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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:P.Obrien@murdoch.edu.au6

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