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A SAFRINET MANUAL FOR PHYTOBACTERIOLOGY
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Sponsored by SDC, Switzerland
Introduction to Practical
Phytobacteriology
A Manual for Phytobacteriology
by
SAFRINET, the Southern African (SADC) LOOP of
BioNET-INTERNATIONAL
Compiled by
T. Goszczynska, J.J. Serfontein & S. Serfontein
Bacterial Diseases Unit
ARC – Plant Protection Research Institute
Pretoria, South Africa
Sponsored by
The Swiss Agency for Development and Cooperation
(SDC)
© SAFRINET 2000
c/o ARC - Plant Protection Research Institute
Private Bag X134, Pretoria, 0001 South Africa
ISBN 0-620-25487-4
First edition, first impression
No part of this publication may be reproduced in any form or by any means, including
photocopying and recording, without prior permission from the publisher.
Layout, design, technical editing & production
Isteg Scientific Publications, Irene
Imageset by Future Graphics, Centurion
Printed by Ultra Litho (Pty) Ltd, Heriotdale, Johannesburg
P
reface
This manual is a guide to a course in practical phytobacteriology for
technical assistants of SADC countries in the SAFRINET-LOOP of
BioNET-INTERNATIONAL.
The course, presented by the staff of the Bacterial Diseases Unit of the
ARC - Plant Protection Research Institute, comprises lectures, practical
sessions and discussions aimed at teaching students to recognise and
identify bacterial diseases of agricultural crops. Techniques to isolate
and identify plant-pathogenic bacteria are presented, as well as
information on how to preserve isolated pathogens for further study.
The manual not only provides technical details but also lists the literature,
including books and manuals, that should be available in laboratories
specialising in phytobacteriology.
A
cknowledgements
•
Sincere thanks are due to Drs Connal Eardley and Elize Lubbe for
guidance and advice and to Dr S.H. Koch for her help in compiling a list
of bacterial diseases of vegetable crops.
(cid:127)
Generous funding by the sponsor, The Swiss Agency for Development
and Cooperation (SDC), is greatly appreciated.
(cid:127) We thank Mr H. Boroko for technical help.
Contributing authors
T. Goszczynska
J.J. Serfontein
S. Serfontein
(cid:127) Illustrated by Teresa Goszczynska and Elsa van Niekerk.
(cid:127) Cover design by Elsa van Niekerk and Nico Dippenaar.
(cid:127) Photographs by Kobus (J.J.) Serfontein and Jacomina Bloem.
C
ontents
Preface ... iii
Acknowledgements ... iv
Introduction .........................................................1
Identification of bacterial plant diseases .....................3
Visual examination and gathering of information . . . . . . . . . . . . . . . . . . . . . . . .3
Testing for bacterial streaming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Colony appearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Microscopic examination of isolated bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . .9
Tests for characterisation of bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
» Utilisation and decomposition of carbon sources . . . . . . . . . . . . . . 13
» Decomposition of nitrogenous compounds . . . . . . . . . . . . . . . . . 14
» Decomposition of macromolecules. . . . . . . . . . . . . . . . . . . . . 15
» Other tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Determination of pathogenicity ................................19
Classification of bacteria ......................................21
Gram-negative bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
» Gram-negative aerobic rods and cocci . . . . . . . . . . . . . . . . . . 24
» Gram-negative facultatively anaerobic rods . . . . . . . . . . . . . . . . 34
Gram-positive bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36
» Actinomycetes and related organisms . . . . . . . . . . . . . . . . . . . 36
Cell-wall-free procaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38
Basic keys for the identification of
phytopathogenic bacteria.......................................39
Key No. 1 — Bean, pea (pod spot, leaf spot and blight). . . . . . . . . . . . . . . . . .41
Key No. 2 — Cowpea (leaf spot or leaf blight). . . . . . . . . . . . . . . . . . . . . . . . . .43
Key No. 3 — Tomato. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44
Key No. 4 — Tomato (canker and wilt) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Key No. 5 — Potatowilt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
Key No. 6 — Soft rots (fruits, tubers, bulbs and leaves). . . . . . . . . . . . . . . . . . . .49
Key No. 7 — Galls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50
Key No. 8 — Crucifers (leaf spot, black rot, soft rot) . . . . . . . . . . . . . . . . . . . . . .51
Other methods to detect and identify
phytopathogenic bacteria.......................................52
Preservation of bacterial cultures ...........................53
Culture collections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
Preservation of bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
» Short-term storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
» Long-term storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Epidemiology and control of bacterial diseases...........57
Inoculum sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57
» Primary sources. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
» Secondary sources. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
» Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Media and diagnostic tests ....................................60
Essential laboratory equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60
Staining of bacteria and KOH solubility test . . . . . . . . . . . . . . . . . . . . . . . . . . . .61
Preparation of culture media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61
General isolation media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
Selective media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64
Media for characterisation of phytopathogenic bacteria. . . . . . . . . . . . . . . . .68
» Utilisation and decomposition of carbon sources . . . . . . . . . . . . . . 68
» Decomposition of nitrogenous compounds . . . . . . . . . . . . . . . . . 70
» Decomposition of macromolecules. . . . . . . . . . . . . . . . . . . . . 71
» Other tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Recommended reading .........................................75
Useful Internet sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77
Index to isolation media and diagnostic tests . . . . . . . . . . . . .78
Glossary ...........................................................80
Appendix — Commonbacterialdiseasesofvegetablecrops.................82
I
ntroduction
Althoughbacteriacausearathersmallproportionofplantdiseases,thisdoesnotmean
thatthesediseasesareunimportant.InNorthCarolina,USA,forinstance,Granvilleor
bacterialwiltoftobaccocausedsomuchdamagefor30yearsafteritsappearancein
1880thatitforcedbankstoclose,farmstobesoldandtownstodecline.Amorerecent
exampleofaseverebacterialdiseaseiswatermelonfruitblotch,whichappearedin
watermelon-productionareasoftheUSA.Pendinglawsuitsandtheriskoffuturelitigation
forcedmajorseedcompaniestosuspendtheirwatermelonseedsalesintheautumnof
1994.
Otherbioticagentsimplicatedinplantdiseasesarefungi,virusesandnematodes;
abioticfactorsmayalsoproducedisease-likesymptoms.Aplantabnormalitycannot
alwaysbediagnosedsolelybysymptomsasdifferentagentscancausesimilar
pathologicalsymptoms(Fig.1).Softrotcanbecausedbyfungiorbacteria;gallsby
A B
C D
Fig.1
Similarsymptomsonbeanplantscausedbydifferentagents: A–virus;B–bacterium;
C–pesticide;D–fungus.
2 Introduction
fungi,bacteriaandinsects;leafspotbybacteria,virusesandfungi,andwiltdiseasesby
fungiandbacteria.Inasingleplantspecies,symptomscausedbydifferentbacteria
mayoverlap,forexamplebacterialblightofbeanandfoliarbacterialdiseasesof
tomato.Symptomexpressionofaparticulardiseasecanvaryconsiderably,andmaybe
influencedbycropcultivar,growthstage,environmentalconditionsandpathogenstrain.
Crop-productionmethods,suchasproductionincontrolledenvironmentslike
greenhousesandinhydroponics,alsoplayaroleinsymptomexpression.Changesin
productionmethodshavealsobroughtpreviouslyunknownandunimportantdiseasesto
theforeground.
+
A preliminary diagnosis of the disease can be made on the basis of symptoms, microscopic
examination and a few diagnostic tests. However, accurate diagnosis of the pathogen is always
essential.
I
dentification of bacterial plant
diseases
Inadiagnosticlaboratorywhereplantmaterialisanalysedforthepresenceofaplant
disease,anumberoflogicalstepsmustbefollowedtoidentifythecausalagentofthe
diseaseorplantabnormality.
Visual examination and gathering of information
•Firststep–besureoftheidentityoftheplanttobeanalysed.
Besidestheplant’sidentity,asmuchinformationaspossibleonthecropmustbe
gathered,forexamplelocationofcrop,methodofcultivation,irrigationmethods,
chemicalsapplied,recentclimatic
information.
(cid:127)Secondstep–familiariseyourself
withthesymptoms(Fig.2).
Gatherinformationaboutthe
distributionofthediseaseinthecrop.
Itisimportanttoexamineasmanyof
thediseasedplantsaspossible,from
earlytoadvancedstagesof
symptomdevelopment.
(cid:127)Thirdstep–obtaininformationabout
allthepossiblediseasesreportedon
thecropinthecountryorsubregion.
TheAppendixtothismanualandthe
IndexofPlantPathogensandthe
DiseasesthattheyCauseinCultivated
PlantsinSouthAfrica(Gorter1977),which
containsthenameofthecrop,alistof
pathogensreportedonthecrop,andthe
commonnamesofthediseases,aregood
startingpointsinallSADCcountries.Notall
diseasesareincludedintheIndexanditis
advisabletoobtainasmuchinformation
aspossiblefromtheliterature.TheDisease
CompendiumSeriespublishedbythe
AmericanPhytopathologicalSocietyis
veryusefulinthisregard.Italsocontains Fig.2
photographsofdiseasesofparticular Symptomscausedbybacteriaonplants.
crops.
