Phytobacteriology: (Cabi)

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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

+

-

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.

Phytobacteriology and diagnosis

71

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.

Phytobacteriology and diagnosis

73

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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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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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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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).

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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.

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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).

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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.

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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

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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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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

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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.

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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.

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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).

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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

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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.

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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.

+ +

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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.

+

+

+ +

+

+

+ + + +

+

+ + + +

+ +

+ +

+ +

+ +

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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).

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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).

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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.

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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).

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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).

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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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Fig. 91

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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

PD

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).

Chapter III

PD

116

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).

Disease and symptoms

117

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.

Epidemiology

119

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).

120

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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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121

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

Epidemiology

123

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

24

Rubber, concrete

> 10

Metal

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).

Damage and losses

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.

144

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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.

Damage and losses

145

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.

146

Chapter V

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.

148

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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).

Prevention and Control

149

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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Chapter VI

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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151

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

PD

STEAM STERILIZATION UNDER PLASTIC AT 80ºC KILLS BACTERIAL PATHOGENS

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

170

Chapter VI

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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171

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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173

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 (