and use of acibenzolar-S-methyl may be helpful in control of (latently) infected seedlings (Werner et al., 2002). Furthermore soil solarization by approximately 6 weeks of soil mulching with transparent polyethylene sheets was effective in reducing disease in plastic tunnels (Antoniou et al., 1995).

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Fig. 192 Top:

Symptoms of pith necrosis, caused by Pseudomonas corrugata on tomato (Lycopersicon esculentum): large, slightly sunken, glassy to black necrotic patches and cracks on the stem. Natural infection. Centre: Scalariform dissolution of the pith and browning as early symptoms of pith necrosis in tomato caused by P. corrugata. Natural infection. Bottom: Advanced stages of pith necrosis in tomato, caused by P. corrugata. In this stage it may be difficult to isolate the causal pathogen. Natural infection.

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10.2 Pseudomonas corrugata Roberts and Scarlett 1981

Tomato pith necrosis Moelle noir, necrose de la moelle Stengelmarknekrose der Tomate

Main hosts: Tomato (Lycopersicon esculentum). Furthermore sweet pepper (Capsicum annuum) and Chrysanthemum spp. (Fiori, 1992). The bacterium has been observed in the rhizosphere of lucerne and wheat and as an endophyte in strawberry and grapevine (Bell et al., 1995; Lukezic, 1979; Roberts and Brewster, 1991). A related bacterium has been isolated from tomato in Italy and was named P. mediterranea (Catara et al., 2002). Symptoms and transmission: First symptom is chlorosis of lower leaves. Thereafter plants wilt and rapidly collapse. On the stem large, slightly sunken, glassy to black necrotic patches develop that also show cracks (Fig. 192 top). When the stem is cut a scalariform dissolution of the pith and browning thereof are the first visible symptoms of pith necrosis (Fig. 192 centre). In later stages the pith of large parts of the plant becomes dark brown and partly hollow, the vascular tissues also turn brown necrotic (Fig. 192 bottom) and plants may totally collapse and die. The pith necrosis may spread up into the petioles and entire stem of the plant. Bacterial slime may ooze from leaf scars. The bacterium has been isolated from seed, is soil-borne and can survive in the soil (Kritzmann, 1991; Scortichini, 1989). Furthermore it has been isolated from irrigation and recirculation water (Scarlett et al., 1978; Sadowska-Rybak et al., 1997). The disease may be spread by manipulations during the growing season. Geographical distribution and importance First described by Scarlett et al. (1978) from the UK. Albania, Argentina, Belarus, Brazil, Canada, Denmark, France, Germany, Greece, India, Israel, Italy, Japan, Latvia, Macedonia, The Netherlands, New Zealand, Norway, Poland, Portugal, Russia, South Africa, Sweden, Switzerland, Syria, Tanzania, Turkey, UK and USA. Economic loss is rare, but may occur under favourable conditions for the disease (Scarlett et al., 1978; Sesto et al., 1996). In southern Italy some (serological) strain variation has been observed. Some related, unnamed Pseudomonas spp. and P. viridiflava can also be involved in pith necrosis (Catara, 1997; Sutra et al., 1997). Control Pith necrosis usually occurs, especially on steam-sterilized soils under high N fertilization and high humidity and low night temperatures. Disease can be controlled, and diseased plants even cured, by reducing N fertilization and proper addition of potassium and calcium to strengthen the plants (Scarlett et al., 1978). Pseudomonas corrugata strain 2140R has been used with success as a biocontrol agent for Take-all disease of wheat caused by Gaeumannomyces graminis var. tritici. The bacterium produces a lipopetide siderophore, named corrugatin (Risse et al., 1998).

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Fig. 193 Top:

Water-soaked, later brown yellow to black necrotic spots, sometimes with a white papery centre and a narrow yellow halo on leaves, petioles and stems of lovage (Levisticum officinale), caused by Pseudomonas levistici. When spots coalesce, larger leaf parts become necrotic and/or show yellowing. Natural infection. The taxonomic position of this bacterium is not certain and needs further research (see text). Bottom: Similar symptoms as in Fig. 193 top on lower side of leaf of lovage (L. officinale).

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10.3 [Pseudomonas levistici] Osterwalder 1909

Bacterial leaf spot of lovage Taches bactérien Bakterielle blattfleckenkrankheit des Liebstöckels

Main hosts: Lovage (Levisticum officinale). Symptoms and transmission: Water-soaked, later brown yellow to black necrotic spots, sometimes with a white, papery centre, are formed on leaves, petioles and stems. Spots on leaves are slightly sunken, sometimes have a narrow yellow halo and may coalesce, killing larger parts of the leaf (Fig. 193). Epidemics occur under cool, wet weather conditions. Geographical distribution and importance Lovage (Levisticum officinale) is a perennial herb native to the mountainous regions of northern Europe and was introduced and naturalized in the eastern United States. It has been grown for centuries for its aromatic fragrance, its fine ornamental qualities, and to a lesser extent, its medicinal values. All parts of the plant, including the roots, are strongly aromatic and contain extractable essential oils. Centres of lovage cultivation are located principally in Germany and central Europe, where the plants are collected and the essential oils extracted by steam distillation. Osterwalder in Switzerland first described the bacterium causing a leaf spot disease on lovage in 1909. On the basis of limited tests he named the bacterium isolated from lovage Pseudomonas levistici but the name did not reappear in the Approved List of Bacterial names January 1, 1980, in the International Journal of Systematic Bacteriology. In the 1980s it was rediscovered in The Netherlands, where an epidemic occurred in some lovage fields in the east of the country. Its true taxonomic position, host range test and pathogenicity test await further research. The Netherlands and Switzerland. Control No specific control measures are known and the disease appears to be only of minor importance and strongly dependent on environmental conditions.

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Fig 194. Top:

Water-soaked spots surrounded by a white-yellow halo on leaves of cucumber (Cucumis sativus), caused by Pseudomonas syringae pv. lachrymans. Natural infection. Bottom left: Brown necrotic leaf spots surrounded by angular or rectangular white halo and whiter necrotic spots, caused by the fungus Pseudoperonospora cubensis causing downy mildew of cucurbits. These symptoms may easily be confused with those of P. s. pv. lachrymans. Bacterial slime, however, is absent. Natural infection. Bottom right: Typical dark-green water-soaked spots on fruit of melon (Cucumis melo), caused by Pseudomonas syringae pv. lachrymans. Natural infection.

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10.4 Pseudomonas syringae pv. lachrymans (Smith and Bryan 1915) Young, Dye and

Wilkie 1978 Syn. Pseudomonas lachrymans

Angular leaf spot of cucumber, cucurbit angular leaf spot Taches angulaires des feuilles du concombre ‘Eckige Blattflecken’-Krankheit der Gurke

Main hosts: Cucumber (Cucumis sativus) and gherkin (Cucumis anguria). Furthermore Benincasa hispida (wax gourd), Citrullus lanatus (watermelon), Cucumis melo (melon), Cucurbita moschata (pumpkin), Luffa acutangula (luffa) and Sechium edule (chayote). Symptoms and transmission: Small water-soaked spots on leaves and petioles often surrounded by a shallow white-yellow to brown halo are the first symptoms of the disease (Fig. 194 top). These spots become angular, because larger veins will hamper extension of the diseased tissue. Spots may coalesce and then larger parts of the leaf become brown and necrotic. On the spots on the lower leaf side a silvery film of bacterial slime may be observed under high humidity conditions. When the slime dries it remains as a papery white layer on the diseased tissues. Leaf spots may be confused with those of the fungus Pseudoperonospora cubensis causing downy mildew of cucurbits (Fig. 194 bottom left). On the fruits, round water-soaked spots are also formed (Fig. 194 bottom right), which may show star-like cracks and yellowish gum and slime exudates in later stages. When the slime runs down the fruit, further (secondary) infections develop on the fruit. From these spots secondary rot often develops rapidly. Young fruits, when affected, easily drop. The bacterium can contaminate the seed (externally or under the seed coat) and be transmitted by the seed (Leben, 1981) and may have already infected cotyledons that show water-soaked to white papery, irregular spots (see Fig. 118). The bacterium can survive for more than a year on plant debris (Atlas de Gotuzzo, 1976) and can be spread by wind-blown sand, hands, insects (Diabrotica undecimpunctata, southern corn rootworm, (Howard et al. (1994)), machines and irrigation water (Kritzmann and Zutra, 1983). Geographical distribution and importance Reported for the first time by E.F. Smith and Bryan in 1915 from the USA. Algeria, Argentina, Australia, Brazil, Canada, China, Colombia, Egypt, widespread in Europe, Gabon, India, Iran, Israel, Japan, Jordan, Kazakhstan, Kenya, Korea, Laos, Mexico, New Zealand, Philippines, Singapore, South Africa, Zimbabwe, Tajikistan, Thailand, Turkey, USA, Uzbekistan and Venezuela. When weather conditions (humid and temperatures of 23-28ºC) or climatic conditions in the greenhouse are conducive for the disease substantial yield loss may occur due to less fruits and less weight per fruit due to assimilation reduction of infected leaves. Control Sixteen pathogenic races of P. s. pv. lachrymans have been discriminated and resistance against these races has been determined in cucumber cultivars (Krivchenko and Medvedeva, 1985). Use of disease-free seed and reduction of humidity in greenhouses as well as crop rotation, strict hygiene and avoidance of overhead irrigation are effective in reducing angular leaf spot. Regular sprays with copper hydroxide (2%) or mancozeb (0.2%) reduce secondary infections in the crop (Marinescu, 1982). Systemic resistance to angular leaf spot was induced in cucumber plants by infection of first leaves with tobacco necrosis virus or the fungus Colletotrichum lagenarium (Jenns et al., 1979) or by application of the biological control fungus Trichoderma asperellum to the root system of plants (Yedidia et al., 2003).

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Fig. 195 Top:

Typical water-soaked spots with droplets of bacterial slime on pod of kidney bean (Phaseolus vulgaris), caused by Pseudomonas syringae pv. phaseolicola. Natural infection. Bottom left: Brown necrotic leaf spots surrounded by a large yellow-green halo as typical symptom of halo blight of kidney bean (Phaseolus vulgaris) by toxin-producing strains of P. s. pv. phaseolicola. Bottom centre: Water-soaked spots on pods of kidney bean (P. vulgaris), caused by P. s. pv. phaseolicola. Bottom right: Chlorosis on leaves of kidney bean (P. vulgaris) as a symptom of systemic infection by P. s. pv. phaseolicola of the plant through seed infection.

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10.5 Pseudomonas syringae pv. phaseolicola (Burkholder 1926) Young, Dye and Wilkie

1978 Syn. Pseudomonas phaseolicola; P. savastanoi pv. phaseolicola

Halo blight or grease spot of bean Maladie de la graisse du haricot, tache auréolée Fettfleckenkrankheit der Bohne

Main hosts: Kidney bean = French bean, snap bean, string bean, frijoles (Phaseolus vulgaris). Furthermore Cajanus cajan (pigeon pea), Desmodium sp. (ticktrefoil), Neonotonia = Glycine wightii (glycine, a tropical forage legume), P. acutifolius (tepary bean), P. atropurpureus = Macroptilium atropurpureum (siratro, a weed), P. bracteatus = Macroptilium bracteatum (burgundy bean, a weed), P. coccineus (runner bean), P. lathyroides = Macroptilium lathyroides (phasey bean, a weed), P. lunatus (butter- or Madagascar bean), P. l. var. macrocarpus (lima bean), P. multiflorus (Dutch case-knife bean), P. mungo = P. aureus = P. radiatus = Vigna radiata (mung bean), P. polyanthus (botil in Spanish), Dolichos lablab (lablab, hyacinth bean or bonavist bean), Pueraria thunbergiana (arrow root or kudzu vine) and Vigna unguiculata = V. sinensis (cowpea). Symptoms and transmission: Small water soaked spots on cotyledons or true leaves are the first symptoms. The spots become red-brown and necrotic and are often, but not always, surrounded by a light green halo, due to the action of phaseolotoxin (see Chapter III.1a and Fig. 195 bottom left and right). Especially under high temperature conditions the halo may be absent; moreover strains that do not have the genetic information for toxin production have also been reported (Oguiza et al., 2004). On stems greasy longitudinal spots are formed. On the pods circular to irregular, water-soaked, greasy spots are formed. Bacterial slime may exude from the spots as grey drops (Fig. 195 top and bottom centre). Symptoms may be confused with those caused by Xanthomonas axonopodis pv. phaseoli (spots more reddish-brown with narrow halo (see Fig. 198) and those of P. s. pv. syringae (causing brown spot of bean, spots brown necrotic with a narrow yellow halo). Infected seeds are symptomless or show shrivelling and discoloration or may rot completely. When plants are systemically infected from the seed they show yellow-green (mottled) chlorosis and often plants remain shorter and may show distortion. The pathogen is spread by infected seed and rain splash, and survives in plant debris and in volunteer bean plants. Infection takes place through wounds and stomata (Fig. 125 and Schwartz, 1989). Geographical distribution and importance First described from the USA by Burkholder in 1926. Worldwide distribution, everywhere where beans are grown under cool, wet climatic conditions. Under these conditions epidemics (from a few infected seeds) and substantial yield loss may occur (Walker and Patel, 1964), but data on losses are scarce. Infection level in seed is often not correlated to subsequent infection level in the field (van den Bovenkamp et al., 1991; Grogan and Kimble, 1967). When weather becomes warmer and drier plants recover and new growth is healthy. Control Use of healthy (tested) seeds and use of low susceptibility or resistant varieties are the most important ways to control halo blight of bean (Beebe and Pastor-Corrales, 1991). Nine races of P. s. pv. phaseolicola have been discriminated. Race 6 is predominant (Taylor et al., 1996). Furthermore removal or deep ploughing of debris, wide crop rotation with cereals or intercropping with maize and copper or streptomycin sprays (if allowed) to reduce epiphytic populations have been shown to be effective in reducing halo blight (Schwartz, 1989).

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Fig. 196 Top:

Diverse stages of leaf spot development on tomato (Lycopersicon esculentum) leaves infected with Pseudomonas syringae pv. tomato, causing bacterial speck. When many lesions occur general yellowing and necrosis become prevalent. Natural infection. Centre: Close up of leaf spots caused by P. s. pv. tomato on tomato (L. esculentum). Yellow halo caused by the toxin coronatine. Natural infection. Bottom: Small, water soaked to black lesions on fruits of tomato (L. esculentum), caused by P. s. pv. tomato. Natural infection.

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10.6 Pseudomonas syringae pv. tomato (Okabe 1933) Young, Dye and Wilkie 1978 Syn. Pseudomonas tomato

Bacterial speck of tomato La moucheture bactérienne Bakterielle Blatt- und Fruchtfleckenkrankheit der Tomate

Main hosts: Tomato (Lycopersicon esculentum). Several weeds serve as a reservoir for epiphytic P. s. pv. tomato populations, such as Arabidopsis thaliana (thale cress), Gnaphalium spp. (cudweed), Lamium amplexicaule (henbit dead-nettle), Oenothera spp. (evening primrose) and Stellaria media (common chickweed) (McCarter et al., 1983). Symptoms and transmission: Small, round, green, dark water-soaked spots with or without a narrow yellow halo on cotyledons and true leaves are first symptoms of bacterial speck. The lesions become grey-black or brown and necrotic with a narrow yellow halo, 2-5 mm in diameter (Fig. 196 top and centre). When many spots occur they may coalesce and leaves show yellowing and necrosis (Fig. 196 top). On stems, petioles, peduncles and pedicels, longitudinal, water-soaked spots are formed that become necrotic in later stages. On fruits small (1-3 mm in diameter) brown-black, slightly raised spots are formed, that look like tar spots (Fig. 196 bottom). Symptoms may be confused with those of X. vesicatoria (see Fig. 201). P. s. pv. tomato is seed transmitted and can survive for a long time in/on seed (over 20 years). Dispersal is also by rain splash and machinery. Survival in soil has also been established for more than 1 year (Bashan et al., 1978). Primary inoculum is usually present on weeds. Infection takes place through wounds (broken hairs) and stomata under conditions of high humidity and relatively low temperatures (18-24ºC). Geographical distribution and importance First described by Okabe in 1933 from Taiwan. Australia, Austria, Belgium, Brazil, Bulgaria, Canada, Chile, China, Czech Republic, France, Germany, Greece, Hungary, Israel, Italy, Jordan, Morocco, New Zealand, Portugal, Romania, Slovakia, South Africa, Switzerland, Taiwan, UK, USA, former USSR, Venezuela, former Yugoslavia. Heavy losses have been reported from Australia, Canada, Israel, New Zealand and the USA (Yunis et al., 1980). Control Use of healthy seeds and use of low susceptibility or resistant varieties are important ways to control bacterial speck. Furthermore crop rotation, separation of production of transplants from production fields, avoiding working in the field when the crop is wet and strict hygiene are helpful in control (Goode and Sasser, 1980). Bacteria can be effectively removed from seed by 1) hot water treatment, 60 min at 48-52 ºC or 30 min at 55 ºC (Devash et al., 1980); 2) hot air treatment, 72 h at 70ºC or dipping in 0.6% HCl or acetic acid for several hours or for 2 min in 1% NaOCl. Copper and streptomycin have been used to control epiphytic populations of P. s. pv. tomato and copper resistance has been observed (Mellano and Cooksey, 1988; Vanneste and Voyle, 2003). Biological control, using the plant growth-promoting bacterium Azospirillum brasilense showed promising results (Bashan and de-Bashan, 2002).

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Fig. 197 Top:

Small brown necrotic spots with a raised centre and a narrow yellow halo on leaf of soybean (Glycine max), caused by Xanthomonas axonopodis pv. glycines causing pustule of soybean. Natural infection. Bottom: The sap-sucking bug Carpocoris fuscispinus that may be of importance in transmission of X. a. pv. glycines (see text).

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10.7 Xanthomonas axonopodis pv. glycines (Nakano 1919) Vauterin, Hoste, Kersters and

Swings 1995 Syn. Xanthomonas glycines; X. campestris pv. glycines; X. phaseoli f. sp. sojense

Bacterial pustule Bactériose du soja Pustelkrankheit

Main hosts: Soybean (Glycine max). Furthermore Brunnichia cirrhosa, Macrotyloma uniflorum = Dolichos biflores (horsegram, Patel et al., 1949), Phaseolus lunatus (lima bean), P. vulgaris (kidney bean) and a number of other Phaseolus and Vigna spp., including Vigna unguiculata (cowpea). Symptoms and transmission: Small, light green to red-brown spots with a raised centre that develop first at the lower side of the leaf are the first symptom of bacterial pustule (Fig. 197 top). In later stages the raised spots shrivel and develop into irregular spots with a yellow halo. The diseased tissue may drop from the leaf and heavily infected leaves turn yellow and may drop as well. Pustules can also occur on the stem and pods. Symptoms may be confused with those caused by Pseudomonas syringae pv. glycinea (angular leaf spot or bacterial blight), which are very similar. In the case of P. s. pv. glycinea spots are more angular in shape. Confusion may also occur with early stages of soybean rust, caused by the fungi Phakopsora pachyrhizi and P. meibomiae but at a later stage the presence of egg-shaped pustules that are filled with rusty-red-coloured spores is a diagnostic differential. Infected seed (Hedges, 1924), rain splash and sometimes insects (Carpocoris fuscispinus in Russia, Fig. 197 bottom) spread the disease. Furthermore the weed hosts Brunnichia cirrhosa (red vine, Jones, 1961) in the USA and Macrotyloma uniflorum (Dolichos biflores, horsegram) in India (Patel et al., 1949) can serve as a reservoir in the field, as well as bacteria surviving in plant debris, soybean volunteers and wheat roots (Graham, 1953). Infection is through stomata and wounds. Bacterial pustule occurs when warm temperatures and frequent rains prevail in the growing season. Geographical distribution and importance Described for the first time in Japan by Nakano in 1919. Argentina, Australia, Austria, Belize, Brunei, Bolivia, Brazil, Bulgaria, Cambodia, Canada, Central African Republic, China, Colombia, Côte d’Ivoire, Cuba, Egypt, Ethiopia, France, India, Indonesia, Japan, Kenya, Korea, Lithuania, Madagascar, Malawi, Malaysia, Mexico, Mozambique, Myanmar, Nepal, Nicaragua, Nigeria, Papua New Guinea, Philippines, Romania, Somalia, South Africa, Sudan, Taiwan, Tanzania, Thailand, Uganda, USA, former USSR, Venezuela, Yugoslavia, Zambia and Zimbabwe. Substantial losses have been reported from the USA (Hartwig and Johnson, 1953). Control Healthy seeds and use of low susceptibility or resistant varieties (Shukla and Prabhakar, 1990) and early spraying with insecticides to reduce vector populations are the only way to prevent or control diseases caused by X. a. pv. glycinea. Crop rotation and avoiding the use of equipment in a field when foliage is wet may help reduce bacterial pustule. Seed treatment with carboxin can reduce disease incidence (Thapliyal and Misra, 1974). Computer-based models have been developed for soybean disease management (Michalski et al., 1983).

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Fig. 198 Top:

Typical symptoms of bacterial blight (large brown-necrotic leaf spots with a bright yellow halo) on kidney bean (Phaseolus vulgaris), caused by Xanthomonas axonopodis pv. phaseoli. Natural infection. Bottom left: Water-soaked and typical reddish-brown older spots on kidney bean (P. vulgaris) pods, caused by X. a. pv. phaseoli. Natural infection. Bottom right: Diverse stages of leaf and pod infection of kidney bean (P. vulgaris), caused by X. a. pv. phaseoli. Natural infection.

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10.8 Xanthomonas axonopodis pv. phaseoli (Smith 1897) Vauterin, Hoste, Kersters and

Swings 1995 Syn. Xanthomonas phaseoli; X. campestris pv. phaseoli; X. c. pv. phaseoli var. fuscans (strains that produce a brown, diffusible pigment)

Common bacterial blight and fuscous blight of bean Brûlure bactérienne du haricot Bakterieller Bohnenbrand

Main hosts: Kidney bean = French bean, snap bean, string bean, frijoles (Phaseolus vulgaris). Furthermore Calopogonium sp., Dolichos lablab = Lablab purpureus (hyacinth bean), Glycine max (soybean), Lupinus polyphyllus (garden lupin), Mucuna deeringianum (velvet bean), P. acutifolius (tepary bean), P. calcaratus = Vigna umbellate (aduki bean), P. coccineus (runner bean), P. lunatus (butter- or Madagascar bean), P. mungo = P. aureus = P. radiatus = Vigna radiata (mung bean), Pueraria sp. (kudzu vine), Strophostyles (Stizolobium, trailing wild bean) helvola and probably Vigna unguiculata = V. sinensis (cowpea) (see Gilbertson and Maxwell, 1992). The weeds Senna (Cassia) hirsuta (senna) and Digitaria scalarum (African couch, Dunn’s finger grass) were reported as symptomless hosts (Opio and Male-Kigiwa, 1995). Symptoms and transmission: Small water-soaked spots on leaves, petioles, pods and stems are the first symptoms. On leaves these spots become reddish-brown and necrotic, surrounded by a bright yellow halo. On pods spots become sunken and reddish-brown (Fig. 198). On stems the yellowing is absent. The bacterium can invade vascular tissues, which may result in wilting. Confusion with halo blight (Fig. 195), is possible. Severely infected seed may be shrivelled and show poor germination or produce weakened plants, but many infected seeds are symptomless (latently infected). On varieties with white seeds, yellow or brown spots can be observed, especially near the hilum. On varieties with dark seed, this discoloration is not visible (Zaumeyer and Thomas, 1957). X. a. pv. phaseoli is seed transmitted (Valarini et al., 1996). Dispersal of the bacterium is also by splash or wind-driven rain. X. a. pv. phaseoli is accidentally dispersed by insects, such as Bemisia tabaci (whitefly), Diaprepes abbreviatus (root weevil), Cerotoma ruficornis (bean leaf beetle) and Epilachna varivestris (Mexican bean beetle) (Kaiser and Vakili, 1978; Zaumeyer and Thomas, 1957). Seed infection can occur internally (including the embryo) and externally. The pathogen can survive in seed, plant debris and epiphytically on hosts and non-hosts (Cafati and Saettler, 1980; Opio and Male-Kigiwa, 1995). Infection is through stomata and wounds at higher temperatures (28-32ºC) and high humidity. Geographical distribution and importance EPPO A2 quarantine pest First described by E.F. Smith from the USA in 1897. Widespread all over the world, where hosts are grown. Bacterial blight is one of the major diseases of bean and yield losses of 2040% are quite common (Yoshii et al., 1976; Allen et al., 1998). Control Healthy (tested) seeds and use of low susceptibility or resistant varieties are important in the control of bacterial blight. Hot water treatment (20 min at 52ºC) or hot air treatment (23-32 h in 60°C dry air at 45-55% RH) of seeds have been shown to be effective (Gondreau and Samson, 1994). Resistance has been found in Phaseolus coccineus and P. acutifolius (Tar’an et al., 2002). Some resistance is present in P. vulgaris (Schuster et al., 1983). Eight races have been described based on reaction in P acutifolius (Opio and Male-Kigiwa, 1995). Sprays with antibiotics or copper compounds reduce epiphytic populations to some extent.

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Fig. 199 Top:

Small water-soaked spots on the upper side of the leaf that later become reddish brown with a water-soaked margin and sometimes show a white papery necrotic centre on cowpea (Vigna unguiculata), caused by Xanthomonas axonopodis pv. vignicola, causing bacterial blight. Loose leaf shows canker on petiole (red arrow). Natural infection. Bottom: Symptoms as for Fig. 199 top, but now on lower side of the leaf. Natural infection.

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10.9 Xanthomonas axonopodis pv. vignicola (Burkholder 1944) Vauterin, Hoste, Kersters

and Swings 1995 Syn. Xanthomonas vignicola; X. campestris pv. vignicola

Cowpea blight, cowpea canker or bacterial pustule of cowpea Chancre bactérien, la bactériose du Niébé Bakterienbrand der Augenbohne

Main hosts: Cowpea (Vigna unguiculata = V. sinensis (cow pea)). Furthermore Dolichos lablab = Lablab purpureus (hyacinth bean), Vigna pubigera (Sudanese weed) and Phaseolus vulgaris (kidney bean). Symptoms and transmission: First symptoms are small, water-soaked spots on cotyledons and leaves that become reddish to yellow-brown and necrotic with a water-soaked margin at later stages. Sometimes the centre of spots becomes light brown to white necrotic (Fig. 199). When many leaf spots occur, the leaf yellows and may drop. The bacterium is often also present in the vascular system of the plant causing cracks and (sometimes swollen) canker-like lesions on the stem and petioles (Fig. 199 top). When cankers occur, especially on young plants, plants may be stunted and show dieback and wilting. Often white bacterial slime occurs on cankers of leaf spots. The bacterium is seedtransmitted and can survive in the seed and volunteer plants (Preston, 1944). In the field the bacterium is dispersed by splash water and wind-driven rain. Infection is through stomata and wounds at higher temperatures (28-32ºC) and high humidity. Geographical distribution and importance Described for the first time by Burkholder in 1944 in the USA. Benin, Brazil, China, India, Mozambique, Niger, Nigeria, South Africa, Puerto Rico, Senegal, Sudan, Thailand, Tanzania, Uganda, USA, Venezuela, Zimbabwe. Substantial losses have been reported from India and West Africa (Sikirou et al., 2000). Control Healthy seeds and use of low susceptibility or resistant varieties, 3-year rotation (Preston, 1944; Wydra et al., 1997; Bua et al., 1998) and intercropping (Bua et al., 1997) are important ways to control bacterial blight of cowpea. Chemical control by spraying of plants (antibiotics or copper compounds) reduces epiphytic populations, but is usually not very effective (Gupta and Singh, 2002).

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Fig. 200 Top:

Typical V-shaped yellow lesions with black veins as symptoms of black rot on rutabaga or swede turnip (B. napus var. napobrassica), caused by Xanthomonas campestris pv. campestris. Natural infection. Bottom left: Black discoloration of vascular bundles in a stem of Brussels sprouts (Brassica oleracea var. gemmifera) caused by Xanthomonas campestris pv. campestris. Natural infection. Bottom right: Typical V-shaped yellow lesions with black veins as symptoms of black rot on Brussels sprouts (Brassica oleracea var. gemmifera) caused by X. campestris pv. campestris. Natural infection.

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10.10 Xanthomonas campestris pv. campestris (Pammel 1895) Dowson 1939 Syn. Xanthomonas campestris

Black rot of cabbage Pourriture noire des crucifères Adernschwärze des Kohls

Main hosts: Cabbage (Brassica oleracea var. capitata), including Brussels sprouts (B. o. var. gemmifera), broccoli (B. o. var. italica), borecole or kale (B. o. var. acephala), cauliflower (B. o. var. botrytis), collards (B. o. var. viridis), Chinese kale (B. o. var. alboglabra), kohlrabi (B. o. var. gongylodes), pe-tsai (B .o. var. pekinensis) and Savoi cabbage (B. o. var. sabauda). Furthermore Arabidopsis thaliana (mouse-ear cress), B. geniculata (shortpod mustard), B. juncea var. juncea (brown mustard), B. napus var. napus (rape), B. napus var. napobrassica (rutabaga or swede turnip), B. nigra (black mustard), B. rapa var. rapa (turnip), B. rapa var. chinensis (Chinese cabbage), Brassica rapa spp. oleifera (turnip rape), Capsella bursa-pastoris (shepherd’s purse), Cardaria pubescens, Coronopus didymus, Erysimum cheiri (wallflower), Lepidium sativum (garden cress), L. virginicum (Virginia pepperweed), Matthiola incana (stock), Raphanus raphanistrum (jointed charlock), R. sativus (radish) and Sinapsis arvensis (wild mustard). Symptoms and transmission: Along the margin of leaves V-shaped yellow sectors with blackened veins develop (Fig. 200 top and bottom right). These sectors enlarge, become brown and necrotic, papery and may cover the whole leaf. On cotyledons yellow spots and distortions may be observed, when the infection develops from infected seed. Sometimes leaf spots occur, that are usually attributed to the closely related X. c. pv. armoraciae. When stems are cut, black vascular bundles are present to a larger or lesser extent, depending on the progress of the disease (Fig. 200 bottom right). In the storage tissues of stems black rot and cavities may develop. In closed types of cabbage secondary soft rot may easily develop under warm humid conditions. Black rot develops in the field especially in late summer under warm (25-30ºC) and humid conditions. Young infected plants show distortions and may die. The bacterium survives in soil, especially in plant debris (Schaad and White, 1974), furthermore in weeds in and around cabbage fields and seed (Bazzi, 1991; Kocks and Zadocks, 1996; Ruissen et al., 1990). Bacteria externally contaminate seed, but penetrate the seed also and can survive in the seed for several years. Dispersal of the pathogen is by (latently) infected seed and seedlings, splash water (irrigation) and winddriven rain. Infection is through infected seeds and wounds, but especially in the field also through hydathodes, hence the development of symptoms along the leaf margin. Geographical distribution and importance First adequately described by Pammel in 1895 in the USA. Widespread all over the world where cabbage is cultivated. Black rot is one of the most important diseases of crucifers (Catara et al., 1999; Eastburn, 1989; Kim, 1986; Nemeth and Laszlo, 1983; Onsando, 1988). Control Healthy (tested) seeds, crop rotation, hygiene (especially for transplants, disinfected containers), control of weeds and volunteer plants, removal of plant debris after harvest and use of low susceptibility or resistant varieties are important ways to control black rot. At least five races of X. c. pv. campestris have been determined and resistance is especially present in B. rapa and B. juncea, but is also present in some cabbage varieties (Vicente et al. 2002; Taylor et al., 2002). Hot water treatment (30 min 50ºC), as well as soaking for 20 min in a solution of 0.5% copper acetate in 0.005 N acetic acid are efficient in eliminating the bacterium from seeds.

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Fig. 201 Top:

Small water-soaked spots with a narrow yellow halo on the upper side of the leaf that later become brown and necrotic on sweet pepper (Capsicum annuum) caused by Xanthomonas vesicatoria. Natural infection. Bottom: Black necrotic spots with a water-soaked margin that become sunken on tomato (Lycopersicon esculentum) fruit, caused by X. vesicatoria. Natural infection.

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10.11 Xanthomonas vesicatoria (Doidge 1920) Dowson 1939 Syn. Xanthomonas campestris pv. vesicatoria. X. c. pv. vesicatoria genetic group A (Stall et al., 1994) named X. axonopodis pv. vesicatoria by Vauterin et al. (1995); group B was named X. vesicatoria.

Bacterial spot, bacterial scab, black spot Gale bactérienne de la tomate et du piment, tache bactérienne de la tomate Bakterielle Fleckenkrankheit der Tomate

Main hosts: Tomato (Lycopersicon esculentum, group B) and sweet pepper (Capsicum annuum, group A). Incidental hosts (mainly weeds) are Argemone mexicana (prickly poppy), Capsicum frutescens (chilli pepper), Datura spp. (thorn-apple), Hyoscyamus spp. (henbane), Lycium spp. (matrimony vine), Lycopersicon pimpinellifolium (currant tomato), Nicandra physaloides (apple of Peru, shoo-fly), Nicotiana rustica (Aztec tobacco), Physalis minima (gooseberry), Solanum dulcamara (bittersweet), S. nigrum (black nightshade), S. rostratum (buffalo bur). Symptoms and transmission: Small irregular green water-soaked spots that become brown-black and necrotic in later stages on cotyledons and true leaves are the first symptoms of bacterial spot (Fig. 201 top). The lesions become grey-black and necrotic with a narrow yellow halo. When many spots occur they may coalesce and leaves show yellowing and necrosis and dead tissue may drop out. Distortion and leaf roll may also occur. On stems and petioles longitudinal spots that have a scabby appearance with fissures can be observed. On fruits small light green to brown-black, oval to irregular blisters with a water-soaked margin develop that burst and show a sunken black necrotic centre surrounded by a cracked epidermis (Fig. 201 bottom). These spots may serve as entry for secondary rotting organisms. Spots may be confused with those caused by Pseudomonas syringae pv. tomato, which are, however, smaller (see Fig. 196). The pathogen is mainly spread by (latently) infected seed and seedlings, but also insects, tools and splashed soil may contribute in the field. The bacterium survives in seed, in volunteer plants and in debris in soil. High humidity and temperatures over 25ºC enhance disease development. Geographical distribution and importance EPPO A2 quarantine pest First described in 1920 and 1921 by Doidge in North America. Antigua and Barbuda, Argentina, Australia, Austria, Azerbaijan, Barbados, Belau, Bermuda, Brazil, Bulgaria, Canada, Chile, China, Colombia, Costa Rica, Cuba, Czech Republic, Dominica, Dominican Republic, Egypt, El Salvador, Ethiopia, Fiji, France, Germany, Greece, Grenada, Guadeloupe, Guatemala, Honduras, Hungary, India, Israel, Italy, Jamaica, Japan, Kazakhstan, Kenya, Korea, Malawi, Martinique, Mexico, Micronesia, Morocco, Mozambique, New Caledonia, New Zealand, Nicaragua, Niger, Nigeria, Reunion, Pakistan, Paraguay, Philippines, Poland, Puerto Rico, Romania, Russia, Saint Kitts and Nevis, Saint Lucia, Saint Vincent and the Grenadines, Samoa, Senegal, Slovakia, Slovenia, Spain, Seychelles, South Africa, Sudan, Suriname, Taiwan, Tanzania, Thailand, Togo, Tonga, Trinidad and Tobago, Tunisia, Turkey, Uruguay, USA, Venezuela, Virgin Islands, Yugoslavia, Zambia, and Zimbabwe. Especially under overhead irrigation, bacterial spot can cause serious damage both on tomato and pepper (Dougherty, 1979; Bashan, 1985). In greenhouses the disease is much less damaging. Control Healthy (tested) seeds, treated seeds (0.8% acetic acid for 24 h or 5% HCl for 5-10 h or 1.05% NaOCl for 30 min. (Dempsey and Chandler, 1963)), volunteer control and use of resistant varieties are ways to control bacterial spot. At least three races within strains from tomato and also from pepper have been discriminated (Pohronezny et al., 1992; Jones et al., 1995).

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Fig. 202 Top left:

Tumours on the crown of an Aster plant (hence the name ‘crown gall’ for this symptom), caused by Agrobacterium tumefaciens. Natural infection. Top centre: Large tumour on rose (Rosa sp.) caused by A. tumefaciens at a pruning wound. Natural infection. Tumour on above-ground plant part (stem) of Chrysanthemum caused by A. tumefaciens at a Top right: pruning wound. Natural infection. Bottom left: Small tumours, caused by A. tumefaciens, that developed on a leaf of Chrysanthemum at wounds made by insects. Natural infection. Bottom right: Left: Isolation plate of selective medium for biovar 1 of A. tumefaciens (so-called Schroth’s crown gall medium, Schroth et al., 1965) showing almost exclusive selectivity for the pathogen and discriminate typical transparent-cream colonies. Right: Non-selective isolation plate (nutrient agar) of the same extract of a tumour as used for the selective medium on the left: a thick carpet of bacteria grows on the plate and typical colonies of the pathogen cannot be discriminated.

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11. Bacterial pathogens that attack many host plants 11.1 Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 Syn. Rhizobium radiobacter. A. rubi, causing tumours on Rubus spp., A. vitis (causing tumours on grapevine (Vitis spp.) and systemic in host, Fig. 94), A. rhizogenes (causing hairy root on different hosts, having a root-inducing or Ri-plasmid) and A. radiobacter (non-pathogenic, no Ti plasmid, see Chapter III.5c, some strains have a Ri-plasmid and cause root mat disease in tomato and cucumber, see Weller et al., 2004) are not discussed here. For. A. tumefaciens at least two biovars have been described that are differently sensitive to biological control with A. radiobacter K84 or its genetically manipulated form K1026 (see Chapter VI.8 and Fig. 147). For detailed taxonomic discussion, and reclassification of Agrobacterium spp. into Rhizobium see Young et al. (2001); classification as A. tumefaciens is retained here.

Crown gall or root knot Galle du collet Wurzelkropf, Wurzelkrebs

Main hosts: Frequently reported natural hosts (also in relation to damage and control) are aster (Aster spp.), apple (Malus spp.), beet (Beta vulgaris), cherry (Prunus avium), peach (P. persica), pear (Pyrus spp.), chrysanthemum (Chrysanthemum spp.), Ficus spp. and rose (Rosa spp.). Host range is very wide, with over 300 species (mostly dicotyledons) listed (DeCleene and De Ley, 1976; Bradbury, 1986 and on http://www.cabicompendium.org/cpc/home.asp), but many hosts are only known through artificial inoculation (Young et al., 2001), e.g. tomato (Lycopersicon esculentum) is a very good indicator host, but has never been found as a natural host. Symptoms and transmission: Excrescences (not galls, but tumours, also see Chapter III.5c and Figs. 90-94) are formed on the crown (ground/air basis), on roots or incidentally (e.g. on chrysanthemum and Ficus) on stems, petioles and leaves (Fig. 202) at temperatures between c. 10 and 33ºC. In some plants, such as chrysanthemum, the bacterium also becomes systemic. Wounds (by grafting, insects, nematodes, etc.) are necessary for infection. The bacterium survives well in soil, especially in the rhizosphere. Distribution of the pathogen is by infected plant material, manipulations during cultivation, splash and irrigation water, nematodes and insects, e.g. chewing insects (see Fig. 202 bottom left) and whiteflies (Schroth et al., 1971; Moore and Cooksey, 1981; Dhanvantari, 1983; Zeidan and Czosnek, 1994). The bacterium is not seed-transmitted. Geographical distribution and importance Worldwide distribution. Occasionally and only in some hosts in some geographical regions or (greenhouse) cultural conditions substantial growth reduction and occasional death of plants occur. This was reported e.g. for rose, aster, chrysanthemum and peach. Otherwise damage is only aesthetical. Severe losses may occur when countries (aberrantly) apply quarantine regulations for this ubiquitous organism. Control Healthy and less susceptible planting material and culling of infected plants (removal of tumours or infected plants), nematode and insect control, hygiene, including disinfection of tools used for grafting/pruning, avoidance of wounding plants if possible and planting clean material in sites previously infested with the bacteria are ways to reduce crown gall infections (Schroth et al., 1971). Biological control with A. radiobacter strain K84 or K1026 is very effective in areas where sensitive (biovar 2) strains of A. tumefaciens occur (Bazzi et al., 1980; Jones and Kerr, 1989; Vicedo et al., 1993).

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Fig. 203 Top:

Erwinia carotovora subsp. carotovora can cause wilting symptoms (top left) in plants when the heart of the plant (top right, red arrow), petiole or (part of) the root system or stem base is rotted and water flow from the roots to the above-ground parts is obstructed, as shown here for courgette or zucchini (Cucurbita pepo) cultivated in a plastic tunnel, where excess water and bad culture conditions enhanced soft rot development. Natural infection. Centre left: Wilting symptoms in Madagascar dragon tree (Dracaena marginata) due to severe soft (internal and invisible) rot of the cane, caused by E. c. subsp. carotovora. Natural infection. Centre right: Typical soft rot symptoms of winter melon (Cucumis melo), caused by E. c. subsp. carotovora: softening and yellow to light brown discoloration of the internal tissues, from which fluids may exude. Natural infection. Bottom: Soft rot of basal stem part with brown discoloration of tissues on Dieffenbachia sp., caused by E. c. subsp. carotovora. No vascular browning as in cases of infection by E. chrysanthemi, see Fig. 205 bottom right. Natural infection.

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11.2 Erwinia carotovora subsp. carotovora (Jones 1901) Bergey, Harrison, Breed, Hammer

and Huntoon 1923 Syn. Erwinia aroideae; Pectobacterium carotovorum var. aroidae; P. carotovorum

Bacterial soft rot Pourriture molle Bakterielle Nassfäule

Main hosts: Very wide host range, especially plants with organs containing soft (storage) tissues. Commonly attacked hosts are Allium cepa (onion), Begonia spp., Brassica spp. (cabbage), Cichorium endivia (chicory), Cucurbita spp. (cucurbits), Daucus carota (carrot), Raphanus sativus (radish), Rheum rhaponticum (rhubarb), Solanum tuberosum (potato), S. melongena (eggplant) and Zantedeschia (calla lily). Symptoms and transmission: Typical soft rot of tubers (see Fig. 101 left), other storage organs and fruits, but also the stem base of plants may show soft rot and plants may show wilting symptoms (Fig. 203). Tissues become soft and fluid may exude from them, stems and petioles may become hollow due to rapid dissolution of the pith and total collapse of plants may occur. Soft rot caused by E. c. subsp. carotovora may be confused with soft rot symptoms caused by Erwinia chrysanthemi (see Fig. 205). In E. chrysanthemi infections, however, brown vascular necrosis up to higher parts of the stem can be observed, as well as rapid wilting. Usually a combination of wounds, high temperatures and a high humidity are necessary to stimulate infection and disease progress. In recent years, however, it has become clear that pathogenicity of this bacterium is complex and less dependent on external factors as was believed for a long time (De Boer, 2003). The bacterium survives in the soil and especially in the rhizosphere and has many hosts, but may also be spread when present externally on seed (tubers). Insects (flies) and their larvae may distribute and transmit E. c. subsp. carotovora (Kloepper et al., 1979). Furthermore (irrigation, rain and wash) water and aerosols may distribute the organism efficiently, but also machines. The bacterium may survive in surface water for some time (Kelman, 1980; Pérombelon and Hyman, 1987; Pérombelon and Pérombelon, 2002). Geographical distribution and importance The bacterium occurs worldwide, but is most prevalent in warm and humid climates. Crop loss under unfavourable conditions (water pooling in the field or greenhouse, damage, harvest in warm weather, followed by (too) rapid cooling and/or bad storage conditions) can be high. Control Healthy planting material, hygiene on the farm or in the nursery, packing stations, etc., use of clean water and especially optimal harvesting and storage conditions (optimal ventilation with avoidance of condensation) are the only ways to prevent or control diseases caused by E. c. subsp. carotovora. Cutting of potato seed and wounds made by machines should be avoided as well as excessive irrigation in combination with high temperatures. Fig. 204 Strong dissolution of middle lamellae (maceration of tissue) of potato due to activity of pectolytic enzymes produced by Erwinia carotovora subsp. carotovora cells that are abundantly present in the intercellular spaces and in fluid surrounding macerated tuber cells. B = intercellular space; IC = intercellular space; S = starch grain; SPC = starch-containing parenchymal cell.

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Fig. 205 Top left:

Typical soft rot of fleshy plant parts, in this case of Gymnocalicium mihanovichii ‘Optima rubra’ cactus, caused by Erwinia chrysanthemi biovar 3. Natural infection. Top right: Fried egg-like colonies of E. chrysanthemi biovar 3, after 3 days’ incubation on an isolation plate (YPG agar). Centre left: Rotting and brown vascular necrosis in Dahlia ‘Golden Wonder’ tubers, caused by E. chrysanthemi biovar 1. Natural infection. Centre middle: Basal stem rot and brown vascular necrosis in Kalanchoe blossfeldiana, caused by E. chrysanthemi biovar 7. Natural infection. Centre right: External symptoms of soft rot in potato (Solanum tuberosum) caused by E. chrysanthemi. The skin of the potato is still firm, but internally half the tuber is a watery mass. Natural infection. Severe rotting of roots and heads of chicory (Cichorium intybus), caused by E. chrysanthemi Bottom left: biovar 5. Natural infection. Bottom right: Water-soaked spots on leaves of Freesia sp., caused by E. chrysanthemi biovar 1, natural infection.

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11.3 Erwinia chrysanthemi Burkholder, McFadden and Dimock 1953 Syn. Pectobacterium chrysanthemi; Dickea chrysanthemi Subdivisions within E. chrysanthemi have been made on the basis of biochemical differences (at least nine biovars were discriminated (see Janse and Ruissen, 1988; Ngwira and Samson, 1990). Biovar 3 occurs mostly in the tropics or in tropical plants kept in greenhouses, biovar 2 is confined to Dieffenbachia, biovars 1 and 4-9 are found in plants grown in temperate climates. Furthermore at least seven pathovars based on original host and host range have been discriminated: pv. chrysanthemi, pv. dianthicola, pv. dieffenbachiae, pv. paradisiaca, pv. parthenii, pv. philodendroni and pv. zeae (Hildebrand et al., 1978; Young et al., 1996). PCR-RFLP has confirmed the biovar/pathovar discrimination (Nassar et al., 1996a). For many hosts mentioned in the literature the biovar or pathovar of E. chrysanthemi has not been determined. Pv. dianthicola is an A2 quarantine pathogen for EPPO, although this pathovar has rarely been reported in recent years. E. chrysanthemi pv. paradisiaca, which is confined to Musa paradisiaca, was renamed Brenneria paradisiaca (Hauben et al., 1998) on the basis of 16S rRNA gene sequences. In a later study all strains of E. chrysanthemi and Brenneria paradisiaca were placed on the basis of a polyphasic taxonomic study into a new genus Dickea and six species were discriminated: Dickeya zeae; D. dadantii; D. chrysanthemi (subdivided into biovar chrysanthemi and biovar parthenii ); D. dieffenbachiae; D. dianthicola and D. paradisiaca (Samson et al., 2005). Synonymy and hosts can be found in the latter article.

Bacterial soft rot, bacterial stem rot of potato Pourriture molle Bakterielle Nassfäule

Main hosts: Very wide host range, see http://www.cabicompendium.org/cpc/home.asp. Commonly attacked hosts are Aechmea fasciata, Aglaonema spp., Allium cepa (onion), Begonia spp., Chrysanthemum spp., Cichorium endivia (chicory), Dahlia spp., Dieffenbachia spp., Dracaena marginata, Kalanchoe blossfeldiana, Musa spp. (banana, plantain), Philodendron spp., Solanum tuberosum (potato), Saintpaulia ionantha, Syngonium podophyllum and Zea mays. Symptoms and transmission: Typically E. chrysanthemi is a soft rot organism, causing soft rot of succulent plant tissues and organs (Fig. 205 top left) or a vascular pathogen causing brown vascular necrosis and wilting (Fig. 205 centre right). Latent infections may occur, especially below 20ºC. Soft rot symptoms may be confused with those caused by E. carotovora subsp. carotovora (Fig. 203). In potato, soft rot of tubers and pith of stems may occur, accompanied by vascular necrosis and wilting (so-called stem rot symptoms). Corn stalk rot is a major disease of maize in tropical and subtropical countries or under conditions of overhead irrigation and warm summers in more temperate climates. E. chrysanthemi is not a true seedborne pathogen, but can be transmitted with transplants and potato seeds and dispersed with irrigation water (Cother et al., 1992), wind-splashed water, aerosols, knives and other instruments or machines and insects. Geographical distribution and importance The bacterium occurs worldwide, especially in warm and humid climates. Crop loss in the field under warm humid conditions and under unfavourable storage conditions can be high. Stalk rot in maize can give up to 98% crop loss (Lopes et al., 1986; Thind and Payak, 1985). Control Healthy planting material (through indexing systems in strict selection and multiplication schemes, Nassar et al., 1996b), hygiene and optimal harvesting and storage conditions are ways to prevent or control diseases caused by E. chrysanthemi. In some hosts (a degree of) resistance has been determined (Thind and Payak, 1985; Haynes et al., 1997; Norman et al., 1997).

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Fig. 206 Top left:

Blossom blight, caused by Pseudomonas syringae pv. syringae on forsythia (Forsythia intermedia). Inflorescence and leaflets turn black and wither completely. Natural infection. Top right: Extensive reddish-brown to black necrosis of young pear fruitlets, caused by P. s. pv. syringae. Natural infection. Centre left: Brown spots with a black rim on the upper side of a leaf of pear (Pyrus communis). Natural infection. Centre right: As centre left on lower side of the leaf. The spots are more blackish and water-soaked. Natural infection. Bottom left: Brown to black leaf spots with a papery centre and water-soaked rim on the lower side of the leaf on mock orange (Philadelphus sp.), caused by P. s. pv. syringae. Natural infection. Bottom right: Light brown necrosis along the veins of a leaf of false spirea (Astilbe sp.), caused by P. s. pv. syringae. Natural infection.

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11.4 Pseudomonas syringae pv. syringae van Hall 1902 Syn. Pseudomonas syringae

Bacterial canker or blast of stone and pome fruits; apoplexy of apricots; bacterial brown spot of bean; bacterial leaf blight of wheat; bacterial spot disease; blister spot of apple; pear blossom blight Chancre bactérien Bakterienbrand des Kern und Steinobstes

Main hosts: Very wide host range, see http://www.cabicompendium.org/cpc/home.asp. Commonly attacked hosts are apple (Malus spp.), apricot (P. armeniaca), kidney bean (Phaseolus vulgaris), European bird cherry (Prunus padus), hawthorn (Crataegus spp.), lilac (Syringa vulgaris); orange (Citrus sinensis), peach (Prunus persica), plum (P. domestica and Japanese plum), poplar (Populus spp.), sweet cherry (P. avium), sour cherry (P. cerasus) and wheat (Triticum aestivum). Some host specialization has been reported (Little et al., 1998). P. s. pv. syringae can also cause stem canker and dieback in a conifer (Pinus radiata, see Dick, 1985). Symptoms and transmission: P. s. pv. syringae causes a diverse range of symptoms on its many hosts (Fig. 206) , including water-soaked, later whitish (on wheat) to brown and black necrotic spots, dieback of shoots and cankers on twigs and branches or bark necrosis (on apple). Leaf spots have no haloes. Spots may coalesce and larger parts of tissue may become necrotic. In other cases as in pear the whole leaf may blacken and die. On stone and pome fruits cankers may develop, also showing gumming, very similar to infections caused by P. s. pv. morsprunorum. Blossom blight may also occur, where flowers, peduncles and pedicels turn black. Symptoms may be very similar to those caused by Erwinia amylovora, and droplets of grey-cream slime may also be formed. Fruits are also attacked, showing large brown to black necrotic areas (Fig. 206, top right), circular spots (ring spot on bean) or wart-like eruptions (Crosse, 1966; Scortichini and Morone, 1997; Sharrock et al., 1997). P. s. pv. syringae can overwinter in cankers and on or in infected leaves and buds in an epiphytically or latent form, and possibly also on weeds or non-susceptible hosts, it can also be seedborne, as in bean (Phaseolus vulgaris). The bacterium survives in the soil, but may also be spread when present externally on the seed. Disease development is enhanced by wet weather, high humidity, and cool temperatures (15-20°C). Bacteria are spread by wind-driven rain and enter host plants through stomata and wounds. Pruning tools, insects and birds are not important in disease transmission (Crosse, 1966). Geographical distribution and importance The bacterium occurs worldwide and was first described from lilac in 1902 in The Netherlands by van Hall, but is most prevalent in temperate climates, where wet and cold springs with frosts prevail. The bacterium is ice-nucleation active (INA) (also see Chapter III.1) and therefore is often associated with (night) frosts, causing frost injury (Crosse, 1966; Mittelstadt and Rudolph, 1998; Young, 1987). In this way substantial damage in orchards and plantations may occur. When the weather becomes warmer and dry, infection often stops. Control Spraying with copper compounds or antibiotics in critical times in spring and autumn can be effective in prevention. Copper resistance, however, may develop (Garrett and Schwarz, 1998; Scheck and Pscheidt, 1998). Use of healthy seeds, budwood or trees, adequate fertilization and pruning and burning of diseased parts (in summer under dry conditions) and proper water management, are factors in the control (Tabilio et al., 1998).

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Fig. 207 Top left:

Bacterial slime oozing from an eye of a potato and soil sticking to this slime as a typical symptom of infection by Ralstonia solanacearum biovar 2, race 3 on potato (Solanum tuberosum) tubers. Natural infection. Top right: Glassy to yellow-brown vascular discoloration and spontaneously protruding bacterial slime (arrow), caused by R. solanacearum biovar 2, race 3 on potato (Solanum tuberosum) tubers. Natural infection. Centre left: Severely rotting rhizomes and stolons of Curcuma longa, caused by R. solanacearum biovar 4, race 1. Natural infection. Centre right: Pelargonim zonale showing wilting, sectorial yellowing and necrosis, 20 days after inoculation with R. solanacearum biovar 2, race 3 strain PD 325 from potato. Plants kept at 21ºC. Bottom left: Severe wilting of a tomato plant, caused by R. solanacearum biovar 2, race 3 in a greenhouse. Natural infection. Bottom right: Strands of bacterial slime protruding from vascular tissues of a cut potato stem infected by R. solanacearum in a glass of water as a rapid presumptive test of brown rot infection.

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11.5 Ralstonia solanacearum (Smith 1896) Yabuuchi, Kosako, Yano, Hotta and Nishiuchi

1996 Syn. Pseudomonas solanacearum; Burkholderia solanacearum. Five pathogenic races and five biovars have been discriminated. Race 1 occurs in tropical areas all over the world and attacks tobacco, many other solanaceous crops and many hosts in other plant families. It has a high temperature optimum (35ºC, as do races 2, 4 and 5). Race 2 occurs mainly in tropical areas of South America and attacks bananas and Heliconia (causing so-called Moko disease), but also in the Philippines (causing so-called bugtok disease on plantains). Race 3 occurs at higher altitudes in the tropics, and in subtropical and temperate areas attacks potato, tomato, occasionally Pelargonium zonale, eggplant and Capsicum, and some solanaceous weeds like black nightshade (Solanum nigrum) and bittersweet (S. dulcamara). A number of non-solanaceous weed hosts have also been found to harbour race 3 infections, often asymptomatically (Pradhanang et al., 2000, Strider et al., 1981; Wenneker et al., 1999; Janse et al., 2004). This race has a lower temperature optimum (27ºC) and appears to be mostly biovar 2A or RFLP group 26 with a worldwide distribution (Cook and Sequira, 1994), RFLP group 27 (found in Chile and Colombia) or 2T (sometimes also called 2N, found in tropical areas in S. America). Race 4 is specialized on ginger, race 5 (biovar 5) on Morus. Another recent subclassification of R. solanacearum, based on RFLP and other genetic fingerprinting studies (Hayward, 2000) is into Division I (biovars 3, 4 and 5 originating in Asia) and II (biovars 1, 2A and 2T, originating in S. America). For a different biotype scheme, based on maltose, mannitol, malonate, trehalose, inositol and hippurate and discrimination of 4 so-called phylotypes using multiplex PCR, based upon the 16S-23S rRNA gene intergenic spacer region (ITS) and 23 so-called sequevars, based upon fingerprinting methods such as rep-PCR, RAPD and AFLP, see Taghavi et al. (1996); Poussier et al. (2000); Fegan and Prior (2005).

Bacterial wilt, bacterial slime disease, potato brown rot, Granville wilt of tobacco; Moko disease of banana, southern bacterial wilt of tomato Flétrissement bactérien, pourriture brune, maladie de moko du bananier Bakterienwelke, Braunfäule, Schleimkrankheit der Kartoffel

Main hosts: Very wide host range, especially race 1; the most important hosts are: Arachis hypogea (peanut), Heliconia, Lycopersicon esculentum (tomato), Musa spp. (banana), Musa paradisiaca (plantain), Nicotiana tabacum (tobacco), Solanum melongena (aubergine) and Solanum tuberosum (potato). For extensive host range see http://www.cabicompendium.org/cpc/home.asp. and for host range of biovar 2, race 3 see Janse et al. (2004). Symptoms and transmission: On potatoes, rapid wilting of leaves and stems occurs (Fig. 208); in later stages plants fail to recover, become yellow and brown necrotic and die. Sometimes brown streaks on the stem may be observed above the soil line, leaves may have a bronze tint and epinasty of the petioles may occur. Grey-white bacterial slime exudes from vascular bundles, which are broken or cut. This slime oozes spontaneously from the cut surface of a potato stem in the form of threads, when suspended in water. Such threads are not formed by other bacterial pathogens of potato. This ‘water streaming’ test is of presumptive diagnostic value in the field. Wilting and other foliar symptoms may not occur under dry, cold conditions. Tubers may show bacterial ooze on the eyes and stolon end attachment. Soil sticks to the tubers at the eyes (Fig. 207 top left). When a diseased tuber is cut, glassy to yellow-browning and eventual necrosis of the vascular ring tissues may be observed. Creamy bacterial slime exudes spontaneously from the vascular ring a few minutes after cutting (Fig. 207 top right). Symptoms may be confused with those of ring rot, caused by Clavibacter michiganensis subsp. sepedonicus. On tomatoes, leaves wilt, usually at the warmest time of day. Wilting of the whole plant may follow rapidly if environmental conditions are favourable for the pathogen. Under less favourable conditions, the disease develops less rapidly, stunting may occur and large numbers of adventitious roots are produced on the stem. The vascular tissues of the stem show a brown

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Fig. 208 Top:

Typical wilting symptoms and epinasty of petioles on potato (Solanum tuberosum), caused by Ralstonia solanacearum biovar 1, race 1. Natural infection. Bottom right: Slimy colonies with a red centre of R. solanacearum biovar 2, race 3 after 5 days’ growth, isolated from surface water on selective SMSA medium. Bottom left: Slimy cream-coloured colonies producing a brown pigment after 3 days’ growth on yeastpeptone-glucose agar of R. solanacearum biovar 2, race 3, after isolation from a diseased potato tuber.

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discoloration and, if the stem is cut crosswise, drops of white or yellowish bacterial slime exude from the vascular tissues. On pelargonium, first symptoms are wilting and subsequent chlorosis of leaves. Stems may blacken and eventually become necrotic. Internally vascular browning is often visible. In a later stage leaves become brown and necrotic and the whole plant desiccates and dies. In the final stages plants collapse totally. On tobacco, a main symptom is unilateral wilting and premature yellowing. Leaves on one side of the plant or even a half leaf may show wilting symptoms. In severe cases, leaves wilt without changing colour and stay attached to the stem. As in tomato, the vascular tissues show a brown discoloration when cut open. The primary and secondary roots may become brown to black. On banana, symptoms caused by R. solanacearum are called Moko disease, which is easily confused with the disease caused by Fusarium oxysporum f. sp. cubense. A clear distinction is possible when fruits are affected - a brown and dry rot is seen only in the case of Moko disease. On young and fast-growing plants, the youngest leaves turn pale-green or yellow and collapse. Within a week all leaves may collapse. Young suckers may be blackened, stunted or twisted. The pseudostems show brown vascular discoloration. R. solanacearum can spread via soil, in which it survives for varying periods of time (often at least more than one season), and in irrigation (drainage) water. In tropical areas, many weeds are alternative hosts. The slow rate of development of the bacterium on the weeds allows them to withstand infection, and so provide a bridge for the pathogen between crops. Natural spread of most of the R. solanacearum races is very limited and slow. However, race 2, which causes Moko disease of banana, is known to be transmitted by insects and has a high potential for natural spread. Race 3 may be spread more easily with surface water when infected S. dulcamara grows with its roots floating in water. The bacterium may subsequently be spread to other hosts when contaminated surface water is used for irrigation (Olsson, 1976; Elphinstone et al., 1998; Janse et al., 1998; Wenneker et al., 1999). The main path for international spread is by (latently) infected seed potatoes and other vegetative propagating materials. Natural infection of true seed has only been firmly established for groundnut (Arachis spp.). Infections of potato tubers may be latent, due to unfavourable weather conditions, partly resistant cultivars or low virulence of certain pathogen strains. Entry into plants is by way of injured roots (e.g. by nematodes), stem wounds or through stomata. Geographical distribution and importance EPPO A2 quarantine pest First described as Bacillus solanacearum by Smith (1896) in the USA. The bacterium occurs worldwide, but is most prevalent in warm and humid climates. For extensive host range see: http://www.cabicompendium.org/cpc/home.asp. For host range of race 3, see Janse et al. (2004). The greatest economic damage has been reported on potatoes, tobacco and tomatoes in the southeastern USA, Indonesia, Nepal, Central and northwestern Uganda, Brazil, Colombia and South Africa and on bananas in South America. Control Control of bacterial wilt has proved to be very difficult, especially for race 1 with its broad host range. Hot air treatment of ginger roots for 30 min at 50ºC has been successful (Tsang and Shintaku, 1998). Several resistant cultivars of potato, eggplant, tobacco and peanut, as well as other crops, are available, but the race and strain diversity of the pathogen make it difficult to utilize these in different countries. Use of healthy (tested) planting material, early and sure detection and reporting of the pathogen, quarantine measures on infected fields and farms, sufficient crop rotation, control of weed hosts and volunteer plants (and in some cases of nematodes), avoidance of surface water for irrigation, cutting of potato seed, and education are key factors in the control of R. solanacearum (Janse, 1996; Pradhanang and Elphinstone, 1996).

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Fig. 209 Top left:

Abnormal sprout formation on phlox (Phlox paniculata), caused by Rhodococcus fascians. Natural infection. Top right: Dry, orange colonies of R. fascians after 4 days’ growth on yeast-peptone-glucose agar. Centre right: Gram-positive cells of R. fascians, 1.5-4 x 0.5-1 µm from a 3-day-old culture grown on nutrient agar. Bottom left: Typical cauliflower-like galls on Dahlia ‘Rembrandt’, caused by R. fascians. Natural infection. Bottom right: Cauliflower-like galls on Angel’s trumpet (Brugmansia sp.), caused by R. fascians. Natural infection.

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11.6 Rhodococcus fascians (Tilford 1936) Goodfellow 1984 Syn. Corynebacterium fascians

Bacterial fasciation, leafy gall, cauliflower disease Fasciation, gale foliare, maladie de chou-fleur Verbänderung Stewart’s krankheit, Bakteriellen welkenkrankheid der Mais

Main hosts: Wide host range (see Bradbury, 1986 and http://www.cabicompendium.org/cpc/home.asp). Main hosts are cabbage (Brassica spp.), carnation (Dianthus spp.), dahlia (Dahlia spp.), gladiolus (Gladiolus spp.), lily (Lilium spp.), mullein (Verbascum spp.), pelargonium (Pelargonium spp.) and sweet pea (Lathyrus spp.). Symptoms and transmission: Fasciation and formation of short, fleshy shoots and cauliflower-like galls under the influence of plant hormones (mainly cytokinins) are produced by R. fascians (Figs. 209 and 210). Galls and malformed shoots often start from the hypocotyls and are found close to soil/air or below the surface. The bacterium lives on the outside or in the superficial tissues of the galls in small pockets. Wounds are not necessary for penetration. Transmission can take place via soil, infected planting material or contaminated tools. The bacterium can survive in the soil, for up to several years, and can be present in seeds (Baker, 1950; Faivre-Amiot, 1967). Geographical distribution and importance Disease first described from sweet pea by Brown in 1927 from the USA and bacterium described by Tilford in 1936. Occurs worldwide, see http://www.cabicompendium.org/cpc/home.asp. Damage for plants is usually low, even when a high number of galls is present, but when young bulbs that are used as planting material are heavily attacked (as is sometimes the case in Lilium), serious crop loss may occur (Miller et al., 1980). Control Healthy planting material, hygiene and (steam) disinfection of growth substrate or soil are ways to control diseases caused by R. fascians. Hot water treatment (2 h at 43ºC) of Lilium bulbs before planting was partly effective (Digat, 1977; Kruyer and Boontjes, 1982).

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Fig. 210 Abnormal sprout formation on cauliflower (Brassica oleracea var. botrytis) caused by Rhodococcus fascians. Natural infection.

Annex 1 – Newer classification of bacteria

ANNEXES Annex 1

Newer classification of bacteria. In many cases not all classes, orders, subranks and genera in the phylum are mentioned. After Boone et al. (2001). Subdivision of Domain of Bacteria

Phylum

Name

I II III IV V VI VII VIII IX X

Aquificae (hyperthermophylic bacteria) Thermotogae ( ,, ,, ) Thermodesulfobacteria (,, ,, ) ‘Deinococcus-Thermus’ Chrysiogenetes Chloroflexi (green non-sulfur bacteria) Thermomicrobia Nitrospirae Deferribacteres Cyanobacteria (‘blue green algae’)

XI XII

Chlorobi Proteobacteria (Gram-negative bacteria) Class I “Alphaproteobacteria” Order I Rhodospirillales Order II Rickettsiales Order III “ Rhodobacterales” Order IV “Sphingomonadales” Order V Caulobacterales Order VI “Rhizobiales”

Class II “Betaproteobacteria” Order I “Burkholderiales” Order II “Hydrogenophilales” Order III “ Methylophilales” Order IV “Neisseriales” Order V “Nitrosomonadales” Order VI “Rhodocyclales” Class III “Gammaproteobacteria” Order I Chromatiales Order II “Xanthomonadales” Order III “Cardiobacteriales” Order IV “Thiotrichacea” Order V “Legionellales” Order VI “Methylococcales” Order VII “Oceanospirillales” Order VIII “Pseudomonadales” Order IX Order X Order XI Order XII

XIII

“Alteromonadales” “Vibrionales” “Aeromonadales” “Enterobacteriales”

Order XIII “Pasteurellales” Class IV “Deltaproteobacteria” Order II “Desulfovibrionales” Order VI “Bdellovibrionales” Class V “Epsilonproteobacteria” Order I “Campylobacterales” Firmicutes (Gram-positive bacteria) Class I “Clostridia” Order I “Clostridiales” Order II “Thermoanaerobacteriales” Order III “Haloanaerobiales”

Representative genera Aquifex, Hydrogenobacter Thermotoga, Fervidobacterium Thermodesulfobacterium Deinococcus, Thermus Chrysiogenes Chloroflexus, Heliothrix, Chloronema Thermomicrobium Nitrospira, Leptospirillum, Magnetobacterium Deferribacter, Geovibrio Oscillatoria, Nostoc, Anabaena, Prochloron, Gloeocapsa Chlorobium, Pelodyction Rhodospirillum, Azospirillum, Acetobacter Rickettsia, Ehrlichia, Anaplasma Rhodobacter, Paracoccus, Rubrimonas Sphingomonas, Rhizomonas, Erythrobacter Caulobacter, Phenylobacterium Rhizobium, Sinorhizobium, Agrobacterium, ‘candidatus Liberobacter’, Phyllobacterium, Beijerinckia, Bradyrhizobium, Hyphomicrobium, Azorhizobium, Xanthobacter Burkholderia, Ralstonia, Janthinobacterium, Alcaligenes, Comamonas, Acidovorax, Leptothrix Hydrogenophilus, Thiobacillus Methylophilus, Methylobacillus Neisseria, Aquaspirillum, Kingella Nitrosomonas, Nitrospira, Spirillum, Gallionella Rhodocyclus, Propionivibrio Chromatium, Lamprocystis, Thiospirillum Xanthomonas, Stenotrophomonas, Xylella, Frateuria Cardiobacterium, Suttonella Thiothrix, Beggiatoa, Leucothrix, Thiobacterium Legionella, Cxiella, Ricketsiella Methylococcus, Methylobacter Oceanospirillum, Marinomonas, Halomonas, Zymobacter Pseudomonas, Azotobacter, ‘Rhizobacter’(now Sphingomonas), Moraxella, Acinetobacter, Sphingomonas Alteromonas, Ferrimonas, Marinomonas Vibrio, Listonella, Salinivibrio Aeromonas, Ruminobacter Escherichia, Brenneria, Citrobacter, Dickeya, Enterobacter, Erwinia, Hafnia, Klebsiella, Pantoea, Pectobacterium, Proteus, Salmonella, Samsonia, Serratia, Xenorhabdus, Yersinia Pasteurella, Actinobacillus Desulfovibrio Bdellovibrio Campylobacter, Thiovulum, Helicobacter Clostridium, Ruminococcus, Eubacterium, Peptococcus, Anaerovibrio, Heliobacterium, Selenomonas, Thermohydrogenium Thermoanaerobacterium, Ammonifex Haloanaerobium, Natroniella, Halobacteroides

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Annex 1

Newer classification of bacteria (continued). In many cases not all classes, orders, subranks and genera in the phylum are mentioned. After Boone et al. (2001). Subdivision of Domain of Bacteria

Phylum

Name Class II Mollicutes Order I Mycoplasmatales Order II Entoplasmatales Order III Acholeplasmatales Order IV Anaeroplasmatales Phytoplasmas Class III “Bacilli” Order I Bacillales Order II

XIV

XV XVI XVII XVIII XIX XX

XXI XXII XXIII

“Lactobacillales”

Actinobacteria Class I Actinobacteria Subclass V Actinobacteridae Order I Actinomycetales

Planctomycetes Chlamidiae Spirochaetes Fibrobacteres Acidobacteria Bacteriodetes Class I “Bacterioides” Class II “Flavobacteriales” Class III “Sphingobacteriales” Fusobacteria Verrucomicrobiae Dictyoglomi

Representative genera Mycoplasma, Ureaplasma Entomoplasma, Spiroplasma Acholeplasma Anaeroplasma, Asteroleplasma Phytoplasma Bacillus, Gracilibacillus, Planococcus, Listeria, Staphylococcus, Paenibacillus, Thermobacillus, Pasteuria, Thermoactinomyces Lactobacillus, Aerococcus, Enterococcus, Leuconostoc, Streptococcus, Lactococcus Acidimicrobium, Rubrobacter, Coriobacterium Actinomyces, Arthrobacter, Brevibacterium, Cellulomonas, Microbacterium, Agromyces, Clavibacter, Curtobacterium, Rathayibacter, Corynebacterium, Mycobacterium, Nocardia, Rhodococcus, Actinoplanes, Streptomyces Nocardiopsis, Frankia, Acidothermus Planctomyces, Gemmata Chlamidia, Simkania Spirochaeta, Borrelia, Treponema, Leptospira Fibrobacter Acidobacterium, Geotrix Bacteroides, Acetomicrobium, Rikenella Flavobacterium, Cellulophaga Sphingobacterium, Cytophaga, Crenothrix Fusobacterium, Leptotrichia, Streptobacillus Verrucomicrobium Dictyglomus

Genera with plant pathogenic species Bacterial genera commonly associated with plants (as endophytes, saprophytes or symbiotic nitrogen-fixing organisms)

Annex 2 – List of plant pathogenic bacteria and their main hosts

Annex 2 Bacterium

List of plant pathogenic bacteria and their main hosts1) Main host(s)

Acidovorax anthurii A. avenae subsp. avenae (syn. Pseudomonas avenae and P. rubrilineans) A. a. subsp. cattleyae (syn. Pseudomonas cattleyae) A. a. subsp. citrulli (syn. Pseudomonas pseudoalcaligenes subsp. citrulli) A. konjaci (syn. Pseudomonas pseudoalcaligenes subsp. konjaci) A. valerianellae

Anthurium spp. Zea mays, Avena sativa

Agrobacterium rhizogenes (syn. Rhizobium rhizogenes) A. rubi (syn. Rhizobium rubi) A. tumefaciens (syn. Rhizobium tumefaciens) A. vitis (syn. Rhizobium vitis) A. larrymoorei (syn. Rhizobium larrymoorei)

apple, rose

Arthrobacter ilicis (syn. Curtobacterium flaccumfaciens pv. ilicis)

Ilex opaca

Brenneria alni (syn. Erwinia alni) B. nigrifluens (syn. Erwinia nigrifluens) B. paradisiaca (syn. Erwinia chrysanthemi pv. paradisiaca) B. quercina (syn. Erwinia quercina) B. rubrifaciens (syn. Erwinia rubrifaciens) B. salicis (syn. Erwinia salicis)

Alnus spp.

Burkholderia andropogonis B. caryophylli B. cepacia B. gladioli pv. alliicola B. g. pv. gladioli B. g. pv. agaricicola B. plantarii

broad host range carnation (Dianthus) broad host range, also human pathogenic strains onion (Allium cepa) Gladiolus Agaricus bisporis rice

Clavibacter michiganensis subsp. insidiosus C. m. subsp. michiganensis C. m. subsp. nebraskensis C. m. subsp. sepedonicus C. m. subsp. tessellarius C. toxicus C. xyli subsp. xyli (syn. Leifsonia xyli subsp. xyli)

lucerne tomato Zea mays potato Triticum aestivum Lolium rigidum Saccharum officinarum

Curtobacterium flaccumfaciens pv. betae C. f. pv. flaccumfaciens C. f. pv. ilicis (syn. Arthrobacter ilicis) C. f. pv. oortii C. f. pv. poinsettiae

Beta vulgaris bean (Phaseolus) Ilex opaca Tulipa Euphorbia pulcherrima

Dickeya: see Erwinia chrysanthemi Dickeya chrysanthemi pv. chrysanthemi (syn. Erwinia chrysanthemi)

For reclassification of Erwinia chrysanthemi into different Dickeya species and their hosts, see

1)

Cattleya Citrullus lanatus Amorphophallus rivieri Valerianella locusta (lamb’s lettuce)

Rubus spp. very broad host range, mainly dicotyledons Vitis Ficus benjamini

Musa paradisiaca Quercus spp. Juglans regia Salix alba, Populus robusta

Also see Vauterin et al. (2000), the exhaustive and frequently updated list (including references) organised by Young et al. at ISPP: http://www.isppweb.org/names_bacterial.asp and http://www.isppweb.org/names_bacterial_new2004.asp. Candidatus species not included except ‘candidatus Liberobacter spp.’ and ‘candidatus Phlomobacter fragariae’.

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Annex 2 – List of plant pathogenic bacteria and their main hosts Samson et al. (2005) D. chrysanthemi pv. parthenii ,, ,, D. dadantii D. dianthicola ,, D. dieffenbachiae D. paradisiaca (syn. Erwinia chrysanthemi pv. ,, paradisiaca, Brenneria paradisiaca) ,, D. zeae Enterobacter cancerogenus (syn. Erwinia cancerogena) E. dissolvens (syn. Erwinia dissolvens) E. nimipressuralis (syn. Erwinia nimipressuralis) E. pyrinus

Populus canadensis

Erwinia alni (syn. Brenneria alni) E. amylovora E. cacticida (syn. Pectobacterium cacticida) E. carnegieana (syn. Pectobacterium carnegieana) E. carotovora subsp. atroseptica (syn. Pectobacterium atrosepticum) E. c. subsp. betavasculorum (syn. Pectobacterium betavasculorum) E. c. subsp. brasiliensis (syn. Pectobacterium brasiliensis) E. c. subsp. carotovora (syn. Pectobacterium carotovorum) E. c. subsp. odorifera (syn. Pectobacterium carotovorum subsp. odoriferum) E. c. subsp. wasabiae (syn. Pectobacterium wasabiae) E. chrysanthemi (syn. Pectobacterium chrysanthemi) E. c. pv. chrysanthemi1) E. c. pv. dianthicola E. c. pv. dieffenbachiae E.c. pv. paradisiaca (syn. Brenneria paradisiaca) E. c. pv. parthenii E. c. pv. zeae E. cypripedii (syn. Pectobacterium cypripedii) E. herbicola pv. gypsophilae E. mallotivora E. milletiae

Alnus spp. Rosaceae e.g. apple, pear, hawthorn, Cotoneaster Carnegiea gigantea

E. nigrifluens (syn. Brenneria nigrifluens) E. papayae E. paradisiaca (syn. E. chrysanthemi pv. paradisiaca) E. persicinus E. psidii E. pyrifolii E. quercina (syn. Brenneria quercina) E. rhapontici (syn. Pectobacterium rhapontici) E. rubrifaciens (syn. Brenneria rubrifaciens) E. salicis (syn. Brenneria salicis) E. stewartii (syn. Pantoea stewartii subsp. stewartii) E. tracheiphila

Zea mays Ulmus spp. Pyrus pyrifolia (Asian pear tree)

potato Beta vulgaris potato (Solanum tuberosum) broad host range Apium graveolens Eutrema wasabi broad host range, different biovars1) Chrysanthemum morifolium Dianthus spp. Dieffenbachia spp. Musa x paradisiaca Parthenium spp. Zea mays, Aglaonema, Solanum tuberosum Cypripedium spp. galls on Gypsophila spp. Mallotus japonicus, Pyrus pyrifolia Wisteria floribunda (galls, in Japan), also classified as E. herbicola Juglans regia Carica papaya Musa x paradisiaca Lycopersicon esculentum, Phaseolus vulgaris. Psidium guajava Pyrus pyrifolia (Asian pear tree) in Korea Quercus spp. Rheum rhabarbarum Juglans regia Salix alba, Populus robusta Zea mays Cucumis sativus

Annex 2 – List of plant pathogenic bacteria and their main hosts Herbaspirillum rubrisubalbicans (syn. Pseudomonas rubrisubalbicans)

Saccharum officinarum

Janthinobacterium agaricidamnosum

Agaricus bisporus (Lincoln et al., 1999)

Leifsonia xyli subsp. cynodontis (syn. Clavibacter Cynodon dactylon xyli subsp. cynodontis) L. x. subsp. xyli (syn. Clavibacter xyli subsp. xyli) Saccharum officinarum ‘Candidatus Liberobacter africanus’ (nonculturable bacterium, vector transmitted, PCR detection) ‘Candidatus L. asiaticus’ (non-culturable bacterium, vector transmitted, PCR detection) ‘Candidatus L. asiaticus subsp. capensis’ (nonculturable bacterium, vector transmitted, PCR detection)

Citrus, Fortunella and Poncirus spp. (citrus greening or Citrus Huanglongbin)

Nocardia vaccinii

Vaccinium spp.

Pantoea agglomerans (syn. Erwinia herbicola, Enterobacter agglomerans) P. a. pv. betae (syn. Erwinia herbicola pv. betae) P. a. pv. gypsophilae (syn. Erwinia herbicola pv. gypsophilae) P. a. pv. millettiae (syn. Erwinia herbicola pv. milletiae) P. ananas (incl. Erwinia uredovora strains, found on Puccinia graminis f. sp. tritici) P. ananatis (syn. E. ananatis) P. dispersa P. stewartii subsp. indologenes P. s. subsp. stewartii

diverse host range, secondary invader

Pectobacterium: see Erwinia Pectobacterium atrosepticum (syn. Erwinia carotovora subsp. atroseptica) P. betavasculorum (syn. Erwinia carotovora subsp. betavasculorum) P. cacticida (syn. Erwinia cacticida) P. carotovorum subsp. atrosepticum (syn. Erwinia carotovora subsp. atroseptica) P. carotovorum subsp. betavasculorum (syn. Erwinia carotovora subsp. betavasculorum) P. carotovorum subsp. carotovorum (syn. Erwinia carotovora subsp. carotovora) P. carotovorum subsp. odoriferum (syn. Erwinia carotovora subsp. odorifera) P. carotovorum subsp. wasabiae (syn. Erwinia carotovora subsp. wasabiae) P. chrysanthemi (syn. Erwinia chrysanthemi) P. cypripedii (syn. Erwinia cypripedii) P. wasabiae (syn. Erwinia carotovora subsp. wasabiae)

Citrus, Fortunella and Poncirus spp. (citrus greening or Citrus Huanglongbin) Calodendrum capense

Beta vulgaris Gypsophila paniculata galls on Wisteria floribunda Ananas comosus onion, Eucalyptus diverse host range Pennisetum americanum, Setaria italica Zea mays potato Beta vulgaris Carnegiea gigantea potato Beta vulgaris broad host range Apium graveolens Eutrema wasabi Cypripedium spp. Eutrema wasabi

‘Candidatus Phlomobacter fragariae’

marginal chlorosis of strawberry

Pseudomonas aeruginosa

onion, lettuce (opportunistic invader)

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Annex 2 – List of plant pathogenic bacteria and their main hosts Agaricus bisporus P. agarici Prunus dulcis P. amygdali Asplenium nidus P. asplenii P. avellanae (syn. Pseudomonas syringae pv. Avellana spp. (hazelnut) avellanae) P. cannabina (syn. Pseudomonas syringae pv. Cannabis sativa cannabina) Carica papaya P. caricapapayae diverse host range, incl. Cichorium, P. cichorii Chrysanthemum Cissus japonica in Japan (genus misnamed) P. cissicola P. coronofaciens (syn. Pseudomonas syringae pv. cereals, incl. Avena, Triticale and grasses incl. coronofaciens and P. s. pv. striafaciens) Lolium tomato P. corrugata Agaricus bisporus P. costantinii Ficus erecta (Japan) P. ficuserectae Phaseolus vulgaris (generically misnamed) P. flectens diverse host range, secondary pathogen P. fluorescens rice P. fuscovaginae P. marginalis pv. alfalfae Medicago sativa (lucerne) P. marginalis pv. marginalis diverse host range, secondary pathogen P. marginalis pv. pastinacae Pastinaca sativa tomato (Lycopersicon esculentum), Italy P. mediterranea Melia azedarach P. meliae rice (Oryza sativa), weakly pathogenic P. palleroniana garlic (Allium sativum) P. salomonii P. savastanoi pv. fraxini) (syn. Pseudomonas Fraxinus excelsior syringae subsp. savastanoi pv. fraxini) P. savastanoi pv. nerii) (syn. Pseudomonas Nerium oleander syringae subsp. savastanoi pv. nerii) P. savastanoi pv. savastanoi (syn. Pseudomonas Olea europaea, Jasminum spp., Ligustrum spp. syringae subsp. savastanoi pv. oleae) P. savastanoi pv. retacarpa Retama sphaerocarpa P. syringae pv. aceris Acer P. s. pv. actinidiae Actinidia deliciosa P. s. pv. aesculi Aesculus P. s. pv. alisalensis broccoli, arugula (Eruca vesicaria subsp. sativa) P. s. pv. anthirrhini Antirrhinum majus P. s. pv. apii Apium graveolens P. s. pv. aptata Beta vulgaris P. s. pv. atrofaciens cereals, mainly Triticum P. s. pv. atropurpurea Agrostis spp., Lolium spp. P. s. pv. avellanae (syn. Pseudomonas avellanae) Avellana spp. (hazelnut) P. s. pv. avii Prunus avium (wild cherry) P. s. pv. berberidis Berberis spp. P. s. pv. broussonetiae paper mulberry (Broussonetia kazinoki x B. papyrifera) P. s. pv. castaneae Castanea crenata P. s. pv. cerasicola galls on Prunus x yedoensis P. s. pv. ciccaronei Ceratonia siliqua P. s. pv. coriandricola Coriandrum P. s. pv. coronafaciens Avena sativa, Triticum P. s. pv. coryli Corylus avellana P. s. pv. cunninghamiae Cunninghamia lanceolata P .s. pv. daphniphylli Daphniphyllum teijsmanni P. s. pv. delphinii Delphinium spp. P. s. pv. dendropanacis Dendropanax trifidus P. s. pv. dysoxyli Dysoxylum spectabile P. s. pv. eriobotryae Eriobotryae japonica

Annex 2 – List of plant pathogenic bacteria and their main hosts P. s. pv. garcae Coffea arabica P. s. pv. glycinea (syn. Pseudomonas savastanoi Glycine max (soybean) pv. glycinea) P. s. pv. helianthi Helianthus annuus (sunflower) P. s. pv. hibisci Hibiscus spp. P. s. pv. japonica Triticum aestivum, Hordeum vulgare P. s. pv. lachrymans Cucumis sativus P. s. pv. lapsa Morus spp. P. s. pv. maculicola Brassica (cauliflower) P. s. pv. mellea Nicotiana tabacum P .s. pv. mori Morus spp. P. s. pv. morsprunorum Prunus spp. P. s. pv. myricae Myrica rubra (galls) P. s. pv. oryzae rice P. s. pv. panici Panicum miliaceum P. s. pv. papulans Malus pumila P. s. pv. passiflorae Passiflora edulis P. s. pv. persicae Prunus persica P. s. pv. phaseolicola (syn. Pseudomonas Phaseolus vulgaris savastanoi pv. phaseolicola) P. s. pv. philadelphi Philadelphus spp. P. s. pv. photiniae Photinia glabra P. s. pv. pisi Pisum sativum P .s. pv. primulae Primula spp. P. s. pv. raphiolepidis Rhaphiolepis umbellata P. s. pv. ribicola Ribes aureum P. s. subsp. savastanoi pv. fraxini (syn. Fraxinus excelsior Pseudomonas savastanoi pv. fraxini) P. s. subsp. savastanoi pv. nerii (syn. Nerium oleander Pseudomonas savastanoi pv. nerii) P. s. subsp. savastanoi pv. oleae (syn. Olea europaea, Jasminum spp., Ligustrum spp. Pseudomonas savastanoi pv. savastanoi) P. s. pv. sesami Sesamum indicum P. s. pv. solidagae tall goldenrod (Solidago altissima) P. s. pv. spinaceae spinach P. s. pv. striafaciens Avena sativum, triticale P .s. pv. syringae broad host range, incl. Syringa, stone fruits P. s. pv. tabaci Nicotiana spp. P. s. pv. tagetis Tagetes spp. P .s. pv. theae Camellia sinensis P .s. pv. tomato tomato P .s. pv. ulmi Ulmus spp. P .s. pv. viburni Viburnum spp. P. s. pv. zizaniae Zizania palustris (cultivated wild rice) P. syzygii (syn. Ralstonia syzygii) Syzygium aromaticum Agaricus bisporus P. tolaasii P. tremae (syn. Pseudomonas syringae pv. Trema orientalis (galls on this host) tremae) diverse host range P. viridiflava Ralstonia solanacearum (syn. Pseudomonas solanacearum) R. syzygii (syn. Pseudomonas syzygii)

race 1, biovars 1, 3-5: broad host range, race 2, biovar 1: banana, Heliconia; race 3, biovar 2: potato, tomato, Pelargonium and some weed hosts; race 4: ginger; race 5: Morus Syzygium aromaticum

Rathayibacter iranicus R. rathayi R. tritici

Triticum aestivum Dactylis glomerata Triticum aestivum

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Rhizobacter dauci Rhizobium: see Agrobacterium Rhizobium larrymoorei (syn. Agrobacterium larrymoorei) R. radiobacter (syn. Agrobacterium tumefaciens) R. rhizogenes (syn. Agrobacterium rhizogenes) R. rubi (syn. Agrobacterium rubi) R. vitis (syn. Agrobacterium vitis)

Daucus carota for reclassification see: Young et al. (2001) and Young (2003) Ficus benjamini very broad host range, mainly dicotyledons apple, rose Rubus spp. Vitis spp.

‘Rhizomonas’ suberifaciens (see Sphingomonas)

Lactuca spp.

Rhodococcus fascians

diverse host range, incl. Lilium, Pelargonium

Samsonia erythrinae

Erythrina sp.

Serratia marcescens

Cucurbita maxima (Cucurbit yellow vine disease)

Sphingomonas melonis S. suberifaciens

spanish melon (Cucumis melo var. inodorus) lettuce (Lactuca sativa)

Spiroplasma citri S. kunkelii S. phoeniceum

Citrus spp., infection in many hosts Zea mays (corn stunt) Catharanthus roseus (periwinkle)

Streptomyces ipomoeae S. acidiscabies S. aureofaciens S. caviscabies S. europaeiscabiei S. reticuliscabiei S. scabiei S. steliiscabiei

Ipomea batatas potato, causing common scab potato, causing russet scab potato, causing deep pitted scab potato, causing common scab potato, causing netted scab potato, causing common scab, Daucus carota potato, causing common scab

Xanthomonas albilineans X. arboricola pv. celebensis X. a. pv. corylina X. a. pv. fragariae X. a. pv. juglandis X. a. pv. poinsettiicola X. a. pv. populi X. a. pv. pruni X. axonopodis pv. alfalfae X. a. pv. allii

sugarcane Musa spp. Corylus spp. Fragaria spp. Juglans regia Euphorbia pulcherrima Populus spp. Prunus spp. lucerne Allium fistulosum (Welsh onion), Allium cepa, garlic Citrus aurantifolia

X. a. pv. aurantifolii (syn. Xanthomonas citri groups B, C, D) X. a. pv. axonopodis X. a. pv. bauhiniae X. a. pv. begonia X. a. pv. betlicola X. a. pv. biophyti X. a. pv. cajani X. a. pv. cassavae X. a. pv. cassiae X. a. pv. citri (syn. Xanthomonas citri group A) X. a. pv. citrumelo (syn. Xanthomonas citri group E)

Axonopus spp. Bauhinia racemosa Begonia spp. Piper betle Biophytum sensitivum Cajanus cajan Manihot esculentum Cassia spp. Citrus spp. Citrus spp.

Annex 2 – List of plant pathogenic bacteria and their main hosts 291 X. a. pv. clitoriae Clitoria biflora X. a. pv. coracanae Eleusine coracana X. a. pv. cyamopsidis Cyanopsis tetragonolobus X. a. pv. desmodii Desmodium dichotomum X. a. pv. desmodiigangetici Desmodium gangeticum X. a. pv. desmodiilaxiflori Desmodium laxiflorum X. a. pv. desmodiirotundifolii Desmodium styracifolium X. a. pv. dieffenbachiae Anthurium spp., Dieffenbachia and other Araceae X. a. pv. erythrinae Erythrina spp. X. a. pv. glycines Glycine max (soybean) X. a. pv. khayae Khaya senegalensis X. a. pv. lespedezae Lespedeza sp. X. a. pv. maculifoliigardeniae Gardenia spp. X. a. pv. malvacearum Gossypium (cotton) X. a. pv. manihotis Manihot esculenta (cassava) X. a. pv. martyniicola Martynia diandra X. a. pv. melhusii Tectona grandis X. a. pv. nakataecorchori Corchorus aestuans X. a. pv. patelii Crotalaria juncea X. a. pv. pedalii Pedalium murex X. a. pv. phaseoli Phaseolus vulgaris, Dolichos lablab X. a. pv. phaseoli var. fuscans Phaseolus vulgaris X. a. pv. phyllanti Phyllanthus niruri X. a. pv. physalidicola Physalis alkekengi X. a. pv. poinsettiicola Euphorbia pulcherrima X. a. pv. punicae Punica granatum X. a. pv. rhynchosiae Rhynchosia memnonia X. a. pv. ricini Ricinus communis X. a. pv. sesbaniae Sesbania sesban X. a. pv. tamarindi Tamarindus indica X. a. pv. vasculorum Saccharum officinarum, Thysanolaena maxima X. a. pv. vesicatoria Capsicum spp. X. a. pv. vignaeradiatae Vigna radiata X. a. pv. vignicola Vigna unguiculata X. a. pv. vitians Lactuca sp. Bromus spp. X. bromi [X. campestris. pv. aberrans] uncertain taxonomic Brassica oleracea position [X. c. pv. alangii] uncertain taxonomic position Alangium salviifolium [X. c. pv. amaranthicola] uncertain taxonomic Amaranthus viridis position [X. c. pv. amorphophalli] uncertain taxonomic Amorphophallus campanulatus position [X. c. pv. aracearum] uncertain taxonomic Colocasia esculenta, Xanthosoma sagittifolium position X campestris pv. allii Allium fistulosum (Welsh onion) X. c. pv. armoraciae Armoracia spp. X. c. pv. barbarae Barbarea vulgaris X. c. pv. campestris Brassica spp., Cheiranthus, Raphanus sativus [X. c. pv. carotae] uncertain taxonomic position Daucus carota [X. c. pv. convolvuli] uncertain taxonomic position Convolvulus arvensis [X. c. pv. coriandri] uncertain taxonomic position Coriandrum sativum [X. c. pv. daturae] uncertain taxonomic position Datura metel [X. c. pv. durantae] uncertain taxonomic position Duranta repens [X. c. pv. esculenti] uncertain taxonomic position Abelmoschus esculentus [X. c. pv. eucalypti] uncertain taxonomic position Eucalyptus citriodora [X. c. pv. euphorbiae] uncertain taxonomic Euphorbia acalyphoides position X. c. pv. fascicularis Corchorus fascicularis

292

Annex 2 – List of plant pathogenic bacteria and their main hosts X. c. pv. fici Ficus benjamina [X. c. pv. guizotiae] uncertain taxonomic position Guizotia abyssinica [X. c. pv. gummisudans] uncertain taxonomic Gladiolus sp. position [X. c. pv. heliotropi] uncertain taxonomic position Heliotropium sp. [X. c. pv. incanae] uncertain taxonomic position Matthiola spp. [X. c. pv. ionidii] uncertain taxonomic position Ionidium heterophyllum [X. c. pv. lantanae] uncertain taxonomic position Lantana camara [X. c. pv. laureliae] uncertain taxonomic position [X. c. pv. lawsoniae] uncertain taxonomic position [X. c. pv. leeana] uncertain taxonomic position [X. c. pv. leersiae] uncertain taxonomic position [X. c. pv. mallotii] uncertain taxonomic position [X. c. pv. mangiferaeindicae] uncertain taxonomic position [X. c. pv. merremiae] uncertain taxonomic position [X. c. pv. mirabilis] uncertain taxonomic position [X. c. pv. musacearum] uncertain taxonomic position [X. c. pv. nigromaculans] uncertain taxonomic position [X. c. pv. obscurae] uncertain taxonomic position [X. c. pv. olitorii] uncertain taxonomic position [X. c. pv. papavericola] uncertain taxonomic position [X. c. pv. passiflorae] uncertain taxonomic position [X. c. pv. paulliniae] uncertain taxonomic position [X. c. pv. pennamericanum] uncertain taxonomic position [X. c. pv. phormiicola] uncertain taxonomic position [X. c. pv. physalides] uncertain taxonomic position X. c. pv. plantaginis X. c. pv. raphani [X. c. pv. sesami] uncertain taxonomic position [X. c. pv. spermacocus] uncertain taxonomic position [X. c. pv. syngonii] uncertain taxonomic position [X. c. pv. tardicrescens] uncertain taxonomic position [X. c. pv. thespesiae] uncertain taxonomic position [X. c. pv. thirumalacharii] uncertain taxonomic position [X. c. pv. tribuli] uncertain taxonomic position [X. c. pv. trichodesmae] uncertain taxonomic position [X. c. pv. uppalii] uncertain taxonomic position [X. c. pv. vernoniae] uncertain taxonomic position [X. c. pv. viegasii] uncertain taxonomic position [X. c. pv. viticola] uncertain taxonomic position [X. c. pv. vitiscarnosae] uncertain taxonomic position [X. c. pv. vitistrifoliae] uncertain taxonomic position [X. c. pv. vitiswoodrowii] uncertain taxonomic position

Laurelia novae-zelandiae Ammania multiflora, Lawsonia inermis Leea edgeworthii Leersia hexandra Mallotus japonicus Mangifera indica Merremia gangetica Mirabilis jalapa Ensete ventricosum Zinnia elegans Ipomoea obscura Corchorus olitorius Papaver rhoeas Passiflora sp. Paullinia cupana Pennisetum americanum Phormium tenax Physalis sp. Plantago lanceolata Raphanus sativus Sesamum indicum Spermacoce hispida Syngonium podophyllum Iris spp. Thespesia populnea Triumfetta pilosa Tribulus terrestris Trichodesma zeylanicum Ipomoea muricata Vernonia cinerea Pachystachys lutea Vitis sp. Vitis carnosa Vitis trifolia Vitis sp.

Annex 2 – List of plant pathogenic bacteria and their main hosts [X. c. pv. zantedeschiae] uncertain taxonomic Zantedeschia aethiopica position [X. c. pv. zingibericola] uncertain taxonomic Zingiber sp. position [X. c. pv. zinniae] uncertain taxonomic position Zinnia sp. Manihot spp. X. cassavae Codiaeum variegeatum X. codiaei Cucurbita maxima X. cucurbitae Cynara scolymus (artichoke) X. cynarae Fragaria spp. X. fragariae X. hortorum pv. hederae Hedera spp. X. h. pv. pelargonii Pelargonium spp. X. h. pv. taraxaci Taraxacum kok-saghyz Hyacinthus orientalis, Scilla spp., X. hyacinthi Cucumis melo X. melonis X. oryzae pv. oryzae Oryza sativa, Leersia spp. X. o. pv. oryzicola Oryza sativa Pisum sativum X. pisi Populus canadensis, Populus spp., Salix alba X. populi X. sacchari (syn. Xanthomonas albilineans) Saccharum officinarum Camellia sinensis X. theicola X. translucens pv. arrhenatheri Arrhenatherum elatius X. t. pv. graminis Dactylis glomerata, Lolium spp. X. t. pv. hordei Hordeum vulgare X. t. pv. phlei Phleum pratense X. t. pv. phleipratensis Phleum pratense X. t. pv. poae Poa trivialis X. t. pv. secalis Secale cereale, Sesamum indicum X. t. pv. translucens Hordeum vulgare, Secale cereale, Triticale X. t. pv. undulosa Secale spp., Triticum spp. X. vasicola pv. holcicola Sorghum spp. X. v. pv. vasculorum Saccharum officinarum Lycopersicon esculentum X. vesicatoria Xylella fastidiosa subsp. agglomerii X. fastidiosa subsp. idiaotraposa X. fastidiosa subsp. piercei Xylophilus ampelinus (syn. Xanthomonas ampelina)

Prunus persica, P. amygdalis, P. domestica, Platanus spp., Ulmus spp., wild Vitis citrus Vitis vinifera, Acer spp., Medicago sativa, Prunus amygdalis Vitis vinifera

293

J. van Vaerenbergh

Annex 2 – List of plant pathogenic bacteria and their main hosts

J. van Vaerenbergh

294

Fig. 211 Diseases of suspected bacterial etiology Top left and right:

Symptoms of so-called bleeding canker of horse chestnut (Aesculus hippocastanum). Severe cracks in the bark and dark spots of dried exudates. Preliminary research indicates that a Pseudomonas syringae pathovar may be involved in this disease syndrome. Centre left and right: Close-up of early symptoms in bark and wood tissues of young branches of horse chestnut with bleeding canker, from which the P. syringae pathovar was isolated repeatedly. Bottom left and right: Brown and necrotic spots with a yellow halo and water-soaked streaks along veins on a leaf of a Marantha sp. From these streaks an Acidovorax sp. was regularly isolated.

IIA1

Oryza spp. (rice) Hordeum vulgare (barley) Vitis vinifera (grapevine), Prunus persica (peach), Citrus sinensis (orange)

Xanthomonas oryzae pv. oryzicola

Xanthomonas translucens pv. translucens

Xylella fastidiosa, vector transmitted. Different forms on grapevine (Pierce’s disease), peach (Phony peach) and Citrus

X

A2

A1

A1

A2

A2

A2

A1

A1

A2

A2

Meaning of A1, A2 or IA1, IA2, IIA1, IIA2 or IIB may slightly differ from the definition by EPPO for A1 and A2. 1) Only main hosts mentioned. 2) Regulated pest on draft list in June 2002 (X = present on the list). 3) Older synonyms in brackets. 4) Species of uncertain taxonomic position. No reference cultures available. NAPPO = North American Plant Protection Organization; EPPO = European and Mediterranean Plant Protection Organization; APPPC = Asia and Pacific Plant Protection Commission; CAN = Comunidad andina; COSAVE = Comite Regional de Sanidad Vegetal del Cono Sur; CPPC = Caribbean Plant Protection Commission; IAPSC = Interafrican Phytosanitary Council; NEPPO = Near East Plant Protection Organization; PPPO = Pacific Plant Protection Organization.

IA1

A2

A1

IIA1

Oryza spp. (rice)

Xanthomonas oryzae pv. oryzae

A2 A2

A2

Anthurium spp., Dieffenbachia spp.

A1

Xanthomonas axonopodis pv. dieffenbachiae

X

IIA1

Citrus, Fortunella and Poncirus spp. (citrus)

Xanthomonas axonopodis pv. citri

X

X

IIA1

IIA1

Citrus, Fortunella and Poncirus spp. (citrus greening or Citrus Huanglongbin)

NAPPO1) APPPC CAN COSAVE CPPC IAPSC NEPPO PPPO

Citrus, Fortunella and Poncirus spp. (citrus greening or Citrus Huanglongbin)

IIA1

EU

Citrus, Fortunella and Poncirus spp.

Main hosts1)

‘candidatus Liberobacter asiaticus’ (nonculturable bacterium, vector transmitted, PCR detection)

a)

295

Bacterial pathogens mentioned in the EPPO quarantine A1 list (2003), lists of other Regional Plant Protection Organizations and the European Union (2002)a)

Citrus variegated chlorosis (probably Xylella fastidiosa) ‘candidatus Liberobacter africanus’ (nonculturable bacterium, vector transmitted, PCR detection)

Bacterium

Annex 3a

Annex 3 – Plant pathogenic bacteria mentioned in quarantine lists

IIA2 IIA2* IA2 IIB IIA2

Medicago sativa (alfalfa) Lycopersicon esculentum (tomato) Solanum tuberosum (potato) Phaseolus vulgaris, Vigna spp. (bean) Malus spp. (apple), Pyrus spp. (pear), Crataegus spp. (hawthorn), Cotoneaster spp.

Clavibacter michiganensis subsp. insidiosus

Clavibacter michiganensis subsp. michiganensis

Clavibacter michiganensis subsp. sepedonicus

Curtobacterium flaccumfaciens pv. flaccumfaciens

Erwinia amylovora

IIA1 IIA2 IA2

IIA2 IIA2* IIA2* IIA2* IIA2*

Zea mays (corn) Prunus persica (peach) Race 1: many (solanaceous) hosts, race 2: Musa spp. (banana) and Heliconia, race 3: mainly Solanum tuberosum (potato), Lycopersicon esculentum (tomato) Coryllus avellanae (hazelnut) Prunus spp. Phaseolus spp. (bean) Fragaria spp. (strawberry) Lycopersicon esculentum (tomato), Capsicum spp. (pepper and chilli pepper), Vitis vinifera (grapevine)

Pseudomonas syringae pv. persicae

Ralstonia (Pseudomonas) solanacearum

Xanthomonas arboricola pv. corylina

Xanthomonas arboricola pv. pruni

Xanthomonas axonopodis pv. phaseoli

Xanthomonas fragariae

Xanthomonas vesicatoria

Xylophilus ampelinus (Xanthomonas ampelina)

X

A1

A2

A2

A1

A1

A1

Race 1: A2

A1

A1

A1

Race 1 and 2: A2

A1

A2

A1

A1

A2

Race 1: A2

A1

A2

A1

A1

A1

A1

Race 2: A2

A2

NAPPO2) APPPC CAN COSAVE CPPC IAPSC NEPPO PPPO

* These bacteria will be transferred from the quarantine list of the EU to a list of regulated non-quarantine pests for which official certification schemes will be developed.

IIA2*

Dianthus caryophylus (carnation)

Erwinia chrysanthemi pv. dianthicola (Dianthus strains only) Pantoea (Erwinia) stewartii subsp. stewartii, vector transmitted

IIA2

Dianthus caryophyllus (carnation)

Burkholderia (Pseudomonas) caryophylli3)

EU

Main hosts1)

Bacterial pathogens mentioned in the EPPO quarantine A2 list (2003), lists of other Regional Plant Protection Organisations and the European Union (2002). Legend: see Annex 3a.

Annex 3 – Plant pathogenic bacteria mentioned in quarantine lists

Bacterium

Annex 3b

296

297

X

Ulmus spp. (elm tree), wood staining Pisum sativum (pea) Triticum aestivum (wheat) Acer spp. (maple) Saccharum officinarum (sugarcane) Cajanus cajan (pigeon pea) Citrumelo rootstock (Citrus paradisi x Poncirus trifoliata) Manihot esculentum (cassava) Saccharum officinarum (sugarcane) Gossypium hirsutum (cotton) Daucus carota (carrot) Populus spp. (poplar tree) Panicum miliaceum (millet), Sorghum spp. (Sorghum), Zea mays (corn)

Leifsonia (Clavibacter) xyli subsp. xyli

Pseudomonas lignicola4)

Pseudomonas syringae pv. pisi

Rathayibacter (Clavibacter) tritici

Xanthomonas acernea

Xanthomonas albilineans

Xanthomonas axonopodis pv. cajani

Xanthomonas axonopodis pv. citrumelo

Xanthomonas axonopodis pv. manihotis

Xanthomonas axonopodis pv. vasculorum

Xanthomonas campestris pv. malvacearum

Xanthomonas hortorum pv. carotae

Xanthomonas populi

Wheat yellowing stripe bacterium (rickettsia-like bacterium, described from China)

Xanthomonas vasicola (campestris) pv. holcicola Triticum aestivum (wheat)

X

Salix spp. (willow tree) Citrullus lanatus (watermelon), Cucumis spp. (cucumber), Cucurbita spp. (cucubits) Saccharum officinarum (sugarcane), ratoon stunting disease

Erwinia salicis

IIA1

Many hosts

Erwinia chrysanthemi pv. chrysanthemi

Erwinia tracheiphila

Solanum tuberosum (potato)

Erwinia carotovora subsp. atroseptica

X

X

X

X

X

Zea mays (corn)

A2

A2

A2

A2

A2

A1

A1

A1

A1

A2

A1

A2

A2

A1

A2

A2

A2

A1

NAPPO1) APPPC COSAVE CPPC IAPSC NEPPO

Clavibacter michiganensis subsp. nebraskensis

EU

Main hosts1)

Other bacteria of quarantine importance in some areas or countries (2003). Legend: see Annex 3a.

Bacterium

Annex 3c

Annex 3 – Plant pathogenic bacteria mentioned in quarantine lists

Annex 4 – List of some (important) Phytoplasmas

298 Annex 4

List of some (important) Phytoplasmas1) After Lee et al. (2000).

Phytoplasma group (RNA group)

Aster yellows in aster, celery, carrot, potato, clover; lettuce yellows; tomato big bud; cabbage witches’ broom; hydrangea phyllody; cyclamen virescence; poplar witches’ broom; eggplant dwarf; cactus virescence; primula yellows; maize bushy stunt; clover phyllody; apricot chlorotic leaf roll Peanut witches’ broom group (16SrII) Peanut, sweet potato and Sunn hemp witches’ broom; "Candidatus Phytoplasma aurantifolia" (on lime) lime witches’ broom faba bean, soybean cotton phyllody Peach, cherry X-disease; pecan bunch; X-disease group (16SrIII) spirea stunt Coconut lethal yellows group Coconut lethal yellows Tanzanian coconut lethal decline (16SrIV) Elm yellows; elm witches’ broom; rubus Elm yellows group (16SrV) "Candidatus Phytoplasma ulmi" (elm stunt; alder yellows yellows) Cherry lethal yellows Jujube witches’ broom Flavescence dorée (grapevine) clover proliferation; tomato big bud; Clover proliferation group (16SrVI) "Candidatus Phytoplasma trifolii" (on potato witches’ broom; alfalfa witches’ clover) broom Ash yellows; lilac witches’ broom Ash yellows group (16SrVII) "Candidatus Phytoplasma fraxini" Loofah witches’ broom Loofah witches’ broom group (16SrVIII) Pigeon pea witches’ broom; Echium Pigeon pea witches’ broom group vulgaris yellows; knautia phyllody (16SrIX) Apple proliferation; hazel decline; Apple proliferation group (16SrX) bindweed yellows; apricot chlorotic 'Candidatus Phytoplasma mali', leafroll; plum leptonecrosis; European 'Candidatus Phytoplasma pyri' and 'Candidatus Phytoplasma prunorum' stone fruit yellows; pear decline; peach (apple proliferation, pear decline and yellow leaf roll European stone fruit yellows) Rice yellow dwarf; sugarcane Rice yellow dwarf group (16SrXI) whiteleaf; grassy shoot "Candidatus Phytoplasma oryzae" Stolbur (pepper, tomato); celery Stolbur group (16SrXII) yellows; grapevine yellows "Candidatus Phytoplasma australiense" (Australian grapevine Australian grapevine yellows yellows) Mexican periwinkle virescence group Mexican periwinkle virescence strawberry green petal (16SrXIII) Bermuda grass white leaf; annual blue Bermuda grass white leaf group (16SrXIV) "Candidatus Phytoplasma grass white leaf cynodontis" Aster yellows group (16SrI) "Candidatus Phytoplasma asteris"

Undesignated groups 1)

Geographical distribution

Disease

Italian bindweed stolbur; buckthorn witches’ broom

Worldwide

Asia Arabian peninsula Africa, Asia North America Florida, Caribbean region Africa North America, Europe China Asia Europe North America North America Taiwan Italy, Central America Europe

Asia Europe Australia Mexico Florida Asia, Italy

Europe

Based on RFLP analysis of 16S rRNA and ribosomal protein sequences. 16S rRNA subgroups and not all diseases and all hosts mentioned, for full list see Lee et al. (2000) and ‘candidatus’ names at http://www.isppweb.org/names_bacterial_new2004.asp . and http://www.bacterio.cict.fr/candidatus.html .

Annex 5 – Subspecific diversity of Ralstonia solanacearum

Annex 5a RACE

299

Subspecific diversity of Ralstonia solanacearum (tests used for determination of different subgroups are mentioned in Annex 5b-d)1) BIOVAR RFLP pattern2) HOST RANGE AREA

1

1

1-7

broad

S. America, USA

1

3

8-14

broad

Mainly S.E. Asia, S. America, Australia, China, some USA

1

4

11, 15-18, 21-23

broad

S.E. Asia, China, Australia, some USA

1

5

19, 20

Morus alba (mulberry) China

2

1

24, 25

Plantain, banana S. and C. America, (Musa spp.), Heliconia Phillipines

3

2A

26A, B

narrow

all continents

3

2A

27A,B,C

narrow

29-33

narrow

west of Andes: Chile, Colombia east of Andes: lowlands Brazil, Peru

Between 1 and 3 2T (2N)

1)

For confirmative AFLP and PCR-RFLP of these subdivisions, see Poussier et al. (2000). Also see Horita et al. (2005). 2) After Cook and Sequiera (1994).

Annex 5 – Subspecific diversity of Ralstonia solanacearum

300

Annex 5b

Tests for biovar determination of Ralstonia solanacearum1)

Utilization of:

Biovar

Maltose Lactose Cellobiose Mannitol Sorbitol Dulcitol

1

2

3

4

5

-

+ + + -

+ + + + + +

+ + +

+ + + + -

1)

For a different biotype scheme, based on maltose, mannitol, malonate, trehalose, inositol and hippurate and discrimination of 4 so-called phylotypes using multiplex PCR, based upon the 16S-23S rRNA gene intergenic spacer region (ITS) and 23 so-called sequevars, based upon fingerprinting methods such as rep-PCR, RAPD and AFLP, see Fegan and Prior (2005).

Annex 5c

Tests for differentiation of subphenotypes of biovar 2 of Ralstonia solanacearum

Test

Biovar 2

Biovar 2A

Biovar 2T or 2N

Utilization of trehalose Utilization of inositol

+

+ -

+ +

Utilization of D-ribose Pectolytic activity

low

low

+ high

Annex 5d Reaction in:

Tomato/eggplant Tobacco plants (stem inoculation) Tobacco leaves (hypersensitivity test) Musa acuminata

Determination of races of Ralstonia solanacearum (race 4, pathogenic to ginger and a few other hosts and race 5, pathogenic to mulberry only, not included) Race 1

2

3

Wilting Wilting

No reaction No reaction

Wilting No reaction

Necrosis (48 h) and wilting (7-8 days) No reaction

HR (12-24 h)

Chlorosis (2-8 days)

Wilting

No reaction

Annex 6a – Scheme for detection of Ralstonia solanacearum in symptomatic material

Annex 6a EU SCHEME FOR DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM IN SAMPLES OF SYMPTOMATIC POTATO, TOMATO OR OTHER HOST PLANTS. Sample of stem pieces

Pathogen extraction and concentration

SCREENING TESTS Perform selective isolation as the core test. Perform optional additional tests: IF test, tomato bioassay, ELISA, PCR and/or FISH

Colonies with typical morphology after isolation

Yes

No

IDENTIFICATION TESTS Confirm identity of pure culture as R. solanacearum

Tests performed all negative

R. solanacearum not detected

No

Yes

R. solanacearum detected

Confirm pathogenicity by host test, sample infected

Full details of methods and procedures, reagents and composition of media are necessary for successful application of the scheme and can be found in Anonymous (1998) or updates issued by the Standing Committee on Plant Health of the European Commission, Brussels, Belgium.

301

302 Annex 6b – Scheme for detection of Ralstonia solanacearum in latently infectedymptomatic material Annex 6b EU SCHEME FOR DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM IN SAMPLES OF ASYMPTOMATIC POTATO TUBERS

Sample of potato tubers Pathogen extraction and concentration

SCREENING TESTS Perform at least one of the following core tests: IF test, selective isolation and/or tomato bioassay Perform optional additional tests: ELISA, PCR and/or FISH

At least one test positive Tests performed all negative

ISOLATION TESTS Perform selective isolation and/or tomato bioassay

Colonies with typical morphology

R. solanacearum not detected

No

Yes

IDENTIFICATION TESTS Confirm identity of pure culture as R. solanacearum

No

Yes

R. solanacearum detected

Confirm pathogenicity by host test, sample infected

Full details of methods and procedures, reagents and composition of media are necessary for successful application of the scheme and can be found in Anonymous (1998) or updates issued by the Standing Committee on Plant Health of the European Commission, Brussels, Belgium. From 2006 onwards two screening tests based on a different biological principle (e.g. IF and PCR test) have to be positive before a sample is designated as suspect and further tests performed.

Suggested reading and literature cited

303

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Suggested reading and literature cited 327 Sletten A, 1985. The effect of Corynebacterium sepedonicum on symptoms and yield of four potato cultivars. Potato Research 28, 27-33. Smith EF, 1896. A bacterial disease of the tomato, eggplant and Irish potato (Bacillus solanacearum n. sp.). Bulletin, Division of Vegetable Physiology and Pathology, United States Department of Agriculture 12, 128. Smith EF, 1897. Description of Bacillus phaseoli n. sp. Botanical Gazette 24, 192 (Abstract). Smith EF, 1898. Notes on Stewart’s sweet corn germ Pseudomonas stewarti n.sp. Proceedings of the American Association for the Advancement of Science 47, 422-426. Smith EF, 1903. Observations on a hitherto unreported bacterial disease, the cause of which enters the plant through ordinary stomata. Science n.s. 17, 456-457. Smith EF, 1904. Bacterial leaf spot diseases. Science (n.s.) 19, 417 Smith EF, 1905a. Bacterial leaf spot diseases. Science (n.s.) 19, 417. Smith EF, 1905b. Bacteria in Relation to Plant Diseases. Volume I. USA: Carnegie Institution of Washington. Smith EF, 1908. Recent studies on the olive-tubercle organism. Bulletin of the Bureau of Plant Industry, US Department of Agriculture 131, 25-43. Smith EF, 1910. A new tomato disease of economic importance. Science (n.s.) 31, 794-796. Smith EF, 1911. Bacteria in Relation to Plant Diseases. Volume II. USA: Carnegie Institution of Washington. Smith EF, 1914. Bacteria in Relation to Plant Diseases. Volume II. USA: Carnegie Institution of Washington. Smith EF, 1920. Bacterial Diseases of Plants. USA, Philadelphia: Saunders. Smith EF, Bryan MK, 1915. Angular leaf spot of cucumbers. Journal of Agricultural Research 5, 465-476. Smith IM, McNamara DG, Scott PR, Holderness M, eds., 1997. Quarantine Pests for Europe. EPPO/CABI. Cambridge, UK: Cambridge University Press. Smidt M, Vidaver AK, 1986. Population dynamics of Clavibacter michiganense subsp. nebraskense in field-grown dent corn and popcorn. Plant Disease 70, 1031-1036. Sneath PH, Sokal RR, 1973. Numerical Taxonomy. San Fransisco, USA: Freeman & Co. Spieckermann A, Kotthoff P, 1914. Untersuchungen über die Kartoffelpflanze und ihre Krankheiten. 1. Die Bakterienringfäule der Kartoffelpflanze. Landwirtschaftliche Jahrbücher 64, 659-732. Stall RE, Civerolo EL, 1991. Research relating to the recent outbreak of citrus canker in Florida. Annual Review of Phytopathology 29, 399-420. Stall RE, Beaulieu C, Egel D, Hodge NC, Leite RP, Minsavage GV, Bouzar H, Jones JB, Alvarez AM, Benedict AA, 1994. Two genetically diverse groups of strains are included in Xanthomonas campestris pv. vesicatoria. International Journal of Systematic Bacteriology 44, 47-53 Stapp C, 1956. Bakteriellen Krankheiten. In: Handbuch der Pflanzenkrankheiten. Appel O, Richter H, eds., IIer Band. Die Virus- und bakteriellen Krankheiten. Berlin, Germany: Paul Parey.

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Suggested reading and literature cited 329 Taghavi M, Hayward C, Sly LI and Fegan M, 1996. Analysis of the phylogenetic relationships of strains of Burkholderia solanacearum, Pseudomonas syzygii, and the blood disease bacterium of banana based on 16S rRNA gene sequences. International Journal of Systematic Bacteriology 46, 10-15. Takikawa Y, Serizawa S, Ichikawa T, Tsuyumu S, Goto M, 1989. Pseudomonas syringae pv. actinidiae pv. nov.: the causal bacterium of canker of kiwifruit in Japan. Annals of the Phytopathological Society of Japan 55, 437-444. Tamponi G, Donati GP, 1990. Walnut cultivars susceptibility to Xanthomonas juglandis. Acta Horticulturae 284, 301-302. Tar’an B, Michaels TE, Pauls KP, 2002. Genetic mapping of agronomic traits in common bean. Crop Science 42, 544-556. Taylor JD, Teverson DM, Allen DJ, Pastor-Corrales MA, 1996. Identification and origin of races of Pseudomonas syringae pv. phaseolicola from Africa and other bean growing areas. Plant Pathology 45, 469-478. Taylor JD, Conway J, Roberts SJ, Astley D, Vicente JG, 2002. Sources and origin of resistance to Xanthomonas campestris pv. campestris in Brassica genomes. Phytopathology 92, 105-111. Teviotdale BL, Schroth MN, 1998. Bark, fruit, and foliage diseases. In: DE Ramos, ed. Walnut Production Manual. Publ. 3373. Division of Agriculture and Natural Sciences, University of California, Oakland, 242– 246. Thapliyal PN, Mishra BC, 1974. Soybean (Glycine max) bacterial pustule (Xanthomonas phaseoli var. sojense). American Phytopathological Society and Nematicide Tests Results of 1973, 29, 147. Thaxter R, 1891. The potato scab. In: 15th Annual Report, Connecticut Agricultural Experimental Station, 153-166. Thind BS, Payak MM, 1985. A review of bacterial stalk rot of maize in India. Tropical Pest Management 31, 311-316. Tilford PE, 1936. Fasciation of sweet peas caused by Phytomonas fascians n. sp. Journal of Agricultural Research 53, 383-394. Treat CL, Tracy WF, 1990. Inheritance of resistance to Goss’s wilt in sweet corn. Journal of the American Society for Horticultural Science 115, 672-674. Trigalet A, Frey P, Trigalet-Demerey D, 1994. Biological control of bacterial wilt caused by Pseudomonas solanacearum: state of the art and understanding. In: Hayward AC, Hartman GL eds. Bacterial Wilt: the Disease and its Causative Agent. Wallingford, UK: CAB International. Tripathi L, Tripathi JN, Tushemereirwe WK, 2004. Strategies for resistance to bacterial wilt disease of bananas through genetic engineering. African Journal of Biotechnology 3, 688-692. Trujillo C, Hernández Y, 1999. Bacterial spot in orchid. Fitopatologia Venezuelana 12, 5-8. Tsang MMC, Shintaku M, 1998. Hot air treatment for control of bacterial wilt in ginger root. Applied Engineering in Agriculture 14, 159-163. Ushiyama K, Kita N, Suyama K, Aono N, Ogawa J, Fujii H, 1992. Bacterial canker disease of wild actinidia plants as the infection source of outbreak of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae. Annals of the Phytopathological Society of Japan 58, 426-430. Valarini PJ, Galvao JAH, Oliveira D de A, 1996. Xanthomonas campestris pv. phaseoli: importance of seed inoculum in the epidemiology of common bacterial blight of French bean. Fitopatologia Brasileira, 21, 261-267.

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Suggested reading and literature cited 331 Vicedo B, Penalver R, Asins MJ, Lopez MM, 1993. Biological control of Agrobacterium tumefaciens, colonization, and pAgK84 transfer with Agrobacterium radiobacter K84 and the Tra- mutant strain K1026. Applied and Environmental Microbiology 59, 309-315. Vicente JG, Taylor JD, Sharpe AG, Parkin IAP, Lydiate DJ, King GJ, 2002. Inheritance of race-specific resistance to Xanthomonas campestris pv. campestris in Brassica genomes. Phytopathology 92, 1134-1141. Vidaver AK, 1983. Bacteriocins: the lure and the reality. Plant Disease 67, 471-475. Vivian A, Gibbon MJ, 1997. Avirulence genes in plant-pathogenic bacteria: signals or weapons? Microbiology 143, 693-704. Vivian A, Murillo J, Jackson RW, 2001. The roles of plasmids in phytopathogenic bacteria: mobile arsenals? Microbiology 147, 763-80. Voloudakis AE, Reignier TM, Cooksey DA, 2005. Regulation of resistance to copper in Xanthomonas axonopodis pv. vesicatoria. Applied and Environmental Microbiology 71, 782-789 Wakker JH, 1883. Vorläufige Mittheilungen über Hyacinthenkrankheiten. Botanisches Centralblatt Bd. XIV, No. 23, 315-316. Walker JC, Patel PN, 1964. Splash dispersal and wind as factors in epidemiology of halo blight of bean. Phytopathology 54, 140-141. Wells JM, Raju BC, Hung HY, Weisburg WG, Mandelco-Paul L, Brenner DJ, 1987. Xylella fastidiosa gen. nov., sp. nov.: Gram-negative, xylem-limited, fastidious plant bacteria related to Xanthomonas spp. International Journal of Systematic Bacteriology 37, 136-143. Wenneker M, Verdel MSW, Groeneveld RMW, Kempenaar C, Van Beuningen AR, Janse JD, 1999. Ralstonia (Pseudomonas) solanacearum race 3 (biovar 2) in surface water and natural weed hosts: First report on stinging nettle (Urtica dioica). European Journal of Plant Pathology 105, 307-315. Werner NA, Fulbright DW, Podolsky R, Bell J, Hausbeck MK, 2002. Limiting populations and spread of Clavibacter michiganensis subsp. michiganensis on seedling tomatoes in the greenhouse. Plant Disease 86, 535-542. White RP, McCulloch L, 1934. A bacterial disease of Hedera helix. Journal of Agricultural Research 48, 807-815. Wiggins BE, Kinkel LL, 2005. Green manures and crop sequences influence potato diseases and pathogen inhibitory activity of indigenous streptomycetes. Phytopathology 95, 178-185. Willems A, Goor M, Thielemans S, Gillis M, Kersters K, De Ley J, 1992. Transfer of several phytopathogenic Pseudomonas species to Acidovorax as Acidovorax avenae subsp. avenae subsp. nov., comb. nov., Acidovorax avenae subsp. citrulli, Acidovorax avenae subsp. cattleyae, and Acidovorax konjaci. International Journal of Systematic Bacteriology 42, 107-119. Woese CR, Kandler O, Wheelis ML, 1990. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proceedings of the National Academy of Sciences USA 87, 4576– 4579. Wullings BA, Van Beuningen AR, Janse JD, Akkermans ADL, 1998. Detection of Ralstonia solanacearum, which causes brown rot of potato, by fluorescent in situ hybridization with 23S rRNAtargeted probes. Applied and Environmental Microbiolology 64, 4546-4554. Wydra K, Fanou A, Sikirou R, Adamou I, Zinsou V, Avocanh A, 1997. Expression of field resistance and tolerance in cassava and cowpea to cassava bacterial blight and cowpea bacterial blight, respectively. DPG Working Group ‘Plant Protection in the Tropics and Subtropics’, Berlin, July 1997. Phytomedizin 27, 4546.

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Suggested reading and literature cited Wysong DS, Doupnik B, Lane L, 1981. Goss’s wilt and corn lethal necrosis - can they become a major problem? In: Proceedings of the Annual Corn Sorghum Research. Conference 36. Washington DC, USA: American Seed Trade Association, 104-130. Yedidia I, Shoresh M, Kerem Z, Benhamou N, Kapulnik Y, Chet I, 2003. Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperellum (T203) and accumulation of phytoalexins. Applied and Environmental Microbiolology 69, 7343-7353. Yoshii K, Galvez GE, Alvarez-Ayala G, 1976. Estimation of yield losses in beans caused by common blight. Proceedings of the American Phytopathological Society 3, 298-299. Young, JM, 1974. Development of bacterial populations in vivo in relation to plant pathogenicity. New Zealand Journal of Agricultural Research 17, 105-113. Young JM, 1987. Orchard management and bacterial diseases of stone fruit. New Zealand Journal of Experimental Agriculture 15, 257-266. Young JM, 2003. Renaming of Agrobacterium larrymoorei Bouzar & Jones 2001 as Rhizobium larrymoorei (Bouzar & Jones 2001) comb. nov. International Journal of Systematic and Evolutionary Microbiology 54, 149. Young JM, Takikawa Y, Gardan L, Stead DE, 1992. Changing concepts in the taxonomy of plant pathogenic bacteria. Annual Review of Phytopathology 30, 67-105. Young JM, Saddler GS, Takikawa Y, De Boer SH, Vauterin L, Gardan L, Gvozdyak RI, Stead DE, 1996. Names of plant pathogenic bacteria 1864-1995. ISPP Subcommittee on Taxonomy of Plant Pathogenic Bacteria. Review of Plant Pathology 75, 721-763. Young JM, Gardan L, Ren XZ, Hu, FP, 1997. Genomic and phenotypic characterization of the bacterium causing blight of kiwifruit in New Zealand. Plant Pathology 46, 857-864. Young JM, Kuykendall LD, Martínez-Romero E, Kerr A, Sawada H, 2001. A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola De Lajudie et al., 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. International Journal of Systematic and Evolutionary Microbiology 51, 89-103. Yunis H, Bashan Y, Okon Y, Henis Y, 1980. Weather dependence, yield losses and control of bacterial speck of tomato caused by Pseudomonas tomato. Plant Disease 64, 937-939. Zaumeyer WJ, Thomas HR, 1957. A monographic study of bean diseases and methods for their control. Technical Bulletin, USDA No. 865, 255 pp. Zeidan M, Czosnek H, 1994. Acquisition and transmission of Agrobacterium by the whitefly Bemisia tabaci. Molecular and Plant Microbe Interactions 7, 792-798. Zreik L, Bové JM, Garnier M, 1998. Phylogenetic characterization of the bacterium-like organism associated with marginal chlorosis of strawberry and proposition of a Candidatus taxon for the organism ‘Candidatus Phlomobacter fragariae’. International Journal of Systematic Bacteriology 48, 257-261.

List of host plants mentioned in Chapter VII List of host plants mentioned in chapter VII Acer rubrum 227 Actinidia chinensis 211 Actinidia deliciosa 211 Aechmea fasciata 273 Aerides japonicum 199 Aesculus hippocastanum 294 Aglaonema commutatum 235 Aglaonema ’pseudobracteatum’ 235 Aglaonema robelinii 235 Aglaonema spp. 273 Agropyron repens 187 Agropyron sp. 191 Agrostis gigantea 185 Allium cepa 177, 203, 271, 273 Allium sativum 177 Amaranthus hybridus 177 Amelanchier spp. 241 Andropogon sorghum, see Sorghum bicolor 193 Anthurium amnicola 235 Anthurium andreanum 235 Anthurium cristalinum 235 Anthurium scherzerianum 235 Arabidopsis thaliana 257, 265 Arachis hypogaea 205, 277 Argemone mexicana 267 Areca catechu 195 Armoracia rusticana 177 Arrhenaterum elatius 191 Asparagus officinalis 203 Aster spp. 269 Avena byzantina 187 Avena sativa 187 Begonia spp. 271, 273 Benincasa hispida 253 Beta vulgaris 205, 269 Beta vulgaris var. esculenta 201 Beta vulgaris var. rubra 205 Brachiaria mutica 189 Brassaia actinophylla 237 Brassica geniculata 265 Brassica juncea var. juncea 265 Brassica napus var. napobrassica 265 Brassica napus var. napus 265 Brassica nigra 265 Brassica oleracea var. acephala 265 Brassica oleracea var. alboglabra 265 Brassica oleracea var. botrytis 203, 265 Brassica oleracea var. capitata 203, 265 Brassica oleracea var. gemmifera 265 Brassica oleracea var. gongylodes 265 Brassica oleracea var. italica 265 Brassica oleracea var. pekinensis 265 Brassica oleracea var. sabauda 265 Brassica oleracea var. viridis 265

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List of host plants mentioned in Chapter VII Brassica spp. 271, 281 Brassica rapa 205 Brassica rapa var. chinensis 265 Brassica rapa var. oleifera 265 Brassica rapa var. rapa 265 Bromus spp. 187, 191 Brunnichia cirrhosa 259 Cajanus cajan 255 Caladium hortulanum 235 Calopogonium sp. 261 Capsella bursa-pastoris 265 Capsicum annuum 247, 249, 267, 277 Capsicum frutescens 267 Cardaria pubescens 265 Carica papayae 199 Cassia hirsuta, see Senna hirsuta Catasetum spp. 197 Cattleya sp. 197 Ceiba pentandra 207 Cenchrus ciliaris 189 Chaenomeles spp. 241 Chrysanthemum spp. 269, 273 Cicer arietinum 177 Cichorium endivia 271, 273 Cichorium intybus 203 Citrullus lanatus 253 Citrus aurantiifolia 223 Citrus aurantium 223 Citrus hystrix 222 Citrus limon 223, 227 Citrus paradisi Citrus reshni 227 Citrus spp. 223 Citrus sinensis 223, 227, 275 Citrus volkameriana 227 Colocasia esculenta 235 Coronopus didymus 265 Corylus avellana 209 Cotoneaster spp. 241 Crataegus spp. 241, 275 Cucumis auguria 253 Cucumis melo 253 Cucumis sativus 253 Cucurbita moschata 253 Cucurbita spp. 271 Cyclamen persicum 177 Cydonia oblonga 241 Cynodon dactylon 189 Cyperus difformis 189 Cyperus rotundus 189 Cypripedium sp. 197, 199 Dactylis glomerata 185 Dactylis sp. 191 Dahlia spp. 273, 281 Datura spp. 267 Daucus carota 203, 205, 271

List of host plants mentioned in Chapter VII Delphinium spp. 233 Dendrobium spp. 197 Desmodium sp. 255 Dianthus caryophyllus 231 Dianthus sp. 177, 281 Dictyosperma album 195 Dictyosperma rubrum 195 Dieffenbachia maculata, see D. picta Dieffenbachia picta 235 Dieffenbachia spp. 273 Digitaria scalarum 261 Dendrobium sp. 197 Dolichos biflores, see Macrotyloma uniflorum Dolichos lablab 255, 261, 263 Doriaenopsis sp. 197 Dracaena marginata 273 Echinogloa crus-galli 183, 189 Epidendrum sp. 197 Epiphronitis veitchii 197 Epipremnum aureum 235 Eriobotrya japonica 241 Erysimum cheiri 265 Eucomis autumnalis 179 Eustoma grandiflorum 231 Fatsia japonica 237 Festuca spp. 191 Ficus spp. 269 Forsythia intermedia 221 Fortunella japonica 223 Fortunella margarita 223 Fragaria x ananassa 225 Fragaria chiloensis 225 Fragaria vesca 225 Fragaria virginiana 225 Fraxinus excelsior 219 Geranium sanguineum 239 Geranium spp. 239 Gladiolus spp. 181, 281 Gladiolus convilli 181 Gladiolus x hortulanus 181 Glycine max 259, 261 Glycine wightii, see Neonotonia wightii Gnaphalium 257 Gossypium barbadense 207 Gossypium herbaceum 207 Gossypium hirsutum 207 Gossypium populifolium 207 Hedera helix 237 Heliconia 277 Hippeastrum 177 Hibiscus rosa-sinensis 207 Hibiscus vitifolius 207

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List of host plants mentioned in Chapter VII Hordeum vulgare 187, 191 Hyacinthus orientalis 177, 179 Hyoscyamus spp. 267 Ionopsis utricularioides 197 Jasminum spp. 221 Jatropha curcas 207 Juglans ailantifolia Juglans ailantifolia var. cordiformis 243 Juglans californica 243 Juglans cinerea 243 Juglans hindsii 243 Juglans nigra 243 Juglans regia 243 Kalanchoe blossfeldiana 273 Lablab purpureus, see Dolichos lablab Lamium amplexicaule 257 Lathyrus spp. 281 Leersia spp. 189 Lens culinaris 177 Lepidium sativum 265 Lipidium virginicum 265 Leptochloa spp. 189 Levisticum officinale 251 Ligustrum japonicum 221 Lillium spp. 281 Limonum sinuatum 231 Lochnera pusilla 207 Lolium sp. 191 Luffa acutangula 253 Lupinus polyphyllus 261 Lycium spp. 267 Lycopersicon esculentum 201, 247, 249, 257, 267, 277 Lycopersicon pimpinellifolium 267 Macroptilium atropurpureum, see Phaseolus atropurpureus Macroptilium bracteatum, see Phaseolus bracteatus Macroptilium lathyroides, see Phaseolus lathyroides Macrotyloma uniflorum 259 Malus spp. 241, 269, 275 Marantha sp. 294 Matthiola incana 265 Medicago sativa 227 Mespilus germanica 241 Miltonia sp. 197 Morus spp. 177 Morus alba 213 Morus alba var. tartarica 213 Morus bombycis 213 Morus kagayamae 213 Morus latifolia 213 Morus multicaulis 213

List of host plants mentioned in Chapter VII Morus nigra 213 Morus rubra 213, 227 Mucuna deeringianum 261 Musa paradisiaca 277 Musa spp. 273, 277 Neonotonia wightii 255 Nerium oleander 221 Nicandra physaloides 267 Nicotiana rustica 267 Nicotiana tabacum 267 Oenothera spp. 257 Olea europaea 221 Oncidium sp. 197 Ornitocephalus bicornis 197 Oryza sativa 189 Oryza spp. 189 Panicum maximum 189 Panicum milliaceum 193 Panicum spp. 185 Paphiopedilum 197, 199 Paspalum scrobiculatum 189 Pastinaca sativa 205 Pelargonium acerfolium 239 Pelargonium x domesticum 239 Pelargonium graveolens 239 Pelargonium x hortorum 239 Pelargonium peltatum 239 Pelargonium scarboroviae 239 Pelargonium spp. 281 Pelargonium tomentosum 239 Pelargonium ‘Torento’ 239 Pelargonium zonale 239, 277 Phalaenopsis 197, 199 Phaseolus acutifolius 255, 261 Phaseolus atropurpureus 255 Phaseolus aureus, see P. mungo Phaseolus bracteatus 255 Phaseolus calcaratus 261 Phaseolus coccineus 255, 261 Phaseolus lathyroides 255 Phaseolus lunatus 255, 259, 261 Phaseolus lunatus var. macrocarpus 255 Phaseolus multiflorus 255 Phaseolus mungo 255, 261, Phaseolus polyanthus 255 Phaseolus radiatus, see P. mungo Phaseolus vulgaris 177, 255, 259, 261, 263, 275 Phillyrea 221 Philodendron oxycardium 235 Philodendron scandens 235 Philodendron selloum 235 Philodendron spp. 273 Phleum pratense 187, 191 Photinia davidiana 241

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List of host plants mentioned in Chapter VII Photinia spp. 241 Physalis minima 267 Pinus radiata 275 Pisum sativum 177 Platanus occidentalis 227 Poa pratensis 185 Poa trivialis 191 Polyscias spp. 237 Poncirus trifoliata 223 Populus spp. 275 Prunus amygdalus 215, 245 Prunus angustifolia 227 Prunus armeniaca 215, 227, 245, 275 Prunus avium 215, 245, 269, 275 Prunus cerasifera 215 Prunus cerasoides 215 Prunus cerasus 215, 245, 275 Prunus dulcis 227 Prunus davidiana 245 Prunus domestica 215, 227, 245, 275 Prunus institia 215 Prunus japonica 245 Prunus laurocerasus 245 Prunus mume 245 Prunus persica 215, 227, 245, 269, 275 Prunus padus 275 Prunus pissardi 215 Prunus salicina 215, 227, 241 Prunus triloba 215 Pueraria sp. 261 Pueraria thunbergiana 255 Puschkinia scilloides 179 Pyracantha spp. 241 Pyrus communis 240 Pyrus pyrifolia 241 Pyrus spp. 241, 269 Quercus rubra 227 Raphanus raphanistrum 265 Raphanus sativus 265, 271 Raphidophora 235 Renanthera sp. 197 Retama sphaerocarpa 221 Rheum hybridum 177 Rheum rhaponticum 177, 271 Ribes aureum 217 Ribes rubrum 217 Rodricidium 197 Rodriguezia 197 Rosa rugosa 241 Rosa spp. 269 Roystonia regia 197 Rubus spp. 241 Rynchostylis spp. 197 Saccharum officinarum 183, 195 Saintpaulia ionantha 273

List of host plants mentioned in Chapter VII Scilla tubergeniana 179 Schefflera arboricola 237 Scindapsis 235 Secale cereale 187, 191 Sechium edule 253 Senna hirsute 261 Setaria lutescens 185 Severinia buxifolia 223 Sinapsis arvensis 265 Solanum douglasii 247 Solanum dulcamara 267, 277 Solanum melongena 201, 271, 277 Solanum nigrum 247, 267, 277 Solanum rostratum 267 Solanum triflorum 247 Solanum tuberosum 201, 203, 205, 271, 273, 277 Sophronitus carnus 197 Sorbus spp. 241 Sorghum almum 193 Sorghum bicolor 183, 193 Sorghum bicolor var. caffrorum 193 Sorghum bicolor var. durra 193 Sorghum bicolor var. technicum 193 Sorghum halepense 193 Sorghum sudanense 183, 185, 193 Sorghum caffrorum, see S. bicolor var. caffrorum 193 Sorghum durra, see S. bicolor var. durra 193 Sorghum technicum, see S. bicolor var. technicum 193 Sorghum vulgare, see S. bicolor 193 Stellaria media 257 Stizolobium helvola, see Strophostyles helvola Stranvesia davidiana, see Photinia davidiana Strophostyles helvola 261 Swinglea glutinosa 223 Syngonium podophyllum 235, 273 Syringa vulgaris 275

Trichocentrum sp. 197 Tripsacum dactyloides 185 Tripsacum laxum 195 Triticale 183 Triticum aestivum 177, 183, 187, 275 Triticum x secale 191 Triticum sp. 191 Tulipa gesneriana 175 Thysanolaena agrostis, see T. maxima 195 Thysanolaena maxima 195 Ulmus americana 227 Vanda spp. 197 Vanilla sp. 197 Verbascum spp. 281 Vigna pubigera 263 Vigna radiata, see Phaseolus mungo Vigna sinensis, see V. unguiculata

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List of host plants mentioned in Chapter VII Vigna umbellate, seePhaseolus calcaratus Vigna unguiculata 255, 259, 261, 263 Vinca minor 227 Vitis labrusca 227 Vitis riparia 227 Vitis vinifera 227, 229 Vuylstekaera sp. 197 Xanthosoma caracu 235 Zantedeschia 271 Zea mays 183, 185, 193, 195, 273 Zea mays subsp. mexicana, see Zea mexicana Zea mexicana 183, 185 Zizania spp. 189 Zoysia spp. 189

Index Index Aceria tristriatus, see Eriophyes erineus Acidovorax avenae subsp. cattleyae 104, 196-197 Acidovorax sp. 294 Actinidia deliciosa 210-211 actinomycetes 17 aerial hyphae, see sporophores aerial transmission 129 Aesculus hippocastanum 294 AFLP-PCR, see amplified fragment length polymorphisms-PCR agar-agar 43 agar media, see media Agaricus bisporus 33, 118 agglutination test 51 agglutinins 91 aggressins, see virulence factors aggressiveness, see virulence Agrobacterium radiobacter 269 strain K84 and K1026 for biocontrol 166-167 Agrobacterium rhizogenes 269 Agrobacterium rubi 269 Agrobacterium tumefaciens 107-111, 268-269 Agrobacterium vitis 111, 269 Agrocin 84 167 Agrocinopine 167 alfalfa dwarf, see Xylella fastidiosa alginate 87 Allium porrum 115 alkaline phosphatase, see also ELISA 53 almond leaf scorch, see Xylella fastidiosa Alnus glutinosa 13 Anthurium andreanum 234 amplified fragment length polymorphisms-PCR 69, 72, 83 amylovoran 87 Anguillulina dipsaci 177 Anguina spp. 127 angular leaf spot of cotton 206-207 of cucumber 252-253 of strawberry 224-225 anthrax 3 Anthurium andreanum 234 antibiotic restistance 25, 169, 171 antibiotics 23 sensitivity test 44 antibodies, see serology antigens, see serology antiserum, see serology API system for biochemical tests 46 apoplexy of apricot 275 Approved List of Bacterial Names 31 archaea 7-10 Areca catechu 194 arginine dihydrolase test 49 ash bark beetle 218 ash twig miner 218 Aster sp. 268 aster yellows 10, 117

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Index Astilbe sp. 274 Avena sativa 186 avirulence 93-97 avirulence genes 93, 97 avr-genes, see avirulence genes Azorhizobium caulinodans 14 Bacillus in biocontrol 167-168 Bacillus anthracis 3 bacteria antibiotics, see antibiotics bacteriocins 22, 25 cell shape 15 cell membrane 21-22 cell wall 21-22 classification 6, 12, 283-284 diversity, see classification flagellar arrangement 19-20 genetics 24-27 Gram-negative 15, 21 Gram-positive 15, 21 growth curve 24 growth media 40-43 metabolism 23-25 minerals 15 morphology 15-20 occurrence 13 physiology 15, 23 plant pathogenic genera 36 plasmids 25 antibiotic resistance 171 biological control 166-167 genetic engineering 110-111 tumour formation 108-111 preservation 78-79 staining reactions 15, 21 taxonomy 27-31 polyphasic taxonomy 29 DNA:DNA hybridization 30 16S rRNA sequencing 32 toxins 23 trace elements 15 bacterial blast of stone and pome fruits 274-275 bacterial blight of Anthurium 234-235 bacterial blight of aroids 234-235 bacterial blight of cereals and grasses 190-191 bacterial blight of rice 139, 188-189 bacterial blight of Pelargonium 238-239 bacterial blight of vine 228-229 bacterial blight of walnut 242-243 bacterial brown spot of bean 275 bacterial canker and leaf spot of kiwifruit 210-211 bacterial canker of ash tree 100-102, 218-219 bacterial canker of stone fruits, Pseudomonas syringae pv. morsprunorum 214-215 bacterial canker of stone fruits, Pseudomonas syringae pv. syringae 274-275 bacterial canker of tomato 246-247 bacterial fasciation 280-282 bacterial knot disease of ash tree 100-102, 218-219

Index bacterial leaf blight of rice, see bacterial blight of rice bacterial leaf blight of wheat 275 bacterial leaf spot Delphinium spp. 232-233 Ribes spp. 216-217 Pelargonium spp. 238-239 bacterial leaf spot of ivy 236-237 bacterial leaf spot of lovage 250-251 bacterial leaf spot of orchids 197 bacterial leaf streak Xanthomonas translucens 191 Xanthomonas vasicola pv. holcicola 193 bacterial pustule of cowpea 263 bacterial pustule of soybean 258-259 bacterial ring rot, see ring rot bacterial scab of tomato 266-267 bacterial slime, see extracellular polysaccharide bacterial slime disease 276-279 bacterial speck of tomato 256-257 bacterial soft rot Erwinia carotovora subsp. carotovora 115-116, 270-271 Erwinia chrysanthemi 272-273 bacterial spot of tomato 266-267 bacterial spot (Pseudomonas syringae pv. syringae) 275 bacterial spot (Xanthomonas arboricola pv. pruni) 244-245 bacterial stem rot of Pelargonium 238-239 bacterial stem rot of potato 272-273 bacterial stripe blight of oat and barley 187 bacterial stripe of cereals and grasses 191 bacterial wilt (Ralstonia solanacearum) 276-279 bacterial wilt of carnation, see Burkholderia caryophylli bacterial wilt of corn, see Pantoea stewartii subsp. stewartii bacterial wilt of Pelargonium, see Xanthomonas hortorum pv. pelargonii bactericides 169-172 bacteriocin 22, 25, 167 bacteriophage lytic cycle 26 temperate phages 27 transduction 27 used for identification 55 in biological control 169 Bactrocera oleae 220-221 bean leaf beetle 261 beet grasshopper 128 Bemisia tabaci 261 Billing’s integrated system 125 biochemical tests 47-49, 81 biofilms 127 BIOLOG system for biochemical tests 47 biological control 166-169 biosecurity 154 bioterrorism 154 bird’s-eyes, on tomato fruits 134, 246-247 bittersweet, see Solanum dulcamara BIS, see Billing’s integrated system black blotch of Delphinium, see Pseudomonas syringae pv. delphinii black chaff 191 blackleg 202-203 black rot of cabbage 264-265 black spot of Delphinium, see Pseudomonas syringae pv. delphinii

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Index black spot of stone fruit 244-245 black spot of tomato 266-267 black spot of walnut 242-243 bleeding canker of chestnut 294 blister spot of apple 275 blossom blast 214-215 BOX-PCR, see repetitive DNA-sequence-based PCR Brassica napus var. naprobrassica 264 Brassica oleracea 112, 145 hydathode infection 87, 94 Brassica oleracea var. botrytis 130 Brassica oleracea var. gemmifera 264 breeding for resistance 162-167 Brenneria paradisiaca, see Erwinia chrysanthemi brown rot of orchids 199 brown rot of potato, see Ralstonia solanacearum brown spot of orchids 197 Brugmansia sp. 280 Burkholderia caryophylli 230-231 Burrill, Thomas J. 35 calcium 90 California vine disease 226-227 Candidatus species 10 canker of hazelnut 208-209 cankers 113-114, 135 Capsicum annuum 266 capsules 15, 18 Carneocephala fulgida, see Xyphon fulgida Carpocorus fuscispinus 258 catalase test 49 Cattleya 104, 196 cauliflower disease 280-282 cell membrane 21-22 cell wall antigens 55 structure 15, 21-22 cellulase 89, 98 cellulose 89 Cerotoma ruficornis 261 chemical compounds for control 169-170 chemical control 169-172 chlamidias 8 cholera 4 Chaetocnema pulicaria 184-185 Chromaphis juglandicola 242-243 Chrysanthemum 144, 268 growth curve of Pseudomonas cichorii 118 Cicadella viridis 227 Cichorium intybus 272 Circulifer tenellus citrus canker 138, 222-223 citrus greening, see Citrus Huanglongbin Citrus Huanglongbin 8 Citrus hystrix 222 citrus leaf miner, see Phyllocnystis citrella Citrus paradisi 222 Citrus sinensis 117, 222, 226 Citrus sp. 138

Index classification of bacteria 6, 12, 283-284 Clavibacter michiganensis subsp. michiganensis 144, 147, 246-247 in test for sensitivity to antibiotics 44 Clavibacter michiganensis subsp. nebraskense 182-183 Clavibacter michiganensis subsp. sepedonicus 200-201 geographical distribution map 142 influence of cutting seed on disease 123 survival 123 Clavibacter xyli, see Leifsonia xyli cluster analysis 64-66, 70-71, 81-83 Cobb’s disease of sugarcane 195 coconut lethal yellowing 117 common bacterial blight of bean 260-261 common scab 204-205 conidia, see exospores conjugate, see serology conjugation 24 control antibiotic resistance 25, 169, 171 bactericides 169-172 biological control 166-169 breeding for resistance 162-167 chemical compounds 169-170 chemical control 169-172 copper compounds 169 copper resistance 169 crop rotation 155 curative treatment 163 disinfectants 170, 172-173 education 155, 161 eradication 153, 154 genetic engineering 165-167 hygiene 155-158, 161 indexing 159-161 integrated control 150, 152, 154-161 meristem culture 160-161 principles of 149 quarantine regulations 149-153 sampling and testing 159-161 sanitation and disinfection 170, 172-173 steam sterilization 168 systemic acquired resistance 165, 167 thermotherapy 163, 168 waste 158 copper compounds 169 copper resistance 169, 171 corn flea beetle, see Chaetocnema pulicaria corn leaf hopper 128 Corylus avellana 114, 208 Corynebacterium michigaense, see Clavibacter michiganensis subsp. michiganensis Corynebacterium nebraskense, see Clavibacter michiganensis subsp. nebraskense Corynebacterium oortii, see Curtobacterium flaccumfaciens pv. oortii Corynebacterium sepedonicum, see Clavibacter michiganensis subsp. sepedonicus Cotoneaster salicifolius var. floccosus 240 Cotoneaster sp. 146 cotton fleahopper 206-207 cotton stainer 206-207 cowpea blight 262-263 cowpea canker, see cowpea blight Crataegus monogyna 240

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Index Crataegus sp. 114, 146 crop loss 143 crop rotation 155 crown gall 268-269 crown rot 177 cryoprotectants 78 Cucumis melo 252, 270 Cucumis sativus 130, 252 cucurbit angular leaf spot 252-253 Cucurbita pepo 270 Cucurbita sp. 144 cultural practices 155 culture collections 78-79 curative treatment 163 Curcuma longa 276 Curtobacterium flaccumfaciens pv. oortii 174-175 Cuscuta 10 cutin 89, 91 cutinase 89 cutting of seed 122 cyanobacteria 8 Cypripedium maudiae 198 cytokinins 86-87, 109 Dacus oleae, see Bactrocera oleae Dahlia sp. 272, 280 Dalbulus maidis 128 damage 143-146 decline of hazelnut 208-209 defense mechanisms 91-97 Delphinium sp. 232 denitrification test 49 deoxyribonuclease 89 detection methods 43-45, 57-64 diagnosis 38-79 steps in diagnosis 38 diagnosis report 79 Diabrotica sp. 127, 253 Dianthus caryophyllus 230 Diaphorina citri 8 Diaprepes abbreviatus 261 Dickeya chrysanthemi, see Erwinia chrysanthemi Dickeya dadantii, see Erwinia chrysanthemi Dickeya dianthicola, see Erwinia chrysanthemi Dickeya dieffenbachiae 273 Dickeya paradisiaca, see Erwinia chrysanthemi Dickeya zeae, see Erwinia chrysanthemi Dieffenbachia sp. 270 disease cycle Erwinia amylovora 135 Pseudomonas syringae pv. phaseolicola 136 Ralstonia solanacearum biovar 2, race 3 137 Xanthomonas axonopodis pv. citri 138 Xanthomonas oryzae pv. oryzae 139 disease free planting material 161, 163 disinfectants 170, 172-173 disinfection 170, 172-173 disposal of waste 158, 173 dissemination 127-134

Index DNA 25, 30, 56-60 DNA:DNA hybridization 30, 56-57 DNA hybridization test 56-57 dot-blot hybridization 56-57 downy mildew of cucurbits 252-253 Dracaena marginata 270 Draeculacephala minerva 227 dwarf diseases 8 Dysdercus suturellus 206 education 155, 161 electrophoresis, see protein electrophoresis elicitors 96 ELISA, see enzyme-linked immuno sorbent assay endophytic bacteria 121 endospores 18, 23 endotoxins, see toxins environmental effects 121-126 enzyme-linked immuno sorbent assay 45, 53 enzymes in pathogenesis 87-89, 98 Epilachna varivestris 261 epiphytic bacteria 120-121, 125-129 EPPO, see European Plant Protection Organisation EPS, see extracellular polysaccharides eradication 153, 155 ERIC-PCR, see repetitive DNA-sequence-based PCR Erwinia phenotypic characteristics 48 Erwinia amylovora 146, 240-241 bacterial strands 115 damage 146 disease cycle 135 epiphytic colonization 125-127 forecasting systems 125 geographical distribution map 140 on Crataegus 114 on Photinia 33 risk assessment 125 streptomycin resistance 169, 171-172 Erwinia ariodeae, see E. carotovora subsp. carotovora Erwinia atroseptica, see Erwinia carotovora subsp. atroseptica Erwinia carotovora subsp. atroseptica 202-203 respiration curve in potato 103 Erwinia carotovora subsp. betavasculorum 203 Erwinia carotovora subsp. brasiliensis 203 Erwinia carotovora subsp. carotovora 116, 144, 270-271 influence of nutrition on disease 122 influence of sowing date 122 Erwinia carotovora subsp. odorifera 203 Erwinia carotovora subsp. wasabiae 203 Erwinia chrysanthemi 272-273 cells and flagella in immuno-fluorescence staining 55 Erwinia cypripedii 198-199 Erwinia herbicola, see Pantoea agglomerans Erwinia papayae 199 Erwinia pyrifoliae 241 Erwinia rhapontici 176-177 Erwinia stewartii, see Pantoea stewartii subsp. stewartii

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Index Eriophyes erineus 243 esterase activity test 49 eubacteria 7 Eucomis autumnalis 178 eukaryotes 7-11 European Community Control Directives 153 European Plant Protection Organisation (EPPO) 153 quarantine lists A1 and A2 295-297 EPS, see extracellular slime excrescences, see galls exospores 17, 23 exotoxins, see toxins experimental error 60, 74-75 extracellular polysaccharides 18, 87, 115-116 extracellular slime, see extracellular polysaccharides FAM reporter dye, see real-time PCR fastidious phloem-limited bacteria 8 fastidious xylem-limited bacteria 8 fat hydrolysis test 49 fatty acid analysis 66-67, 81 Ficus 107 fire blight 114, 135, 240-241 FISH test, see fluorescent in situ hybridization FITC, see fluorescein isothiocyanate flagella 19-20 flagellar antigens 55 fluorescein isothiocyanate 51-52 fluorescent in situ hybridization 57-59 fluorescent pigments, see pigments, bacterial forecasting systems for disease development 125-126 Forsythia intermedia 274 FPLB, see fastidious phloem-limited bacteria Fragaria x ananassa 224 Frankia 13 Frankia alni 13 Fraxinus excelsior 100-102, 218 Freesia sp. 272 freeze-drying, see lyophilization frost damage 89 fuscous blight of bean 260-261 FXLB, see fastidious xylem-limited bacteria galls 104-109 rhizotamnia on Alnus glutinosa 13 root nodules of Rhizobium 14 gas chromatography, see fatty acid analysis gelatin hydrolysis test 49 gel electrophoresis, see protein electrophoresis and PCR gel scan software 65 gene-for-gene relationship 93, 97 genetic engineering 110-111 for control of disease 165, 167 herbicide resistance 111 genetic manipulation, see genetic engineering geographical distribution map Clavibacter michiganensis subsp. sepedonicus 142 Erwinia amylovora 140

Index Ralstonia solanacearum biovar 2, race 3 142 Xanthomonas axonopodis pv. citri 141 Xanthomonas oryzae pv. oryzae 141 germplasm 159-161 Gladiolus 106 Gladiolus convilli 180 Glycine max 258 Goss’s bacterial wilt and blight of corn 183 Gossipium hirsutum 206 Gram stain in differentiation of cell structure 15, 21 Granville wilt of tobacco 277-279 Graphocephala atropunctata 227 grease spot of bean 254-255 growth stimulating substances, see hormones gummosis of stone fruits 214-215 gummosis of sugar cane 195 Gymnocalicium mihanovichii 272 halo blight of bean 116, 136, 254-255 halo blight of oats 187 H-antigens 55 hazelnut canker 208-209 Hedera helix 236 hell fire of tulip 174 hemicellulose 89 histotropism 87 honeybees 127, 135, 241 horizontal resistance 93 hormones 86-87 hrp genes 95-96 Huanglongbin, see Citrus Huanglongbin Hyacinthus orientalis 34, 176 hydathode 74, 99 hydathode infection 87, 94 hydrogen sulphide test 49 hygiene 155-158, 161 risk factors 157 hyperplasia 91 hypersensitivity test on tobacco 78-79 hypertrophy 91 hypoplasia 91 IAA, see indole acetic acid ice-nucleation activity 89, 119 identification methods 47-56, 63-75 biochemical tests 46-49 physiological tests 44, 47 serological tests 51-55 IF-test, see immuno-fluorescence immuno-electrophoresis 65 immuno-fluorescence 45, 51-55 INA, see ice-nucleation activity incubation, in the disease process 99 indexing 159-161 indole acetic acid 86-87, 109 infection process 95-97, 99 insect transmission 10, 127-128

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Index insect vectors 10, 127-128 in situ hybridization, see fluorescent in situ hybridization test integrated control 150, 152, 154-161 interaction host pathogen 93 molecular interaction 95-98 internal browning of hyacinth 177 International Code of Nomenclature of Bacteria 27 International Plant Protection Convention (IPPC) 151 International Standard for naming pathovars 31 International Standards on Phytosanitary Measures (ISPM) 151 IPPC, see International Plant Protection Convention (IPPC) irrigation 131-134 isolation of bacteria from plant material 38, 40-43 ISPM, see International Standards on Phytosanitary Measures Janthinobacterium agaricidamnosum 118 Juglans regia 242 K-84, Agrobacterium radiobacter biological control strain 166-167 K-1026, Agrobacterium radiobacter biological control strain 166-167 Kalanchoe blossfeldiana 272 Kalanchoe daigremontiana 108 kasugamycin 169 Kasumin 169 Koch, Robert 3 Koch’s postulates 3-5 see also II.3 Diagnosis of bacterial plant diseases Kresek disease, of rice 189 large walnut aphid 242-243 latent infections 121, 127, 302 leaf freckles (Nebraska) and wilt 182-183 leaf miners 138 leaf scars 99 leaf scorch 8, 181 leaf spot of Gladiolus 181 leaf spot of kiwifruit 210-211 leaf spot of stone fruits 214-215 leaf spots 104-105 leaf streak, see bacterial leaf streak leafy gall 280-282 lectins 91 Leersia sp. 139 Leeuwenhoek, Antoni van 3 Leifsonia xyli 8 lenticels 99 Leperisinus varius 218 levan 42 levan production on nutrient sucrose agar 32 Levisticum officinale 250 Liberobacter africanus 8 Liberobacter asiaticus 8 life cycle 120 lignin 91 Lilium 106 lipids, see fatty acid analysis Lolium perenne 190

Index long distance movement 120, 149-150 losses 143-147 Lycopersicon esculentum 112, 114, 117, 134, 144, 147, 246, 248, 256, 266, 276 anatomy of stem under influence of nutrition 88 Lygus vosseleri 207 lyophilization 78-79 Malus 126, 146, 240 epiphytic colonization by Erwinia amylovora 126 Manihot esculentum 92 Maranthe sp. 294 Maryblyt TM 125 media 40-44 melanin, see pigments, bacterial meristem culture 160-161 metaplasia 91 Mexican bean beetle 261 Microbial Identification System, see fatty acid analysis MIS-system, see fatty acid analysis Moko disease of banana 277-279 molecular detection methods 57-62 molecular identification methods 65-75 molecular host-pathogen interaction 95-97 monoclonal antibodies, see serology Morus latifolia 212 mulberry blight 212-213 mulberry leaf spot 212-213 multidimensional scaling, see cluster analysis mycoplasmas 10 Nebraska leaf freckles and wilt 183 necrosis 113-114 nectaries 99, 126 nematodes 127 Anguillulina dipsaci 177 Nerium oleander 220 netted scab of potato 205 nitrate reduction test 49 nitrogen fixation 13-14 nitrogen-fixing bacteria 11, 13-14 nomenclature 27, 31 nopaline 108-111, 166-167 nutrition effect of calcium 90 effect on disease development 88, 122 O-antigens 55 octopine 18-111, 166-167 OEPP, see European Plant Protection Organisation Olea europaea 104, 220 epiphytic populations of Pseudomonas savastanoi 129 oleander knot 104, 220-221 oligogalacturonase 89 olive fly 220-221 olive knot 104, 220-221 Oncometopia nigricans 128, 227 opines 108-111, 166-167

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Index organotropism 87 Oryza sativa 188 oxidase test 49 oxytetracyclin, see tetracyclin PAGE test, see protein electrophoresis Pantoea agglomerans in biological control 167 growth curve in bean 103 Pantoea stewartii subsp. stewartii 184-185 Paphiopedilum sp. 198 Pasteur, Louis 3 pathogenic bacterium 85-89 pathogenesis phases in 99-103 pathogenicity test 76-77, 84 PCR test, see polymerase chain reaction pear blossom blight 275 pear decline 117, 298 pectate lyase 89, 98 pectic substances 89 pectin, see pectic substances pectin esterase 98 pectin hydrolysis test 49 pectin lyase 89 pectin methylesterase 89 pectinases 89 Pectobacterium atrosepticum, see Erwinia carotovora subsp. atroseptica Pectobacterium carotovorum var. aroideae, see Erwinia carotovora subsp. carotovora Pectobacterium subsp. carotovorum, see Erwinia carotovora subsp. carotovora Pectobacterium chrysanthemi, see Erwinia chrysanthemi Pectobacterium cypripedii, see Erwinia cypripedii Pelargonium diagnosis of Ralstonia solanacearum biovar 2, race 3 80-84 Pelargonium hortorum 238 Pelargonium peltatum 238 Pelargonium zonale 145, 276 section through tumour of Agrobacterium tumefaciens 107 pest-free-area 151 pH influence on potato scab 121 Phaseolus vulgaris 145, 254, 260 phenotypic tests 28, 47-49 Philadelphus sp. 274 Philaenus spumarius 227 Phlox paniculata 280 phony disease of peach, see Xylella fastidiosa phosphatidase 89 phospholipase 89 phospholipids 22, 67 Photinia davidiana 33 Phyllocnistis citrella 138, 222-223 physiological tests 47 phytoalexins 91 phytohormones, see hormones phytopathogenic bacteria antibiotic resistance 25, 169, 171 as human and animal pathogens 116 bacterium-host interaction 99-103

Index biological control 166-169 breeding for resistance 162-167 control 149-173 damage 143-146 diagnosis 38-79 steps in diagnosis 38 disease cycle 120, 135-140 disinfection and sanitation 170, 172-173 dissemination 127-134 education 155-156 enzymes 87, 89-90 eradication 153, 155 genera 36 genome analysis, total 97 geographical distribution 140-142 hygiene 155-158, 161 indexing 161, 163 integrated control 150, 152, 154-161 isolation media 40, 42 latent infections 120 life cycle 120 long distance movement 120, 149-150 losses 143-147 names 285-293 phytoplasmas 10 prevention 149-161 quarantine measures 149-153 seed transmission 37, 127, 130 survival 121, 123-124, 127 symptoms 105-117 transmission 127-139 thermotherapy 163 virulence 85-89 water transmission 127, 131-134 phytoplasmas 10, 117 phytotoxicity 163 Pierce’s disease of grapevine, see Xylella fastidiosa pigments, bacterial 43, 45 fluorescent 43-44 pyoverdins 43 xanthomonadins 43 pink seed 177 Pisum sativum 104, 130 symptoms of fungal disease and Pseudomonas syringae pv. pisi 92 resistance genes for Pseudomonas syringae pv. pisi 97 pith necrosis, of tomato 248-249 plant debris 155 plant pathogenic bacteria, see phytopathogenic bacteria plasmids, see bacteria, plasmids plum leaf scald, see Xylella fastidiosa p-nitrophenylphosphate, see enzyme-linked immuno sorbent assay polyacrylamide gel electrophoresis, see protein electrophoresis polyclonal antibodies, see serology polygalacturonase 89, 98 β-polyhydroxybutyrate 15, 67 polymerase chain reaction 59-60 control samples in PCR test 60, 74 real-time or Taqman PCR 59, 61-62 polyphasic taxonomy, see bacteria, taxonomy

353

354

Index Populus 33, 114 potato blackleg, see blackleg potato brown rot 276-279 disease cycle biovar 2, race 3 137 potato scab 204-205 Prays fraxinella 218 precipitation test 51-52 preservation of bacteria 78-79 prevention 149-161 Primula sinensis hydathode 94 prokaryotes 7-12 protease 89, 98 protected zone 151 protein electrophoresis 63-65 Prunus avium 214 Prunus persica 244 Prunus salicina 226 Pseudatomoscelis seratius 206 Pseudomonas aeruginosa 116 Pseudomonas avellanae 114, 208 Pseudomonas caryophylli, see Burkholderia caryophylli Pseudomonas cattleyae, see Acidovorax avenae subsp. cattleyae Pseudomonas cichorii 42, 118, 144 Pseudomonas coronofaciens 186-187 Pseudomonas corrugata 114, 248-249 Pseudomonas delphinii, see Pseudomonas syringae pv. delphinii Pseudomonas fluorescens in biocontrol 167-168 Pseudomonas lachrymans, see P. syringae pv. lachrymans [Pseudomonas levisitici] 250-251 Pseudomonas mediterranea 249 Pseudomonas mori, see P. syringae pv. mori Pseudomonas mors-prunorum, see P. syringae pv. morsprunorum Pseudomonas phaseolicola, see P. syringae pv. phaseolicola Pseudomonas ribicola, see P. syringae pv. ribicola Pseudomonas savastanoi pv. fraxini 218-219 pure culture 41 (early) infection stages, microscopy 100-102 Pseudomonas savastanoi pv. nerii 220-221 Pseudomonas savastanoi pv. phaseolicola, see P. syringae pv. phaseolicola Pseudomonas savastanoi pv. savastanoi 104, 220-221 epiphytic populations on olive 129 in gall tissue of olive 106 in Gram stain 11 Pseudomonas sp., fluorescent, in biological control 167-168 Pseudomonas syringae 294 Pseudomonas syringae pv. actinidiae 210-211 Pseudomonas syringae pv. coronofaciens, see P. coronofaciens Pseudomonas syringae pv. coryli 209 Pseudomonas syringae pv. delphinii 232-233 Pseudomonas syringae pv. lachrymans 130, 252-253 Pseudomonas syringae pv. mori 212-213 Pseudomonas syringae pv. morsprunorum 214-215 Pseudomonas syringae pv. phaseolicola 254-255 disease cycle 136 fluorescent pigment production 44 growth curve in bean 103 levan production 42 Pseudomonas syringae pv. pisi 92, 104, 130

Index resistance in pea, avirulence genes 97 Pseudomonas syringae pv. porri 115 Pseudomonas syringae pv. ribicola 216-217 Pseudomonas syringae pv. striafaciens, see P. coronofaciens Pseudomonas syringae pv. syringae 209, 240, 274-275 fluorescent pigment production 44 growth curve in bean 103 in glucose metabolism test 46 toxin production, demonstration of 44 Pseudomonas syringae pv. tomato 256-257 Pseudomonas syringae subsp. savastanoi, see Pseudomonas savastanoi Pseudomonas tolaasii 33 Pseudomonas tomato, see P. syringae pv. tomato Pseudoperonospora cubensis 252 psyllid vectors 8 pure culture, technique 43, 41 Puschkinia scilloides 178 pyoverdins, see pigments, bacterial Pyrus communis 146, 240, 274 Pyrus pyrifolia 241 Pyrus spp. quarantine legislation 149-153 quarantine measures 149-153 quarantine regulations 149, 151, 153 quorum sensing 109 Ralstonia solanacearum 276-279 biological control 168 biovar tests 300 diagnosed in Pelargonium 80-84 disease cycle 137 dispersal by water 129, 131-132, 134 EU scheme for detection of symptomatic and latent infections 301-302 geographical distribution map 142 in FISH test 58 in Koch’s posulates 5 in spiral vessel of potato 112 on selective SMSA medium 278 phenotypic characteristics 28 population dynamics in water 131-132 race determination 300 subspecific diversity 299-300 survival 124, 132 water streaming test 276 weed hosts, Solanum dulcamara 129, 137 Ralstonia syzigii 8 random amplified polymorphic DNA analysis 69 RAPD analysis, see random amplified polymorphic DNA analysis REA test, see restriction enzyme analysis of PCR product real-time PCR 60-62 receptors 96 red-headed sharpshooter 226 reference cultures 78-79 regional plant protection organisation (RPPO) 153 quarantine lists 295-297 reidentification 79 reisolation 79

355

356

Index REP-PCR, see repetitive DNA-sequence-based PCR repetitive DNA-sequence-based PCR 68-69, 82 resistance 93, 95-97 breeding for 163-167 horizontal 93 vertical 93, 95-97 resistance genes 95-97 resting spores, see endospores restriction enzyme analysis of PCR product 67-72, restriction fragment length polymorphism analysis 67-68 RFLP, see restriction fragment length polymorphism analysis Rhizobium 13 Rhizobium radiobacter, see Agrobacterium tumefaciens Rhizobium trifolii 14 rhizosphere 121, 127 Rhodococcus fascians 106, 280-282 Ribes rubrum 216 Ribonuclease 89 ribosomes 25 ribosomal RNA 25, 29, 32 rickettsias 8 ring rot 122-124, 200-201 Ri-plasmid 111 risk assessment 125 rhizosphere 120-121 rhizothamnia 13 RNA 25, 29, 32 root knot 268-269 root nodule 14 root weevil 261 Rosa sp. 268 RPPO, see regional plant protection organisation russet scab of potato 205

sampling and testing 159-161 sampling factor 161 Sanitary and Phytosanitary Agreement (SPS-agreement) 151 Sanitation 173 SAR, see systemic acquired resistance Scilla tubergeniana 178 scorch of Gladiolus Scroth’s crown gall medium 268 Secale cereale 190 seed infection 37 seed transmission 37 selective media, see media selective medium Agrobacterium tumefaciens 268 sequencing, see ribosomal RNA serology 45, 50-55, 80 agglutination test 51-52 conjugate 51 cross-reactions 50, 55 immuno-fluorescence test 45, 51-53 monoclonal antibodies 57 polyclonal antibodies 50-55 precipitation test 51

Index Sesbania rostrata aerial root nodules 14 sharpshooters 128, 227 shot-hole of stone fruit 244-245 slot-blot hybridization 56-57 Smith, Erwin F. 34-35 soft rot, see bacterial soft rot sodium dodecyl sulfate, see protein electrophoresis Solanum dulcamara 129, 131, 137 Solanum melongena 200 Solanum tuberosum 106, 116, 200, 202-204, 272, 276-278 Sorghum bicolor 192 southern bacterial wilt of tomato 277-279 sowing date 122 Spiroplasma citri 10, 117 insect vectors 127-128 Spiroplasma kunkelii 10, 128 insect vectors 127-128 spiroplasmas 10, 117 spores, see exospores or endospores sporophores 17 SPS, see Sanitary and Phytosanitary Agreement steam sterilization 168 stem crack of carnation 230-231 stewartan 87 Stewart’s disease 184-185 stolbur 117 stomata 99-100 Streptomyces acidiscabiei 205 Streptomyces aureofaciens 205 Streptomyces caviscabiei 205 Streptomyces europaeiscabiei 205 Streptomyces luridiscabiei 205 Streptomyces niveiscabiei 205 Streptomyces puniciscabiei 205 Streptomyces reticuliscabiei 205 Streptomyces scabiei 42, 106, 204-205 cell and colony morphology 17 influence of pH 121 Streptomyces scabies, see S. scabiei Streptomyces stelliscabiei 205 Streptomyces turgidiscabiei 205 streptomycin resistance 169, 171-172 streptomycin sulphate 169, 171 suberin 89, 91 suberin esterase 89 surface water 127, 129, 131-134 survival 121, 123-124 systemic acquired resistance (SAR) 165, 167 Syzigium aromaticum, see Ralstonia syzigii TAMRA reporter dye, see real-time PCR tarnished bug 207 Taqman-PCR, see real-time PCR Taq-polymerase 59-60 taxonomy, see bacteria, taxonomy teratomata 108 tetracycline 169 thermotherapy 163, 168

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358

Index titre, of antiserum 50-51 tip burn of Anthurium 234-235 Ti plasmid 108-111, 166 tomato pith necrosis 248-249 toxigenicity 85 toxins 23, 44, 85-87 coronatine 85 endotoxins 85 exotoxins 85 phaseolotoxin 85 syringomycin 85 syringostatin 85 syringotoxin 85 tabtoxin 85 tagetitoxin 85 thaxtomin 85 test for production of 44 tolaasin 85 toxoflavin 87 transmission 127-134 trichomes 99, 134 Trioza erytreae 8 Trifolium repens root nodules of Rhizobium 14 Tulipa gesneriana 174-175 tumour formation process 107-111 Ti plasmid 110-111 UPGMA cluster analysis, see cluster analysis variegated chlorosis of citrus, see Xylella fastidiosa vascular disease 112-113 vertical resistance 93-97, 163-167, Verticillium albo atrum 246 Vibrio cholerae 4 dispersal and survival in water 133 Viburnum sp. 240 Vigna unguiculata 262 virulence 87, 95-98 virulence factors 87, 89, 95-98 Vitis 228 Vitis vinifera 111, 228 volunteer plants 127, 155 Vuylstekaera 196 walnut bacterial blight 242-243 walnut leaf gall mite 243 Wakker, Jan Hendrik 35 water pore, see hydathode water, transmission and survival of pathogens 127, 131-134 survival of Ralstonia solanacearum 129, 131-132 waste 158 waste disposal 158 weed hosts 129, 131, 139 survival in weeds 129 whitefly 261

Index wilting, see vascular disease witches’ broom 10, 117 xanthan 87 xanthomonadins, see pigments, bacterial Xanthomonas ampelina, see Xylophilus ampelinus Xanthomonas arboricola pv. fragariae 224-225 Xanthomonas arboricola pv. juglandis 242-243 Xanthomonas arboricola pv. pruni 244-245 Xanthomonas axonopodis pv. citri 222-223 disease cycle 138 geographical distribution map 141 Xanthomonas axonopodis pv. aurantifolii 223 Xanthomonas axonopodis pv. dieffenbachiae 234-235 Xanthomonas axonopodis pv. glycines 258 Xanthomonas axonopodis pv. malvacearum 206-207 dispersal by irrigation water 134 Xanthomonas axonopodis pv. manihotis 92 Xanthomonas axonopodis pv. phaseoli 145, 260-261 Xanthomonas axonopodis pv. vasculorum 194-195 Xanthomonas axonopodis pv. vesicatoria 267 Xanthomonas axonopodis pv. vignicola 262-263 Xanthomonas campestris, see X. campestris pv. campestris Xanthomonas campestris pv. campestris 112, 130, 145, 264-265 hydathode infection in cabbage 87 Xanthomonas campestris pv. citromelo 223 Xanthomonas campestris pv. dieffenbachiae, see X. axonopodis pv. dieffenbachiae Xanthomonas campestris pv. glycines, see X. axonopodis pv. glycines Xanthomonas campestris pv. gummisudans 180-181 Xanthomonas campestris pv. hederae, see X. hortorum pv. hederae Xanthomonas campestris pv. holcicola, see X. vasicola pv. holcicola Xanthomonas campestris pv. juglandis, see X. arboricola pv. juglandis Xanthomonas campestris pv. oryzae, see X. oryzae Xanthomonas campestris pv. pelargonii, see X. hortorum pv. pelargonii Xanthomonas campestris pv. phaseoli, see X. axonopodis pv. phaseoli Xanthomonas campestris pv. phaseoli var. fuscans, see X. axonopodis pv. phaseoli Xanthomonas campestris pv. pruni, see X. arboricola pv. pruni Xanthomonas campestris pv. translucens, see X. translucens Xanthomonas campestris pv. vasculorum, see X. axonopodis pv. vasculorum and X. vasicola pv. vasculorum Xanthomonas campestris pv. vesicatoria, see X. vesicatoria Xanthomonas campestris pv. vignicola, see X. axonopodis pv. vignicola Xanthomonas citri, see X. axonopodis pv. citri Xanthomonas dieffenbachiae, see X. axonopodis pv. dieffenbachiae Xanthomonas fragariae 224-225 Xanthomonas glycines, see X. axonopodis pv. glycines Xanthomonas gummusidans, see X. campestris pv. gummisudans Xanthomonas hederae, see X. hortorum pv. hederae Xanthomonas holcicola, see X. vasicola pv. holcicola Xanthomonas hortorum pv. hederae 236-237 Xanthomonas hortorum pv. pelargonii 145, 238-239 Xanthomonas hyacinthi 34-35, 178-179 Xanthomonas juglandis, see X. arboricola pv. juglandis Xanthomonas malvacearum, see X. axonopodis pv. malvacearum Xanthomonas oryzae pv. oryzae 188-189 disease cycle 139 geographical distribution map 141 Xanthomonas pelargonii, see X. hortorum pv. pelargonii Xanthomonas phaseoli, see X. axonopodis pv. phaseoli

359

360

Index Xanthomonas phaseoli f.sp. sojense, see X. axonopodis pv. glycines Xanthomonas populi 33, 114 Xanthomonas pruni, see X. arboricola pv. pruni Xanthomonas translucens 190-191 pv. arrhenateri 191 pv. cerealis 191 pv. graminis 190-191 pv. hordei 191 pv. phlei 191 pv. phleipratensis 191 pv. poae 191 pv. secalis 191 pv. translucens 191 pv. undulosa 191 Xanthomonas vasculorum, see X. axonopodis pv. vasculorum and X. vasicola pv. vasculorum Xanthomonas vasicola pv. holcicola 192-193 Xanthomonas vasicola pv. vasculorum 195 Xanthomonas vesicatoria 266-267 Xanthomonas vignicola, see X. axonopodis pv. vignicola xylanase 89 Xylella fastidiosa 3, 6, 8, 117, 226-227 insect vectors 127 Xylophilus ampelinus 228-229 Xyphon fulgida 226 yellow disease of hyacinth 179 yellow pustule of tulip 175 yield loss 143-147 Zea mays 182, 184