RESEARCH REPOSITORY
This is the author’s final version of the work, as accepted for publication following peer review but without the publisher’s layout or pagination.
The definitive version is available at:
https://doi.org/10.1007/s13313-017-0481-4
O’Brien, P.A. (2017) Biological control of plant diseases. Australasian Plant Pathology, 46 (4). pp. 293-304.
http://researchrepository.murdoch.edu.au/id/eprint/37568/
Copyright: © 2017, Australasian Plant Pathology Society Inc.
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1
BiologicalControlofPlantDiseases1
PhilipA.O’Brien2
SchoolofVeterinaryandLifeSciences,MurdochUniversity,MurdochWA6150,3
Australia4
5
Email:[email protected]
7
8
Abstract 9
10
Biologicalcontrolisthecontrolofdiseasebytheapplicationofbiologicalagents11
toahostanimalorplantthatpreventsthedevelopmentofdiseasebyapathogen.12
Withregardtoplantdiseasesthebiocontrolagentsareusuallybacterialor13
fungalstrainsisolatedfromtheendosphereorrhizosphere.Virusescanalsobe14
usedasbiocontrolagentsandthereisaresurgentinterestintheuseofbacterial15
virusesforcontrolofplantdiseases.Thedegreeofdiseasesuppressionachieved16
withbiologicalagentscanbecomparabletothatachievedwithchemicals.Our17
understandingofthewaysinwhichbiocontrolagentsprotectplantsfrom18
diseasehasdevelopedconsiderablyinrecentyearswiththeapplicationof19
genomicsandgeneticmodificationtechniques.Wehaveuncoveredmechanisms20
bywhichbiocontrolagentsinteractwiththehostplantandothermembersof21
themicrobialcommunityassociatedwiththeplant.Understandingthese22
mechanismsiscrucialtotheisolationofeffectivebiocontrolagentsandthe23
developmentofbiocontrolstrategiesforplantdiseases.Thisreviewlooksat24
recentdevelopmentsinourunderstandingofbiocontrolagentsforplant25
diseasesandhowtheywork.26
27
28
29
Introduction 30
31
2
Plantdiseasesareamajorconstraintoncropproductioninallagriculturaland32
horticulturalsystems.Allcropsaresusceptibletodiseasescausedbyavarietyof33
pathogens(bacteria,fungi,andviruses).Ingenerallossesofcropsduetodisease34
amountto25%ofworldcropproductionperannumLugtenberg(2015).Of35
course,lossesarenotdistributedevenlybutinsomecasesmaybemuchhigher36
resultinginlossoftheentirecrop.Attheveryleastthiscanhavesevere37
financialimplicationsatthelocal,regionalornationallevels.Atworstitcanlead38
tofaminewithconsiderablelossoflife.39
40
Managementofplantdiseasesisasignificantcostcomponentincropproduction.41
Traditionallytheapproachestodealingwithdiseaseinagriculturalecosystems42
includesbreedingresistantvarietiesofthecropsspecies,hygienetopreventthe43
spreadofcontaminatedsoilorseed,andfungicidestokillpotentiallyinfecting44
fungi.Howeverincreasingconcernsabouttheeffectsoffungicidesinthe45
environmentandresiduesinfoodhaveresultedinderegistrationofanumberof46
fungicides.Theneedtoreplacethesehasincreasedinterestinbiologicalcontrol47
ofplantdiseasesinrecentyears.Biologicalcontrolisthesuppressionofdisease48
bytheapplicationofaBiocontrolAgent(BCA)usuallyafungus,bacterium,or49
virus,oramixtureofthesetotheplantorthesoil.TheBCAactstoprevent50
infectionoftheplantbythepathogen,orestablishmentofthepathogeninthe51
plant.ThemainadvantageofusingaBCAisthattheyarehighlyspecificfora52
pathogenandhenceareconsideredharmlesstonon‐targetspecies.Overthepast53
decadetherehavebeenmanyreportsoftheidentificationofeffectiveBCAsfor54
fungalandbacterialdiseasesincropsandanumberofBCAsareincommercial55
production(Table1).InrecentyearsourunderstandingofhowBCAsprotect56
theplantfrominfectionhaschangeddramaticallywiththeapplicationof57
genomics.Inordertoimplementaneffectivebiocontrolprogramit’sessentialto58
understandhowBCAsworktopreventdiseasedevelopment.Thepurposeof59
thisarticleistoreviewthevariousmechanismsbywhichBCAsprotectplants60
againstpathogens.61
62
3
How effective is biocontrol 63
64
ThelevelofdiseasecontrolachievedbyapplicationofBCAstoacropcanbe65
closetoorequivalenttothatachievedbyapplicationofafungicide.Application66
ofafungicidetoPhytophthoracactoruminfectedappleresultedin100%disease67
suppressionwhilstapplicationofvariousBCAssinglyresultedinlevelsof68
diseasesuppressionbetween79%‐98%dependingontheBCA(Alexanderand69
Stewart2001)(Table2).InanotherstudyapplicationofaBacillus70
amyloliquefaciensBCAtomandarinfruitsuppressedP.digitatuminfectionby71
77%whichcomparesto96%afterapplicationofthefungicideimazalil(Table72
2).TheefficacyofaBCAcanbeenhancedbymixingwithafungicideprovided73
thefungicidedoesnotadverselyaffecttheBCA.Infectionofstrawberryby74
BotrytiscinereawasreducedtolowlevelsbyapplicationofaTrichoderma75
atroviridaeBCA,butwaseliminatedbyapplicationoftheBCAwithafungicide76
(Table2).Interestinglyinthiscasethefungicidealonewaslesseffectivethan77
theBCAalone.NakayamaandSayama(2013)reportedasimilarenhancement78
indiseasecontrolusingaBCA‐fungicidemixtoinhibitpowderyscabofpotato79
(Table2).Wheretherearecomparativeresultsfordiseasesuppressionin80
glasshouseandfieldtrials,thedegreeofsuppressiontendstobelowerinthe81
fieldtrialse.g.,inthestudyofFuetal.(2010)thedegreeofsuppressionwas24%82
lowerinthefield(Table2).Thisisconsideredtoreflectthemorediverse83
environmentinthefield.Anumberofstudieshavedemonstratedthat84
biocontrolcanalsobeusedeffectivelyagainstpostharvestdiseases(Table2).85
86
Someendophytesprotectagainstmultiplepathogens.AnendophyticstrainG387
withpotentialasabiocontrolagentwasisolatedfromthestemsofTriticum88
aestivumL.Itwasclassifiedby16SrDNAsequencingasamemberofSerratia.89
AlthoughstrainG3displayedabroadspectrumofantifungalactivityinvitro90
againstanumberofphytopathogenssuchasBotrytiscinerea,Cryphonectria91
parasitica,RhizoctoniacerealisandValsasordidaishasnotbeentestedfor92
diseasesuppression(Liuetal.2010).AstrainofBacilluspumilisisolatedfrom93
theendosphereofpoplarsuppressedthegrowthofthreepathogensCytospora94
4
chrysosperma,PhomopsismacrosporaandFusicoccumaesculiingreenhousetests95
(Renetal.2013).96
97
98
Host Genotype Effect 99
100
Oneoftheproblemswithbiocontrolisthelackofconsistencyindisease101
suppressionbyaBCA.Differencesinhostgenotypecontributetodifferencesin102
responsestoaBCA.IncontrolofPhytophthorameadiiinfectionofHevea103
brasiliensisbyAlcaligenesspthedegreeofcontroldifferedbetweentwocultivars104
ofthehost(Table2)(Abrahametal.2013).Acultivareffectwasalsoobserved105
instudiesonbiocontrolofdiseasesinstrawberry(Cardetal.2009)andpepper106
(Leeetal.2008).Thespecificityeffectmayberelatedtotheproductionofplant107
moleculesthatactivatetranscriptionalactivatorsoftheLuxRfamilyinthe108
bacterium.TheproductsoftheLuxRgenesactasglobalregulatorscontrolling109
suchprocessesasbiofilmformationandantibioticproductionamongothers.110
AlthoughLuxRregulatorsnormallyoperateinquorumsensingsystemswhereby111
bacteriacommunicatewitheachother,somesuchasthePsoRgeneofP.112
fluorescens(Subramonietal.2011)andtheOryRgeneofXanthomonasoryzae113
(FerlugaandVenturi2009)respondtoplantcompoundstherebyfacilitating114
plant‐BCAcommunication.Alternativelycommunicationcouldbemediatedby115
secondarymetabolitesproducedbytheBCA.Endophytesproducealargearray116
ofdifferenttypesofsecondarymetabolitesmanyofwhichhavenotbeen117
detecteddirectlybuthavebeeninferredfromgenomicanalysis(Braderetal.118
2014).Thereareexampleswherethesynthesisofsecondarymetabolites119
stimulateschangesinplantmetaboliteproductionandvice‐versa(Ludwig‐120
Muller2015).121
122
Mixtures of BCAs 123124
SeveralresearchershavereportedthatusingmixturesofBCAshasincreasedthe125
consistencyofbiocontrolacrosssiteswithdifferentconditions.Instudieson126
5
infectionofpotatobyPhytophthoracapsicigreaterdiseasecontrolwasachieved127
usingamixtureofthreebacterialBCAscomparedtousingthesinglestrains(Kim128
etal.2008)(Table2).Sliningeretal.(2001)intheirinvestigationinto129
postharvestdryrotofpotatofoundthatformulationsofmixedBCAsperformed130
moreconsistentlyacross32storageenvironmentsvaryingincultivar,washing131
procedure,temperature,harvestyear,andstoragetime.Enhancedbiocontrol132
usingmixturesofBCAshasbeenreportedforcontroloflateblightinpotato133
(Sliningeretal.2007),diseasesofpoplar(Gyenisetal.2003),chilli134
(Muthukumaretal.2011),andcucumber(RaupachandKloepper1998;Roberts135
etal.2005).Itisalsopossiblethatdifferentmixturesmayneedtobeusedin136
differentclimaticareas.Thusthereisaneedtoidentifyanumberofpotential137
biocontrolagents.Mixturesdonotalwaysgiveincreasedcontrol.Insomecases138
theremaybeantagonismbetweentheBCAsthatresultsinreducedcontrol139
comparedtosinglestrains.Inevaluatingagentsforcontroloffireblightinpear,140
Stockwelletal.(2011)foundthatmixturesofPseudomonasfluorescensA506,141
PantoeavagusC9‐1andPantoeaagglomeransEh252werelesseffectivethatthe142
individualstrains.ItwasfoundthatthePantoeastrainsexerttheireffect143
throughtheproductionofpeptideantibiotics.Inthemixturethesewere144
degradedbyanextracellularproteaseproducedbyP.fluorescensA506.Roberts145
etal.(2005)havealsoreportedantagonismbetweenBCAstrains.They146
observedthatpopulationsofTrichodermavirensGL3orGL321wereboth147
substantiallyreducedafterco‐incubationwithBacilluscepaciaBC‐1orSerratia148
marcescensisolatesN1‐14orN2‐4incucumberrhizospheres.Thesereports149
highlighttheimportanceofconsideringpossibleantagonismbetweenstrains150
whendevelopingabiocontrolformulation.Co‐cultivationinvitrocansometimes151
revealinhibitoryeffects(Robertsetal.2005)butnotalways.Inthestudyby152
Stockwelletal.(2011)antagonismbetweenthespeciesinthemixturewouldnot153
havebeenevidentfromco‐cultivationofthethreespecies,itwouldonlyhave154
beenevidentifthemixturewastestedinaconfrontationassaywiththe155
pathogen.156
157
158
159
6
Where do BCAs come from. 160
161
MostcommonlyBCAsareisolatedbyscreeningorganismsfromtherhizosphere162
orendophytepopulationforinhibitionofgrowthofthetargetpathogeninvitro.163
Thosethatshowinhibitionareassessedfurtheralthoughitshouldbestressed164
thatinvitroinhibitionisnotalwaysasuccessfulindicatorofasuccessfulBCAas165
thereareothermechanismsofdiseasesuppression(stimulationofhostgrowth;166
inductionofhostdefence;occlusionofpathogen;competitionfornutrients;toxin167
inactivation)thatdonotinvolvegrowthinhibition,andthereareother168
characteristicsrequiredforasuccessfulBCAsuchabilityformassproduction169
andpersistenceunderfieldconditions(Elliottetal.2009;Martinetal.2015;170
Melnicketal.2008).Prominentamongthosespeciesofrhizosphereand171
endophyticbacteriathatareeffectiveBCAsaretheactinomycetesandspecies172
fromthegeneraPseudomonasandBacillus.Amongthefungithatconstitute173
effectiveBCAsspeciesofthegenusTrichodermaarewellrepresented(Table2).174
Allofthesearecapableofsynthesizinganarrayofsecondarymetabolites.175
176
ActinomycetesmakeverygoodBCAs.Endophyticactinobacteriaisolatedfrom177
healthycerealplantswereassessedfortheirabilitytocontrolfungalroot178
pathogensofcerealcropsbothinvitroandinplanta.Thirtyeightstrains179
belongingtothegeneraStreptomyces,Microbispora,Micromonospora,and180
Nocardioidieswereassayedfortheirabilitytoproduceantifungalcompoundsin181
vitroagainstGaeumannomycesgraminisvar.tritici(Ggt),thecausalagentoftake‐182
alldiseaseinwheat,RhizoctoniasolaniandPythiumspp.Sporesofthesestrains183
wereappliedascoatingstowheatseed,withfivereplicates(25plants),and184
assayedforthecontroloftake‐alldiseaseinplantainsteamedsoil.The185
biocontrolactivityofthe17mostactiveactinobacterialstrainswastested186
furtherinafieldsoilnaturallyinfestedwithtake‐allandRhizoctonia.Sixty‐four187
percentofthisgroupofmicroorganismsexhibitedantifungalactivityinvitro,188
whichisnotunexpectedasactinobacteriaarerecognizedasprolificproducersof189
bioactivesecondarymetabolites.Seventeenoftheactinobacteriadisplayed190
statisticallysignificantactivityinplantaagainstGgtinthesteamedsoilbioassay.191
TheactiveendophytesincludedanumberofStreptomyces,aswellas192
7
MicrobisporaandNocardioidesspp.andwerealsoabletocontrolthe193
developmentofdiseasesymptomsintreatedplantsexposedtoGgtand194
Rhizoctoniainthefieldsoil(Coombsetal.2004;Costaetal.2013;Doumbouet195
al.2001).196
197
HypovirulentisolatesofapathogenspeciescanalsoactasBCAs(Sneh1998).A198
naturallyoccurringhypovirulentisolateofPhytophthoranicotianaewasfoundto199
effectivelycontrolcitrusrootrotcausedbyP.nicotianaeandP.palmivora200
(ColburnandGraham2007).InanotherstudybinucleateisolatesofRhizoctonia201
solaniwereeffectiveatcontrollingdampingoffdiseasesinpeppercausedby202
RhizoctoniasolaniorPthyiumultimum(Harris2000).Hypovirulentisolatesof203
theChestnutBlightdiseasepathogenCryphonectriaparasiticawerewidelyand204
successfullyusedtocontrolthediseaseinchestnuttrees(Nuss2005).205
206
Viruses as BCAs 207
208
Duetothepaucityofeffectivebactericidalcompoundsformanagementof209
bacterialphytopathogensthereisrenewedinterestintheuseofbacterialviruses210
(bacteriophageorphage)asBCAsforcontrolofbacterialdiseases.Thehistory211
ofphageuseformanagementofplantdiseasesisreviewedinJonesetal.(2007).212
Recentstudieshavedemonstratedsignificantreductionindiseaseseverityfora213
rangeofpathogensincluding,Agrobacterium,Xanthomonas,Ralstonia214
solanacearum,Erwiniaamylovora,andStreptomycesonavarietyofcrops.The215
advantagesofusingphageare:a)easeofproduction;b)highspecificityforthe216
targetorganism;c)longshelflife.Thephagecanbegrowninthefieldusingan217
avirulentformofthepathogeninfectedwiththephageappliedasadressingto218
thecrop.Theavirulentstrainactsasavehicleforproductionofthephagebutis219
notabletodamagethecrop(Dialloetal.2011).Ineffectthiscreatesaself‐220
perpetuatingbiocontrolsysteminthefield.Inthestudiesonthesuppressionof221
Ralstoniawiltoftomatousingphage,infectivephageparticlesweredetected222
fourmonthsaftertreatment(Fujiwaraetal.2011)(Table2).Oneproblem223
associatedwiththeuseofphageBCAsisthedevelopmentofresistanceinthe224
8
hostbacterialpopulation.Theuseofacocktailcontaininganumberofhost225
rangemutantsisrecommendedtoovercomethis.Suchmutantscanbeevolved226
inthelab(Jackson1989).ThepersistenceofphageBCAsinthefieldmaybe227
enhancedbymicroencapsulationoftheBCAinaninertpolymermatrixandthe228
slowreleaseofphagefromthismatrix(Choinska‐Pulitetal.2015;Vonaseketal.229
2014).230
231
Fungalviruses(mycoviruses)havealsobeenusedasBCAs.Mycovirusesare232
presentinallmajortaxaoffungi(Ghabrialetal.2015;Nuss2005)Theydonot233
appeartohavemechanismsoftissueinfectionbutratheraretransmittedby234
hyphalanastomosis,andthuscanonlybeexchangedbetweenvegetatively235
compatiblestrains.Inthemajorityofcasesinfectiondoesnotappeartocause236
anysymptomsalthoughinsomecasesmycovirusinfectionresultsina237
hypovirulentphenotype.ThemostfamousexampleistheChestnutBlight238
pathogenCryphonectriaparasiticawhichhasdevastatedchestnutpopulationsin239
theUSAandEurope(Nuss2005).Applicationofvirusinfectedhypovirulent240
strainstochestnuttreesresultedintransmissionofthevirustovirulentstrains241
byhyphalanastomosiswithattenuationofvirulenceandprotectionofthetrees.242
WhilstthisstrategywassuccessfulinEurope,itdidnotworkintheUSAbecause243
ofvegetativeincompatibilitybetweenthestrainspreventedtransmissionofthe244
virustothepathogenicstrains.Hypovirulenceinducingmycoviruseswiththe245
abilitytoinfecthostfungaltissuewhenappliedexternallywithouttheneedfor246
anastomosishavebeenidentifiedinthefungalpathogensSclerotinia247
sclerotiorumandRosellinianecatrix.Thesearelikelytobeparticularlyusefulas248
BCAsastheirspreadwillnotbelimitedbyvegetativeincompatibility(Ghabrial249
etal.2015).250
251
252
How do endophytes protect plants? 253
254
Stimulation of plant growth 255
256
9
AcommoneffectoftheapplicationofarhizosphericorendophyticBCAtoaplant257
isacceleratedgrowthoftheplant.ManybacterialandfungalBCAsproduce258
analoguesofplantgrowthregulatoryhormonesandvolatilecompoundsthat259
stimulategrowth(Harmanetal.2004;Taghavietal.2009).Thegrowthincrease260
canbequitesubstantial.Inoneexperimentinoculationoflettucewithgrowth261
promotingstrainsofBacillusresultedina30%increaseinplantweighttwo262
weeksafterinoculation(Santoyoetal.2012).Thusbesidesdiseasesuppression,263
anotheradvantageofbiocontrolisincreasedyieldevenintheabsenceofdisease.264
Volatilessuchas2,3‐butanediol,acetoin,andaldehydesandketonesare265
producedbybacteriaandmayplayapartinpromotingplantgrowth.266
Inactivationofgenesforsynthesisofthevolatiles2,3‐butanediolandacetoinin267
theB.subtilisbiocontrolstrainsBSIP1173andBSIP1174disruptedstimulation268
ofthehostplantgrowth(Santoyoetal.2012).FungalBCAsalsostimulate269
growthofthehostplant.Trichodermaharzianumproducesabutenolide270
metabolitecalledharzianolidethatbothstimulatesgrowthandinducesdefence271
mechanisms(Caietal.2013).272
273
Analogsofplanthormonesproducedbyendophyticbacterianotonlypromote274
growthoftheplantsbuttheyalleviateotherstressessuchasdrought.For275
example,abscisicacidandgibberellinsproducedbythebacterialendophyte276
Azospirillumlipoferumhavebeenshowntobeinvolvedinalleviatingdrought277
stresssymptomsinmaize(Braderetal.2014).278
279
Induction of Host Defence Mechanisms 280
281
AnothermechanismcommonlyassociatedwithprotectionofplantsbyBCAsis282
inductionofthehostdefencepathways.Thisoccursasaresultofthereleaseof283
elicitors(proteins,antibioticsandvolatiles)bytheBCAthatinduceexpressionof284
thegenesofthesalicyclicacidpathwayorthejasmonicacid/ethylenepathway285
(NawrockaandMalolepsza2013;Pieterseetal.2014).Adifferentdefence286
mechanism,InducedSystemicResistance(ISR),characterisedbybroadspectrum287
resistanceagainstpathogensofvarioustypesaswellasabioticstressesisalso288
10
induced(Anetal.2010;Shoreshetal.2010).InductionofISRusuallyinvolvesa289
primedstateforanenhancedreactiontoabioticorabioticstimulusratherthan290
fullinduction(Conrathetal.2006;Pieterseetal.2014).Becausethisisnotfull291
inductionitisconsideredtorequirelessenergythanfullinductionand292
consequentlyhavelessofanegativeimpactongrowth(Perazzollietal.2011).293
Bacterialvolatileshavealsobeenimplicatedininductionofsystemicresistance294
inthehostplantviaanethylenedependentpathway(Kloepperetal.2004).In295
additiontovolatilesISRisinducedbysiderophoresandcycliclipopeptide296
antibiotics(Janetal.2011).297
298
299
Secretion of polysaccharide degrading enzymes 300
301
Secretionofavarietyofpolysaccharidedegradingenzymesincludingchitinases,302
glucanases,proteasesandcellulasesisacommonfeatureofbacterialandfungal303
BCAs(Janetal.2011;Quecineetal.2008).Theseenzymesare capableof304
degradingthecellwallsoffungal(oroomycete)hyphae,chlamydospores,305
oospores,conidia,sporangia,andzoosporesresultinginlysisandthuscontribute306
totheprotectionoftheplant.Theoligosaccharidesreleasedfromdegradationof307
thefungalcellwallsactassignalingmoleculestoinducethehostdefence308
mechanisms.Howevertheproductionofenzymescapableofdegradingthe309
hyphalcellwallsofpathogenicfungiinvitrodoesnotconstituteproofthatthese310
enzymesareresponsibleforbiocontrolactivityinplanta.Michelsenand311
Stougaard(2011)showedthatalthoughPseudomonasfluorescensIn5produces312
chitinaseandbeta‐1,3‐glucanasethebiocontrolactivityexhibitedbythisstrainis313
notduetotheseenzymesbuttotheproductionofthenon‐ribosomalpeptide314
antibioticsnunamycinandnunapeptin(Michelsenetal.2015).Inotherstudies315
Kimetal.(2008)foundthatbacterialchitinaseproductionisnotresponsiblefor316
biocontrolofphytophthorablightofpepper,whilstWorasatitetal.(1994)317
showedthattherewasnorelationshipbetweenthebiocontrolactivityof318
Trichodermakoningiiandtheproductionofchitinase,glucanase,orcellulaseby319
thefungus.However,contrastingresultswereprovidedbyCherninetal.(1995)320
11
whoshowedbygeneinactivationthatchitinaseproductionisresponsiblefor321
biocontrolactivityofEnterobacteragglomerans,andbyDowningandThomson322
(2000)whotransformedaPseudomonasstrainwithachitinasegenethus323
creatingaBCA.324
325
Production of antibiotics 326
327
Manybiocontrolbacteriaandfungiproducemultipleantibiotics(including328
biosurfactantswithantibioticpropertiessuchaslipopeptides)thatconfera329
competitiveadvantagebyeliminatingotherbacteriaandfungi.Singlestrains330
canproducemultiplevariantsofeachtype(reviewedinRaaijmakersetal.331
(2010)andJanetal.(2011)).Inadditiontotheirantibioticproperties,332
lipopeptidesareimportantsignallingmoleculesandaffectprocessessuchas333
motility,inductionofhostplantdefencemechanisms,andformationofmicrobial334
biofilmsontheinnerandoutersurfacesofplants.ThefungusTrichoderma335
whichiswidelyusedasabiocontrolagentandwhichformsthebasisofseveral336
commercialproductsforbiocontrol(Table1)alsosynthesizesanarrayof337
secondarymetaboliteswithantibioticactivity(Druzhininaetal.2011).Among338
thesearethenon‐ribosomalpeptideswhichformvoltagedependention339
channelsinmembranes;polyketidesofunknownfunction;isoprenoid340
derivativesthatarehighlyfungitoxicandphytotoxic;andpyroneswith341
antifungalactivity.342
343
Variousstudieshaveattemptedtoprovideevidenceforaroleforthese344
antibioticsinpathogensuppressionbyenhancingtheirsynthesisordisrupting345
thegenesfortheirsynthesis.Inactivationofantibioticsynthesisgenesinvarious346
speciesofPseudomonas,orBacillushasprovidedstrongevidencefortheroleof347
antibioticsinbiocontrolbythesespecies(Wuetal.2015).Initialobservations348
showedthatatryptophanauxotrophicmutantofP.aeruginosadeficientin349
phenazinesynthesiswasineffectiveatsupressinginfectionofcocoyamby350
Pythiummyriotylumincontrasttothewildtypestrainwhicheffectively351
suppressedinfection(TambongandHöfte2001).Disruptionofrhamnolipidand352
Deleted: ins
Deleted: biosurfactins
12
phenazinesynthesisgenesinthespeciesPseudomonasaeruginosaand355
Pseudomonaschlororaphissignificantlyreducedtheabilityofthisspeciesto356
suppressVerticilliummicrosclerotia. Howeveritdidnotcompletelyremovethe357
suppressionsuggestingthatthereareothermechanismsofpathogen358
suppression (Debodeetal.2007).SubsequentexperimentsinwhichthedarA359
anddarBgenesresponsibleforthebiosynthesisoftheantibiotic2‐hexyl,5‐360
propylresorcinol(HPR)inPseudomonaschlororaphiswereinactivated361
confirmedtheroleoftheantibioticinantagonism(Calderonetal.2013).362
Similarly,genedisruptionwasusedtoprovideevidenceforrolesforfengycin363
(Yanez‐Mendizabaletal.2012)anditurininbiocontrolofpeachandcurcubit364
diseasesrespectivelybystrainsofBacillussubtilis(Zeriouhetal.2011)andof365
iturininbiocontroloffruitdiseasesbyBacillusamyloliquefaciens(Arrebolaetal.366
2010).Morerecentworksuggeststhatdifferentantibioticsfromthesamestrain367
interactsynergisticallytoachievediseasesuppression.APseudomonasstrain368
producingphenazineandtwotypesofcycliclipopeptideantibiotics(sessilins369
andorfamides)suppressedinfectionofChinesecabbagebyR.solaniAG2‐1370
(Olorunlekeetal.2015).Althoughproductionofphenazinealonewassufficient371
toachievediseasesuppression,intheabsenceofphenazinebothsessilinsand372
orfamideswererequired.InsuppressionofrootrotofbeancausedbyR.solani373
4‐HG1bothphenazinesandeithersessilinsororfamideswererequired.This374
studyalsodemonstratesthatthelackofaneffectuponinactivationofthe375
synthesisofasingleantibioticinabiocontrolstraindoesnotprecludearolefor376
thatantibioticinbiocontrol.377
378
Despitetheevidenceproducedbytheabovestudiesshowingthatantibiosisis379
thebasisforbiocontrolactivityinanumberofspecies,anumberofstudieshave380
producedconflictingresults.Poritsanosetal.(2006)reportedthataGacS381
mutantofP.chlororaphiswasgreatlyreducedintheproductionofphenazine382
andshowedtenfoldreductioninbiocontrolefficacy..However,theGacS383
mutationalsoaffectedtheproductionofprotease,lipase,andbiofilmformation384
allofwhichwouldcontributetobiocontrolefficacy.Thebiocontrolyeast385
Pseudozymaflocculosa(syn:Sporothrixflocculosa)isaneffectivebiocontrol386
agentforcontrolofpowderymildew(Bélangeretal.2012).Theyeastproduces387
13
apowerfulantibioticthatinducesarapidcellcollapseinthepathogen.Despite388
theinitialindicationsthattheantibioticisresponsibleforthebiocontrolactivity,389
itturnedoutnottobeinvolved.Incontrasttoexperimentsshowingthat390
disruptionofantibioticsynthesisgenesinspeciesofPseudomonasandBacillus391
reducedbiocontrolefficacy(Arrebolaetal.2010;Calderonetal.2013;Debodeet392
al.2007;TambongandHöfte2001;Yanez‐Mendizabaletal.2012),Mazzolaetal.393
(2007)foundthatdisruptionofsynthesisofthecycliclipopeptideantibiotic,394
massetolideinPseudomonasfluorescensbyTn5insertiondidnotaffect395
biocontrolactivity.Thedemonstrationoftheinvolvementofantibiosisasa396
mechanismofbiocontroliscomplicatedbytheplethoraofantibioticsproduced397
byindividualbacterialstrains.Inaddition,manyantibioticsynthesisgenesare398
onlysynthesizedathighcelldensity,orwhenthebacteriumformspartofa399
biofilm(RutledgeandChallis2015).Manysuchcrypticgeneshavebeen400
detectedinthegenomesoffilamentousfungi,inparticularAspergillusspp.and401
actinomycetes.Demonstratingtheinvolvementofantibiosisinbiocontrolis402
furthercomplicatedbythefactthatantibioticsoftenhaveadditionalrolesother403
thaninhibitingthegrowthofmicroorganisms.Surfactinsforexampleare404
importantinmotilityofcellsontheplantsurfaces,intriggeringtheformationof405
biofilmsandinductionofhostdefences.Thusinactivationofcycliclipopeptide406
antibioticgenesleadsnotonlytodecreasedantibiosis,andimpairedhost407
inductionbutalsodecreasedabilitytoformbiofilms(Raaijmakersetal.2010).408
Thusantibioticsactinmultiplewaystosuppresspathogens.409
410
Biofilms 411
412
Onplantsurfacesbacteriararelyexistassinglecells,butformlargemulticellular413
assemblagescalledbiofilms Boginoetal.2013;Flemmingetal.2016 .Biofilms414
typicallycontainmultiplebacterial,ormixedbacterialandfungalspecies415
Flemmingetal.2016;Frey‐Klettetal.2011 .Inabiofilmthecellsarecovered416
byamatrixthatprotectsthemfromdesiccation,UV,predation,andbactericidal417
compoundssuchasantibiotics.Thematrixconsistsofsolublegelforming418
polysaccharides,protein,lipidandDNAaswellasinsolubleamyloids,fimbriae,419
Deleted: contian
Deleted: forming
14
piliandflagellaandispermeatedbychannelsthatactasacirculatorysystemfor422
exchangeofnutrients,water,enzymes,signallingmoleculesandremovaloftoxic423
metabolites.Biofilmsarecomplexsorbentsystemswithbothanionicand424
cationicexchangers,whichmeansthataverywiderangeofsubstancescanbe425
trappedandaccumulatedforpossibleconsumptionbycellsinthebiofilm.The426
nutrientcaptureefficiencyofthematrixexceedsthatoffreelivingcells427
Flemmingetal.2016 .Notonlynutrients,butalsotoxicsubstances,can428
accumulateinbiofilmsbybindingtothematrix.Inthiswaythematrixsoaksup429
toxicsubstancesthatwouldotherwisebeinhibitorytothecells.These430
substancesareeitherretainedinthematrixuntilitdecomposes,orreleased431
fromthematrixintothewaterphaseandexudedfromthematrix.Biofilm432
formationalsofacilitatestheexchangeofgeneticinformationbetweencells.433
Conjugationhasbeenshowntobe700timesmoreefficientinbiofilmsthanin434
free‐livingcells Flemmingetal.2016 .Biofilmsaidinplantprotectionby435
preventingaccesstothesurfaceoftheplantbythepathogen,andbythe436
productionofantibiotics,manyofwhichareonlyproducedwhengrowingina437
biofilm.Justasbiofilmsmayaidthesurvivalandproliferationofbiocontrol438
speciesonplantsurfaces,somaytheyaidthesurvivalandproliferationof439
pathogenicspecies MorrisandMonier2003 .Additionally,cellwalldegrading440
enzymessecretedbythepathogenbindtothebiofilmmatrixleadingto441
increasedheattoleranceandprotectionagainstenzymaticdegradation442
Flemmingetal.2016 .443
444
Biofilmformationinitiateswiththeaggregationofcellsontheplantsurfacea445
processthatistriggeredbythesecretionofAHLSsignalingmoleculesby446
neighbouringcells.Theaggregationisfacilitatedbycomponentssuchas447
surfactinwhichmodifythesurfacepropertiestoenhancemotilityandadhesins448
Boginoetal.2013;Flemmingetal.2016 .Onceaggregationhasinitiatedthe449
cellssynthesizethecomponentsforthematrix.450
451
Competition for nutrients 452
453
15
Competitionfornutrientson,orproximaltotheplantsurface(rhizosphere)is454
anothermechanismusedtoprotectplantsfrompathogens.BCAscompetefor455
sugarsontheleafsurfacesorrootexudatesintherhizosphere.Thesesamefood456
sourcesarerequiredforinitialestablishmentofthepathogenpriortoinfection.457
ByutilisingthesefoodsourcestheBCApreventsestablishmentofthepathogen458
(Cardetal.2009;Ellisetal.1999).Forthesereasonshypovirulentvariantsof459
thepathogenmakeeffectiveBCAs.Theyoccupythesamenichesasthe460
pathogen,utilisethesamenutrients,andcanoccupyentrypointstotheplant461
tissuesthatwouldbeusedbythepathogentherebypreventinginfectionbythe462
pathogen(Sneh1998).Biocontrolspeciesareabletosequesterironfortheir463
ownusebytheproductionofironbindingsiderophores.Thisreducesthe464
availabilityofirontootherorganismssuchaspathogens(Santoyoetal.2012).465
Becausebacterialsiderophoreshaveahigheraffinityforironthanfungal466
siderophores,theyareeffectiveatdeprivingfungiofiron(Janetal.2011).467
468
Inactivation of pathogen phytotoxins. 469470
Manyplantpathogensproducephytotoxinsthatcontributetopathogenicityby471
disruptingprocessinthehostplant(Strange2007).Thesetoxinseitheractas472
enzymeinhibitors(HCtoxinofHelminthosporiumcarbonum),interferewith473
membranefunction(syringomycinofP.syringae),orpreventinductionofhost474
defences(coronatineofP.syringae).BCAscanprotectplantsfromphytotoxins475
byinactivatingthemorpreventingtheirproduction.ThepotentBCA476
BurhholderiaheleiaPAK1‐2preventssynthesisofthephytotoxintropoloneby477
thericepathogenBurkholderiaplantarii(Wangetal.2016).Abiocontrolstrain478
ofBacillusmycoidesinactivatesthetoxinsthaxtominA(1)andB(2)producedby479
thepotatocommonscabpathogenStreptomycesscabei(Kingetal.2000).The480
ricesheathblightpathogenR.solaniproducesahostspecifictoxin,theRStoxin481
thatispartofit’spathogenicity.KnownbiocontrolstrainsofT.viridaethat482
produceanalpha‐glucosidasethatinactivatesthetoxinhavebeenisolated483
(Shanmugametal.2001).Thealphaglucosidaseisdifferentfromotherknown484
alpha‐glucosidasesandisspecificforthetoxin.StrainsofFusariumand485
TrichodermacapableofinactivatingthetoxinsEutypine,4‐486
16
hydroxybenzaldehyde,and3‐phenyllacticacidproducedbythepathogens487
causingEutypadiebackandescadisease,twotrunkdiseasesofgrapevine(Vitis488
vinifera)havebeenisolated(Christenetal.2005).489
490
491
Genetically Modified BCAs 492
493
Techniquesforgeneticengineeringofallorganismshavebeendevelopedtoa494
highdegreeofprecisionandhavebeenappliedtotheimprovementofstrainsof495
bacteria,andfungiforindustrialprocesses.Thesetechniquescanbeappliedto496
improvetheefficacyofBCAs.Inoneexperimentthetransferofachitinasegene497
fromSerratiatoaPseudomonasendophytecreatedastrainwithagreatly498
increasedabilitytosuppressR.solaniinfectionofbean(DowningandThomson499
2000).SimilarlytheadditionofaglucanasegenetoTrichodermaresultedina500
strainthatsecretedamixtureofglucanasesandshowedgreatlyenhanced501
protectionagainstthepathogensPythium,Rhizoctonia,andRhizopus(Djonovicet502
al.2007).Zhouetal.(2005)assembleda2,4‐diacetylphloroglucinol (2,4‐DAPG)503
biosynthesislocusphlACBDEclonedfromstrainCPF‐10intoamini‐Tn5504
transposonandintroducedintothechromosomeofthenon2,4‐DAPGproducing505
strainPfluorescensP32.Theresultantstrainsprovidedsignificantlybetter506
protectionofwheatagainsttake‐allcausedbyGaumannomycesgraminisvar507
triticiandtomatoagainstbacterialwiltcausedbyRalstoniasolanacearum.In508
spiteoftheresultsofthesestudiesthesenewlycreatedBCAsaresubjecttothe509
regulationsthatgoverntheuseoforganismsthataregeneticallymodified510
throughtheuseofrecombinantDNA.Giventhestiffoppositionthathasfaced511
theuseoftransgenicplantsandtheevengreaterdifficultiesofcontainment512
facedwithgeneticallymodifiedmicroorganismsitisunlikelythatBCAscreated513
byrecombinantDNAtechnologywillbeapprovedforgeneraluseinthenear514
future.515
516
Amorerealisticapproachwouldbetousenon‐recombinantDNAtechnologyto517
enhanceBCAs.Clermontetal.(2011)usedgenomeshufflingtogenerate518
17
improvedbiocontrolstrainsofStreptomycesmelanosporofaciensEF‐76.Two519
roundsofgenomeshufflingresultedintheisolationoffourstrainswith520
increasedantagonisticactivityagainstthepotatopathogensStreptomycesscabies521
andPhytophthorainfestans.Chemicalmutagenesishasbeenusedtoenhance522
biocontrolactivity,e.g.,nitrosoguanidinemutagenesisofPseudomonas523
aurantiacaB‐162resultedintheisolationofastrainwiththreefoldelevated524
levelsofphenazineproductionandenhancedbiocontrolactivity(Feklistovaand525
Maksimova2008).Marzanoetal.(2013)isolatedstrainsofT.harzianumwith526
greatlyenhancedbiocontrolactivityafterUVmutagenesis.Becausethegenetic527
techniquesusedinthesestudiesdonotinvolverecombinantDNA,theysimply528
mimicwhathappensnaturallytheydonotfallundertheregulationsgoverning529
theuseofgeneticallymodifiedorganismsandhencetheyshouldbemore530
acceptabletobeingusedfordiseasecontrol.Howeveroneofthepotential531
problemswithsuchagentsisthatasidefromthedesiredmutationtheremaybe532
additionalmutationsinothergenesthatcanresultinundesirableconsequences..533
Morerecentlydevelopedtechniquesofgenomeeditingcanovercomethese534
limitations.UsingtoolssuchasCrispr/Caswecanwithgreatprecisionintroduce535
mutationsintospecificlocationsinthegenomewithgreatefficiency(Barrangou536
andvanPijkeren2016).Anadditionaladvantageisthatmutationscanbe537
inducedinmultiplegenessimultaneouslyandthiswillbeanadvantageis538
identifyingtheroleofdifferentgenesinbiocontrol.539
540
Conclusions 541
542
ThetraditionalmethodofsearchingforaBCAisbasedontheassumptionthat543
theBCAwilldirectlyantagonisethepathogeneitherbyantibioticproductionor544
predation.Suchantagonismisdetectedbyconfrontationassaysonagar545
medium.AswehaveseentherearemultiplemechanismsbywhichaBCAmay546
protectplantsfrompathogens,anddifferentBCA’smayusedifferent547
combinationsofthese,ormayusedifferentmechanismsunderdifferent548
circumstances.Onlysomeofthesewouldbedetectedbyconfrontationassays.549
OthersrequirecommunicationbetweentheBCAandthehostandother550
18
endophytesorgrowthoftheBCAinbiofilms.Itisapparentthatthe551
confrontationassaysareaninadequatescreeningmethodastheydonottake552
intoaccountallmechanismsofantagonism,anddonotreplicatethe553
environmentinwhichtheBCAmustfunction.Theythereforeidentifyonlya554
subsetofpossibleBCAs.Itisconsideredthattheuseofinappropriatescreening555
methodsisamajorcontributortothefailureofbiocontrolstrategies(Pliegoetal.556
2011).ScreeningforBCAsmustbedonewithaninplantaassayoranassaywith557
tissueexplant.Withthecontinuedapplicationofgenomicstoidentificationof558
genesresponsibleformaintainingtheendophyticstateitispossiblethatwewill559
beabletoidentifyeffectiveBCAsbasedonageneticprofile(Benítezand560
McSpaddenGardener2009).Inaddition,geneidentificationopensup561
possibilitiesforgeneticmodificationsothatinsteadofscreeningfornewBCAs562
wesimplymakenewonesbymodificationofpre‐existingones.563
564
HavingidentifiedasuitableBCAtheassumptionisthatitcanbeproducedin565
liquidcultureandusedasaseeddressing,soildrenchorfoliarspray.Whatis566
crucialtoeffectivediseasecontrolisthepersistenceoftheBCA.Ithasto567
competewithothermicrobialspeciesintherhizosphere,endosphereand/or568
phyllospheresothatitcanestablishandofferprotectionoverareasonable569
timeframe.Inthisregardthemethodofproductioniscrucialasitdetermines570
thetypeofpropagules(spores,conidia,vegetativecells)producedandthusthe571
shelflife,andpersistenceoftheproductintheenvironment(Bisuttietal.2015;572
Hanitzschetal.2013;Kakvanetal.2013;MocellinandGessler2007).573
574
Despitethefactthatalotoforganismswithbiocontrolpotentialhavebeen575
identifiedagainstalargenumberofpathogenstherehavebeenrelativelyfew576
developedcommercially.Toremedythisandtakefulladvantageofthebenefits577
inbiologicalcontroltheresearchfocusneedstoshiftfromidentificationof578
antagonisticorganismstowardsproduction,formulationanddelivery.579
580
581
19
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Table1.Examplesofbiologicalcontrolagentsincommercialproduction.BIOCONTROLAGENT CROP SUPPLIER COUNTRYAmpelomycesquisqualisM‐10 Powderymildews EcoGen USAB.subtilisMB1600 Fungalpathogensofcotton,
LargeseededlegumesSoybeans
BekerUnderwood USA
B.subtilisMB1600+Rhizobium Fungalpathogensofsoybeans,peanut
BekerUnderwood USA
Bacillusamyloliquefaciens GB99+B.subtilisGB122
Beddingplantsinpottingmixes
Gustafson,Inc. USA
Bacilluslichenformis SB3086 TurfGrass,Sclerotinia NovozymesBiologicals,USA USABacilluspumillusGB34 Soybeanfungaldiseases Gustafson,Inc. USABacillussubtilisGB03 Cotton,legumefungal
diseasesGustafson,Inc. USA
BacillussubtilisGB03,otherB.subtilisB.lichenformis,B.megaterium
Fungalpathogensofgreenhouseandnurseryplants.
GrowthProductsLtd
BacillussubtilisQST713 Vegetables,Fruits AgraQuest USAConiothyriumminitans Rootrot ProphytaBiologischer Germany
Coniothyriumminitans Rootrot Bioved HungaryEscherichiacoliphage Bacterialpathogensoffruit
andvegetablesIntralytix USA
Fusariumoxysporum non‐pathogenic
Wilt S.I.A.P.A. Italy
Fusariumoxysporum non‐pathogenic
Wilt NaturalPlantProtection France
Gliocladiumcatenulatum Vegetables,Fruits ArgaQuestGliocladiumcatenulatum Rootrotwilt Verdera FinlandListeriamonocytogenes phage Bacterialpathogensoffruit
andvegetablesMicreos The
Netherlands
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Listeriamonocytogenes phage Bacterialpathogensoffruitandvegetables
Intralytix USA
P.fluorescensA506,and1629RSPsyringae742RS
Certainfruits,almond,potato,tomato
FrostTechnolCorp.
Pseudomonasaureofaciens Tx‐1 FungalopathogensofturfGrass
TurfScienceLaboratories
Pseudomonaschlororaphis63‐28 WiltdiseasesofornamentalsandvegetablesinGH
TurfScienceLabs
Pseudomonassyringae Pomefruit,citrus,cherries,potatoes
JETHarvestSolutions
Pseudozymaflocculosa powderymildew PlantProducts CanadaPythiumoligandrum Rootrot Bioreparaty Czech
RepublicSalmonellaphage Bacterialpathogensoffruit
andvegetablesIntralytix USA
Streptomycesgriseoviridis Vegetables,Fruits ArgaQuestStreptomycesgriseoviridis K61 Fieldornamental,vegetable
fungalpathogensAgBio
T.harzianum Greymold MakhteshimChemicalWorks IsraelT.harzianumATCC20476TrichodermapolysporumATCC20475
Binab Sweden
T.harzianumT‐22 Rootrot Bioworks USA
Trichodermaharzianum Vegetables,Fruits ArgaQuestTrichodermaharzianum Rootrot EfalAgri IsraelTrichodermaspp Rootrotwilt Binab SwedenTrichodermaspp. Rootrotwilt Bioplant Denmark
Trichodermaspp. Rootrot AgrimmTechnologies NewZealandTrichodermavirens GL‐21 Rootrot Certis USA
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Trichodermaviride Rootrotwilt EcosenseLaboratories IndiaXanthomonascampestris phagePseudomonassyrinagephage
Bacterialpathogensoffruitandvegetables
Omnilytics USA
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Table2.Suppressionofpathogensonvarioushostspeciesbybiologicalandchemicalcontrolagents.PLANTHOST PATHOGEN DISEASECONTROLAGENT YEAR DEGREE
OFCONTROL
ASSAYTYPE
SOURCE
Apple Phytophthoracactorum Flavobacterium 79% GH (AlexanderandStewart2001)
Apple Phytophthoracactorum Oidiodendron 85% GH (AlexanderandStewart2001)
Apple Phytophthoracactorum Microsphaeropsis 98% GH (AlexanderandStewart2001)
Apple Phytophthoracactorum Trichodermaharzianum 89% GH (AlexanderandStewart2001)
Apple Phytophthoracactorum Trichodermakoningii 93% GH (AlexanderandStewart2001)
Apple Phytophthoracactorum Paecilomyces 93% GH (AlexanderandStewart2001)
Apple Phytophthoracactorum Metalaxyl+Mancozeb 100% GH (AlexanderandStewart2001)
Banana Pseudocercosporamusae Bacillussubtilis B106 72% GH (Fuetal.2010)
Banana Pseudocercosporamusae Bacillussubtilis B106 48% F (Fuetal.2010)
Chinesecabbage Plasmodiophorabrassica Bsubtilis >80% F (Pengetal.2011)
Chinesecabbage Plasmodiophorabrassica Gliocladiumcatenulatum >80% F (Pengetal.2011)
HeveabrasiliensisRRII105
Phytophthorameadii Alcaligenes spEIL‐2 63% GH (Abrahametal.2013)
HeveabrasiliensisRRIM600
Phytophthorameadii Alcaligenes spEIL‐2 30% GH (Abrahametal.2013)
mandarinfruit Penicilliumdigitatum B.amyloliquefaciens HF‐01 77% PH (Haoetal.2011)
mandarinfruit Penicilliumdigitatum Imazalil 96% PH (Hao etal.2011)
Pepper Phytophthoracapsici singlebacterium 32‐73% F (Kimetal.2008)
Pepper Phytophthoracapsici mixof3bacteria 99% F (Kimetal.2008)
Pepper Phytophthoracapsici B.subtilis R33 87% F (Leeetal.2008)
Pepper Phytophthoracapsici B.subtilis R13 71% F (Lee etal.2008)
Potato Fusariumsambucinum Serratiaplymuthica 5‐6 75% PH (Gouldetal.2008)
Potato S.subterranea Aspergilliusversicolor lm6‐50
70% T (NakayamaandSayama2013)
Potato S.subterranea Aspergilliusversicolor lm6‐50+fluazinam
93% T (NakayamaandSayama2013)
30
StrawberrycvYolo
B.cinerea LU829 TrichodermaatroviridaeLU132
2004 77% F (Cardetal.2009)
StrawberrycvPajero
B.cinerea LU829 TrichodermaatroviridaeLU132
2004 88% F (Cardetal.2009)
StrawberrycvPajero
B.cinerea LU829 Fenhexamide 2004 71% F (Cardetal.2009)
StrawberrycvPajero
B.cinerea LU829 TrichodermaatroviridaeLU132+Fenhexamide
2004 100% F (Cardetal.2009)
Tomato Ralstoniasolanacearum PhagePhiRSL1 100% P (Fujiwara etal.2011)
*AssayType:F,fieldtrial;GH,greenhouse;P,pot;PH,post‐harvest;SD,seeddressing;TC,tissueculture