Fungal communities associated with almond throughout crop development

15 Pages·2017·2.15 MB·English

RESEARCHARTICLE
Fungal communities associated with almond
throughout crop development: Implications
for aflatoxin biocontrol management in
California
AlejandroOrtega-Beltran1,2¤a*,JuanMoral1,2¤b,RyanD.Puckett1,2,DavidP.Morgan1,2,
PeterJ.Cotty3,ThemisJ.Michailides1,2*
a1111111111 1 DepartmentofPlantPathology,UniversityofCaliforniaDavis,Davis,California,UnitedStatesofAmerica,
2 KearneyAgriculturalResearchandExtensionCenter,Parlier,California,UnitedStatesofAmerica,
a1111111111
3 USDA-ARS,SchoolofPlantSciences,UniversityofArizona,Tucson,Arizona,UnitedStatesofAmerica
a1111111111
a1111111111
¤a Currentaddress:InternationalInstituteofTropicalAgriculture,Ibadan,Nigeria
a1111111111 ¤b Currentaddress:DepartamentodeAgronom´ıa,UniversidaddeCo´rdoba,Co´rdoba,Spain
*[email protected](TJM);[email protected](AOB)
Abstract
OPENACCESS
Interactionsbetweenpathogenicandnonpathogenicfungalspeciesinthetreecanopyare
Citation:Ortega-BeltranA,MoralJ,PuckettRD,
MorganDP,CottyPJ,MichailidesTJ(2018)Fungal complexandcandetermineifdiseasewillmanifestintheplantandinotherorganismssuch
communitiesassociatedwithalmondthroughout ashoneybees.SeasonaldynamicsoffungiwerestudiedinanalmondorchardinCalifornia
cropdevelopment:Implicationsforaflatoxin
whereexperimentalreleaseoftheatoxigenicbiopesticideAspergillusflavusAF36todis-
biocontrolmanagementinCalifornia.PLoSONE13
(6):e0199127.https://doi.org/10.1371/journal. placetoxigenicAspergillusstrainshasbeenconductedforfiveyears.Thepresenceofthe
pone.0199127 vegetativecompatibilitygroup(VCG)YV36,towhichAF36belongs,intheblossoms,and
Editor:RichardA.Wilson,UniversityofNebraska- thehoneybeesthatattendtheseblossoms,wasassessed.Inblossoms,A.flavusfrequen-
Lincoln,UNITEDSTATES ciesrangedfrom0to4.5%,dependingontheyearofstudy.Frequenciesofhoneybeescar-
Received:February26,2018 ryingA.flavusrangedfrom6.5to10%.OnlyoneA.flavusisolaterecoveredfromablossom
in2016belongedtoYV36,whilemembersoftheVCGwerenotdetectedcontaminating
Accepted:June3,2018
honeybees.ExposureofpollinatorhoneybeestoAF36wasdetectedtobeverylow.The
Published:June20,2018
densityofseveralAspergillusspecieswasfoundtoincreaseduringalmondhullsplitand
Copyright:Thisisanopenaccessarticle,freeofall throughoutthefinalstagesofmaturation;thisalsooccurredinpistachioorchardsduringthe
copyright,andmaybefreelyreproduced,
maturationperiod.Additionally,wefoundthatAF36effectivelylimitedalmondaflatoxincon-
distributed,transmitted,modified,builtupon,or
otherwiseusedbyanyoneforanylawfulpurpose. taminationinlaboratoryassays.Thisstudyprovidesknowledgeandunderstandingofthe
TheworkismadeavailableundertheCreative seasonaldynamicsofAspergillusfungiandwillhelpdesignaflatoxinmanagementstrate-
CommonsCC0publicdomaindedication.
giesforalmond.TheevidenceofthelowlevelsofVCGYV36encounteredonalmond
DataAvailabilityStatement:Allfilesareavailable blossomsandbeesduringpollinationandAF36’seffectivenessinlimitingaflatoxincontami-
fromthepublicrepositorywebsiteOpenScience
nationinalmondprovidedadditionalsupportfortheregistrationofAF36withUSEPAtouse
Framework,DOI10.17605/OSF.IO/MBGD5.
inalmondinCalifornia.
Funding:ThisworkwassupportedbytheAlmond
BoardofCalifornia(15-AFLA1-Michailidesand16-
AFLA1-Michailides(TJM);ConsejoNacionalde
CienciayTecnologia-Mexico,Estancia Introduction
postdoctoralenelextranjero,contractnumber
InCalifornia,almondsaregrownonoveramillionandaquarteracresproducingover80%of
237422(AOB);andEuropeanUnion’sH2020,
MarieSkłodowskaCuriefellowship,contract theannualglobalalmondproduction[1].AlmondisconsideredasthetopUSagricultural
PLOSONE|https://doi.org/10.1371/journal.pone.0199127 June20,2018 1/15
Fungiinfectingalmondthroughoutcropdevelopment
number658579(JM).Thefundershadnorolein exportproductdestinedtonutconsumption[2].Thecropanditsassociatedindustriesand
studydesign,datacollectionandanalysis,decision by-products(almondmilk,almondbutter,etc.)areofprimaryimportancetotheUSeconomy
topublish,orpreparationofthemanuscript.In
generatingover21billionUSDofgrossrevenue[3].
addition,noneoftheauthorsareemployed,have
Farmersareabletoproduceprimequalityalmonds,inpart,throughsuccessfulcontrolof
relationshipasconsultants,orareworkingin
almondpathogens,particularlyfungi[4].Unfortunately,therearenoeffectivestrategiesto
patents,productdevelopmentnormarketingof
productsfortheAlmondBoardofCalifornia. controlcertainpathogens,suchasaflatoxin-producingfungi,whichcanoccasionallyinfect
andcontaminatealmondnutswithaflatoxins.Aflatoxins,producedprimarilybyAspergillus
Competinginterests:Havingfundingsupportfrom
theAlmondBoardofCaliforniadoesnotalterour flavusandA.parasiticus,aremycotoxinsthatcontaminateseveraleconomicallyimportant
adherencetoPLOSONEpoliciesonsharingdata cropsinCalifornia,includingalmond,fig,andpistachio[5,6].Therearefourmajoraflatoxins:
andmaterials. B ,B ,G ,andG .BothA.flavusandA.parasiticusarecapabletoproduceBaflatoxinswhile
1 2 1 2
fromthesetwospeciesonlyA.parasiticuscanproduceGaflatoxins[4].Humansandanimals
arepronetochronicandacutedetrimentalhealtheffects,includingdeath,duetoconsumption
ofaflatoxin-contaminatedcrops[7–10].Commoditiesthatexceedaflatoxinthresholdsas
determinedbygovernmentalregulationscannotenterdomesticandinternationalmarkets.
Rejectionofcommoditiescancausesevereeconomiclossestogrowers,packers,anddistribu-
tors[11,12].
AflatoxincontaminationinCalifornianalmondswasaminorconcernuntilrelatively
recently.Increasedalmondinfestationbythenavelorangeworm(Amyeloistransitella)insect
pestandseveredroughtthroughoutCaliforniafavoredbothcropinfectionbyaflatoxin-pro-
ducingfungiandsubsequentaflatoxinformation[5,13,14].Theriskofalmondsbecoming
contaminatedwithaflatoxinsisnowaconstantandseriousconcern.
Useofseveralpre-andpost-harvesttechnologiesmaylimitalmondaflatoxincontamina-
tion;theseincludeagriculturalpractices,useofinsect-resistantvarieties,insectcontrol,and
sorting,amongothers[15,16].However,thesetechnologiesmaynotbesufficienttoresultin
renderingalmondssafefromaflatoxins.Atechnologynotyetexploitedinalmondproduction
istheuseofatoxigenic(non-toxinproducing)strainsofA.flavusasbiocontrolagentstoout-
compete(displace)thetoxigenicAspergillusfungi.Aflatoxinbiocontrolisasafe,commercially
proventechnologythatsuccessfullyreducesaflatoxinaccumulationbefore,during,andafter
harvestinallcropswhereitisused[6,17–19].Twoaflatoxinbiocontrolproductsareregis-
teredbytheUnitedStatesEnvironmentalProtectionAgency(USEPA)intheUSandoneof
them,AspergillusflavusAF36,isusedincotton,maize,andpistachiogrowninArizona,Cali-
fornia,NewMexico,andTexas[6,18];theotherbiocontrolproduct,afla-guard1,isusedin
maizeandgroundnutinseveralUSstates[20].In2015theUSEPAgrantedCalifornia’sfig
industryanemergencyexemption(Section18)toutilizeAF36becausehighaflatoxinlevels
wereexpectedasaresultofdroughtconditions.TheCaliforniaFigAdvisoryBoardrequested
aSection18undertheclauseoftheFederalInsecticide,Fungicide,andRodenticideActfor
usein2016,butitwasnotapprovedinasecond-timerequest.However,boththeAlmond
BoardofCaliforniaandtheCaliforniaFigAdvisoryBoardsubmittedapetitionforthefullreg-
istrationofAF36(newformulationAF36Prevail)foruseinthosetwocrops.InMarch2017
USEPAapprovedtheregistrationofAF36Prevailforuseinbothalmondandfigs[21]and
presentlyitispendingapprovalbytheCaliforniaDepartmentofPesticideRegulations.
ThepistachiosproducedintheUSarerenownedworldwidefortheirexcellentquality
whichincludessafeaflatoxinlevelsthatareprimarilyachievedthroughapplicationsofAF36
[6].ThisencouragedthealmondindustrytopursueAF36’sregistrationwithUSEPAtotreat
thealmondcrop.UseofAF36inalmondisexpectedtodecreasetheaflatoxincontamination
ofalmondinasimilarwayasinpistachio.
OurlaboratoryhasinvestigatedwhetherAF36isappropriateforuseinalmond.Initialstud-
iesrevealedthatmembersofthevegetativecompatibilitygroup(VCG)towhichAF36belongs,
YV36,arealsoendemictoallalmond-growingregionsofCalifornia(range=3.5to12.6%of
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Fungiinfectingalmondthroughoutcropdevelopment
recoveredA.flavusisolates)[22].ThenaturalpresenceofmembersofYV36inalmond
orchardsisparticularlyimportantbecauseourlaboratoryembracestheideathatatoxigenic
strainsusedinbiopesticideformulationsshouldbebothnativetotheregionwherethebiocon-
troltreatmentwillbeappliedandsuperiorly-adaptedtothetargetcrop.Furthermore,useof
nativestrainsdiscardsenvironmentalconcernsposedbytheuseofexoticfungi[23–25].
ExperimentalreleaseofAF36inalmondresearchplotsrevealedthatuseofAF36doesnot
increaseeitherkernelrotnorAspergilluspropaguledensitywithinandoutsideoftheorchard
[22].Amulti-yearstudyrevealedthatdensitiesofAspergillussporesinAF36-treatedandnon-
treatedcommercialpistachioorchardsremainthesamethroughouttheyear,withmaxima
peaksduringtheharvestperiodandlowlevelsduringtherestoftheyear[26].Aspergillusden-
sitiesinAF36-treatedalmondorchardsthroughouttheyearareexpectedtobethesameasin
theabsenceoftreatment.Ontheotherhand,theabilityofAF36toreduceaflatoxincontami-
nationinalmondlaboratoryassaysneedstobeinvestigatedtodeterminewhetherAF36suc-
cessfullylimitsaflatoxinformationonalmondnutswhenchallengedwithhighlytoxigenic
Aspergillusstrains.
EventhoughusingtheAF36biopesticidewouldimprovealmondquality,deliberaterelease
ofanA.flavusstraininalmondorchardshasraisedconcernstocertainprivate,public,and
academicsectorsdespiteplentifulpublicinformationonthesafetyofthisUSEPA-approved
biologicalcontrolagent[27,28].Honeybee(ApismelliferaL.)populationsacrosstheUShave
drasticallydecreasedduringthelastdecade[29].Honeybeesarecritically-neededforpollina-
tionofmanycrops,includingalmond,andgrowersplacebeehivesattheborderoftheir
orchards,inaratiooftwohivesperacre.ThereisthenotionthattheuseofAF36mayaccentu-
atehoneybeepopulationdecline.ConcernsstemprimarilybecauseA.flavuscausesstone-
broodandaspergillosisofhoneybeelarvaeandadults,respectively[30].However,neitherA.
flavusnorthegenusAspergillushavebeenimplicatedasmajorpathogensofhoneybeespolli-
natingalmondsinCalifornia[31,32].
VirtuallyallalmondcropsinCaliforniaarepollinatedbycommercially-managedhoney
beecolonieswhichremainintheorchardsonlyduringthe3–4weekblossomperiod(lateFeb-
ruary–earlyMarch)beforemovingtootherlocationswheretheirservicesareneeded[31];
mostofthebeehivesaremovedtootherstateswhilethefewbeehivesremaininginCalifornia
willbemovedtoforageinothercropssinceflowersinandaroundalmondorchardsarescarce
afterthepollinationperiod.Aspergillusdensitiesdonotbecomealteredafterapplicationof
AF36incommercialfieldsofmaize,cottonseed,pistachio,orexperimentalalmondplots[6,
22,23,26].Itcanthusbeexpectedthat,afterAF36applicationsonalmondorchards,honey
beesbroughtinthenextseasontopollinatethealmondcropwillnotbecomeexposedtoA.fla-
vuspropagulesinadifferentmannerasintheabsenceoftreatment.Nevertheless,influences
ofAF36’sreleaseduringcropdevelopmentincontributingtobothalmondblossominfection
andhoneybeecontaminationinthefollowingseasonhavenotbeenformallyinvestigated.
TheobjectivesofthisstudyweretoexamineA.flavuscommunities,particularlyofYV36,
throughoutthealmondcroppingseasoninorderto(i)determinefrequenciesofYV36in
almondblossoms,(ii)assessfrequenciesofYV36inhoneybeescollectedwhilepollinating
almondblossoms,(iii)determinetheoptimalperiodforapplicationofAF36inalmond
orchardsbasedontheperiodinwhichAspergillusspp.increaseindensityinalmondorchards,
and(iv)reportAF36abilitiesinlimitingaflatoxincontaminationinlaboratorycompetition
experimentswithhighlytoxigenicAspergillusstrains.Resultsfromthisstudyprovidedsup-
portiveinformationforregistrationofthebiopesticideAF36withtheUSEPAforusein
almond.Nowthatitisregistered,theuseofAF36byalmondgrowerswillsignificantlyreduce
aflatoxin-contaminationofalmondsandthiswillallowtomeetdomesticandinternational
standardswithalowcost,commercially-provenandenvironmentally-safetechnology.
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Fungiinfectingalmondthroughoutcropdevelopment
Materialsandmethods
Almondorchard
AnalmondresearchplotlocatedattheUniversityofCaliforniaKearneyAgriculturalResearch
andExtensionCenterinParlier,California,wasusedforthesestudies.Theexperimentalplot
wasplantedwiththecultivarsButte,Carmel,Nonpareil,andPadreinJanuary2006.The
experimentaldesignoftheplotis12randomizedrow-blockswith12almondtreesperrow-
block.Ineachrow-block,threealmondtreesofthesamecultivarareplantedtogether.
ExperimentalreleaseofAspergillusstrains,bothatoxigenic(i.e.AF36)andtoxigenic(vari-
ousA.flavusandA.parasiticusstrains),occursonayearlybasisinthisplot,includinginboth
2014and2015(theimmediatepreviousyearsinwhichthedifferentsamplesevaluatedinthe
currentstudywerecollected),aspartofdifferentexperimentsthatincludeevaluationofatoxi-
genicstrainstodecreaseaflatoxincontent[22],determiningperiodofalmondsusceptibilityto
aflatoxincontamination[33],orassessinginfluencesofbothfungicidesandinsecticidesin
decreasingaflatoxincontent,amongothers.
Fungalcommunitiesofalmondblossoms
Fungalcommunitiesofblossomsofthefouralmondcultivarswereexaminedinboth2015
and2016toinvestigatefrequenciesofA.flavus,particularlybelongingtotheVCGYV36.Four
treespercultivarwererandomlyselectedinfourdifferentrowsand30blossomspertreewere
collectedandplacedintopaperbags.Inbothyears,30blossomspertreewerecollectedtwice
duringthethirdweekofFebruaryandthesecondweekofMarch.Immediatelyaftercollection,
blossomswereplacedinsterileplasticscreensinsidesterileplasticcontainersusedashumid
chambers.Sterilewater(300ml)wasaddedtoeachcontainertomaintainhighhumidity
(approx.100%).Containerswereincubatedat31˚Cfor7days.Fungifoundgrowinginthe
examinedblossomswereassignedtotheircorrespondingspeciesbasedonmacroscopicand
microscopiccharacteristics[34,35].AllAspergillusspeciesisolatesweresub-culturedon5–2
agar[5%V8juice(CampbellSoupCompany,Camden,NJ),2%Bacto-agar,pH6.0)[36]]for7
days(31˚C).Isolatesweresavedasagarplugsofsporulatingculturesin8mlvialscontaining4
mlsteriledistilledwateruntilfurthercharacterization.
Fungicontaminatinghoneybees
FrequenciesofA.flavus,particularlyoftheVCGYV36,contaminatinghoneybeeswere
investigatedbycollectinghoneybeesattendingblossomsofthealmondtreesmentionedabove
inboth2015and2016.Ineachyear,120honeybeeswerecollectedrandomlybyplacingone
beeperaplasticbagduringthefirstweekofMarch.Honeybeeswereimmediatelytransferred
tothelaboratoryandsacrificedbystoringthemat-2˚Cfor24h.Honeybeeswereplated
directlyonmodifiedroseBengalagar[37],incubatedat31˚Cduring3daysinthedark,and
thefungalisolateswereidentified.Aspergillusfungiweresub-culturedandsavedasdescribed
above.
Fungalcommunitiesduringalmonddevelopment
DensitiesofA.flavuswereexaminedingreenalmondfruitsofthefourcultivarsfromAprilto
Augustin2015.Fourtreespercultivarwereusedoneachcollectiondateandthesametrees
weresampledthroughoutthestudy.Fruitswerecollectedonthe1stand15thdayofeach
monthfromAprilthroughJuly.InAugust,fruitswerecollectedonthe1st,8th,15th,22nd,and
28thday.CollectiondatesinAugustweredoneonaweeklybasisbecauseinpreviousexperi-
mentsitwasnoticedthatAspergillusspp.increaseindensityafterthesecondweekofthis
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Fungiinfectingalmondthroughoutcropdevelopment
month.Foreachtree,duringeachcollectiondate,20randomfruitswerecollectedandplaced
inplasticbags.Then,oneachdate,fiverandomlyselectedfruitswerewashedwith100mlster-
ilewatercontainedinsterile500mlErlenmeyerflasks.Aftercontinuousagitation(10min)in
anEberbach6010reciprocatingshaker(AnnArbor,MI),100μlofthewashwerecombined
with9.9mlofsterilewater.Dilutionswerevortexedand100μlaliquotswereplatedonacidi-
fiedpotatodextroseagar(APDA)[38],sixplatesperdilution.Almostall(>98%)ofthecon-
taminatingfungiwereassignedtotheircorrespondinggenus.
Fungalcommunitiesduringpistachiodevelopment
Becausepistachioandalmondplotsarefrequentlyplantedincloseproximity,fungalcommu-
nitiesassociatedwithpistachiothroughoutthegrowingseasonwereexaminedincommercial
orchardsin2015.PistachiofruitswereperiodicallycollectedfromApril18thtoSeptember8th.
Oneachdate,threesamplesof10fruitclusterswererandomlycollected.Tenrandomly
selectedfruitswerewashedasdescribedabove.Dilutionplatingandincubationwerecon-
ductedasdescribedabove.Most(>95%)ofthecontaminatingfungiwereassignedtotheir
correspondingspeciesbasedonmacroscopicandmicroscopiccharacteristics.
Vegetativecompatibilityanalyses
AllA.flavusisolatesrecoveredfrombothalmondblossomsandhoneybeesweretestedfor
membershipinYV36,theVCGtowhichthebiopesticideAF36belongs.Nitratenon-utilizing
(nit)mutantswereobtainedbyplatingsporesuspensions(approximately1,000sporesin
15μl)ofisolatesonSELagar(Czapek-Doxbroth,25gKClO ,50mgroseBengal,and2%
3
Bacto-agar(DifcoLaboratoriesInc.,Detroit,MI),perliter,pH7.0)into3mmcentralwellscut
intoagar.Plateswereincubatedforuptoonemonth(31˚C).Spontaneousauxotrophicsectors
weretransferredontoMITagar(Czapek-Doxbroth,15gKClO ,and2%Bacto-agarperliter,
3
pH6.5),andincubatedfor3days(31˚C)tostabilizethemutants.Mutantswerethengrown
on5–2agarfor7days(31˚C).VCGmembershipwasdeterminedbyusingpreviouslydes-
cribed[39]testersofVCGYV36,ATCC96045andATCC96047,andfollowingpreviously
describedprotocols[6].Briefly,fungalsuspensions(15μl)ofthetwotestersandthenitmutant
fromtheisolateunderevaluationwereseededindependentlyintooneofthree3mmwells
spaced1cmapartinatriangularpatternonstarchagar(36gdextrose,20gsolublestarch,3g
NaNO ,2%Bacto-agarperliter,pH6.0[40])andincubatedfor7days(31˚C,dark).Forma-
3
tionofprototrophicgrowthinthezoneofhyphalinteractionbetweenanitmutantandeither
ofYV36testersindicatedmembershipinVCGYV36.
Co-inoculationofAF36’sactiveingredientwithtoxigenicAspergillus
isolatesonviablealmondkernels
TheabilityofAF36’sactiveingredientinlimitingaflatoxinaccumulationwhenco-inoculated
independentlywithhighlytoxigenicisolatesofeitherA.flavusorA.parasiticus,isolates2A1L-
11and4C1P-11,respectively,wasinvestigatedincompetitionlaboratoryassays.Bothtoxi-
genicisolatesproducelargequantitiesofaflatoxininchemicallydefinedmedia,aswellasvia-
blealmondkernels(PicotandMichailides,unpublishedresults).Forinoculumpreparation,
isolatesweregrownon5–2agarfor7daysat31˚C.Conidiawerecollectedwithasterilecotton
swabandsuspendedinsteriledeionizedwater.Conidialsuspensionswerequantifiedusinga
hemocytometeranddilutedtoafinalinoculumconcentrationof1.75×106conidia/ml.Sterile
glassvials(20ml)containingapproximately5g(about6to7kernels)ofmature,livingalmond
kernelspreviouslysurface-disinfestedbysubmersioninhotwater(80˚C,45s)wereeitherco-
inoculatedwithacombinationofatoxigenicandAF36isolateorinoculatedwithatoxigenic
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Fungiinfectingalmondthroughoutcropdevelopment
isolatealone.Eachvialwasinoculatedwithafungalsuspensioncontainingapproximately
350,000conidiapergofalmonds,whichwaspreviouslycombinedwiththeappropriate
amountofdistilledwatertobringalmondmoisturecontentto25%.Theinitialalmondmois-
turecontentwas6%.Theadjustedsuspensionswerevortexedanddistributedevenlyonthe
surfaceofalmondkernelsinsidethesterileglassvials.Whenco-inoculationoccurred,equal
conidiaamountsfromthetwoisolateswereused.Almond-containingvialsinoculatedwith
sterilewaterservedasnon-inoculatedcontrols.Afterinoculation,flaskswerecoveredwith
sterileplasticcaps,positionedinacrisperintoarandomizeddesign,andincubatedat31˚Cfor
7days.Aftertheincubationperiod,theexperimentwasterminatedbyadding30mlof60%
methanol.AflatoxinswereextractedfollowingtheofficialanalysismethodoftheAssociation
ofOfficialAnalyticalChemists[41].AflatoxinswerequantifiedusinganHPLCHewlettPack-
ard1050aspreviouslydescribedinotherstudiesbyourlaboratory[6].Fourreplicatesper
treatmentwereused.Eachreplicateconsistedofasingleglassvialcontainingalmonds.The
experimentwasrepeatedtwice.
Statisticalanalysis
DataweresummarizedandanalyzedusingStatistix10(AnalyticalSoftware,Tallahassee,FL).
Theeffectofthecultivaronthepercentageofisolationofeachfungalspeciesorthedifferences
onisolationspercentageamongfungalspecieswereevaluatedusingZar’stestofmultiplecom-
parisonsofproportions[42].Non-parametricFriedman’stestwasusedtostudythepopula-
tionoffungalspeciesfromalmondfruits.Afterthat,differencesamongalmondcultivarswere
determinedusingDunn’stestwithaBonferroniadjustmentatP=0.05.
Speciesdiversityforeachalmondcultivaroneachcollectiondatewascalculatedbasedon
P
Shannon-Weinerdiversityindex(H)withPivalues:H ¼(cid:0) S Pi(cid:3)lnPiwherePiisthepro-
i¼1
portionfortheithspeciesandSisthetotalnumberofspeciesinthepopulation(speciesrich-
ness).Equitability(J)wascalculatedas: H.Jassumesvaluesbetween0and1.ThecloserJgets
lnS
to1,themoreequitableisthepopulation[43].
TheWelch’st-test,orunequalvariancest-test,wasusedtocompareaflatoxinconcentra-
tionsbetweenalmondkernelsinoculatedwiththetoxigenicisolatesaloneandalmondkernels
co-inoculatedwithbothtoxigenicandatoxigenicisolates.
AflatoxinreductionsbyAF36werecalculatedas[1–aflatoxincontentinalmondsco-inocu-
latedwithatoxigenicisolateandAF36isolate/aflatoxincontentinalmondsinoculatedbythe
toxigenicisolatealone]x100asdescribedbyProbstandco-workers[24].
Results
Blossoms
Ineachyear,resultsofthetwocollectiondateswererelativelysimilar(Df=1;Chi-Square=
0.1230;P=0.7257)andwerecombinedforanalyses.Intotal,900blossomswereexamined
duringbothyears.Overall,Alternariaspp.wasthemostcommonlyidentifiedgroup(77.8%),
followedbyA.flavus(2.1%),Fusariumspp.(2.0%),A.niger(1.5%),andPenicilliumspp.
(0.2%);therewasnofungalgrowthin16.4%ofblossoms.ThepopulationofAlternariaspecies
wassignificantlylower(Df=3;OverallChi-Square=33.3;P=0.0001)inblossomsofthe
cultivarsPadreandNonpareilthanintheblossomsoftheothercultivars.Furthermore,the
cultivarButtehadhigher(Df=3;OverallChi-Square=15.75;P=0.0013)frequenciesofA.
niger.FrequenciesofA.flavus,Fusariumspp.,andPenicilliumspp.weresimilar(Df=3;Chi-
Square<7.8147;Pvalues>0.05)amongallthecultivars.Noteworthy,in2015noneofthe
examinedblossomsharboredA.flavus,oranyotheraflatoxin-producingspecies.In2016,
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Fungiinfectingalmondthroughoutcropdevelopment
4.0%ofblossomswerecontaminatedwithA.flavus.Vegetativecompatibilityanalysesrevealed
thatonlyoneoutof18A.flavusisolatesassociatedwiththeblossomsbelongtoVCGYV36.
Honeybees
Resultsofthetwohoneybeecollections,oneperyear,wererelativelysimilarandcombined
foranalyses.Alternariaspecieswasthemostcommonlyidentifiedgroup(21.4%),followedby
Rhizopusspp.(20.8%),A.niger(20.0%),Fusariumspp.(19.4%),A.flavus(8.5%),Penicillium
spp.(5.3%);A.fumigatus(2.4%),Trichodermaspp.(1.2%),andBotrytisspp.(1.2%).Overall,
foralldominantgenera(i.e.,Alternaria,Rhizopus,Aspergillus,andPenicillium)therewereno
significantdifferences(Df=2;Chi-square<5.9915,Pvalues>0.05)infrequenciesbetween
thetwoexaminedperiods.VCGtestingrevealedthatnoneoftherecoveredA.flavusisolates
belongtoVCGYV36.
Developingalmonds
Duringthecourseofthestudyover45,000fungalisolatesbelongingto17generawereenu-
merated.ThemostcommonlyidentifiedgenerawereAcremonium(52.9%),Cladosporium
(5.4%),Aureobasidium (4.1%),Aspergillus(3.1%),Fusarium(1.0%),Alternaria(0.3%),Penicil-
lium(0.3%),anddifferentspeciesofyeasts(32.5%).TheBotryosphaeriaceaespeciesandspe-
ciesbelongingtothegeneraBotrytis,Coniothyrium,Epicoccum,Colletotrichum,Ophiostoma,
Phomopsis,Rhizopus,andTrichodermaweredetectedatfrequenciesof(cid:20)0.2%.Only1.1%of
therecoveredfungiwerenotassignedtoacorrespondingspecies,mainlybecausetheydidnot
produceanysporesinculturemedia.RelativelylowdensitiesofthegenusAspergillus(0to
6CFU/g),themainsubjectofthecurrentstudy,weredetecteduntilAugust8th(Fig1).Densi-
tiesofAspergillusspp.increasedlinearlyovertimeduringAugust(Y=284.5X–237.9;R2=
0.9516;P=0.0008).Significantly(Friedman’stest;F=17.24;Df=3.3;P=0.0001)moreAsper-
gillusspp.fungiwererecoveredfromNonpareilcultivarwhileButteandPadrecultivars
Fig1.FungaldensitiesofAspergillusspp.inalmondandpistachioorchardsattheKearneyAgriculturalResearch
andExtensionCenterinParlier,CA.Inbothcrops,Aspergillusspp.fungiincreaseafterthesecondweekofAugust.
DevelopingalmondfruitswerenotexaminedinSeptember8th.
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Fungiinfectingalmondthroughoutcropdevelopment
harboredsignificantly(Dunn’stest;P>0.05)lessAspergilluspropagules.Carmelformedan
intermediategroup(Dunn’stest;P<0.05)betweentheothertwoaccordingtothepercentage
ofAspergillusisolation.ThistestalsoshowedthattheAspergillusinoculumincreasedacross
theseasonwithmaximapeaksduringAugust.
TheoverallShannon-Weinerdiversityindiceswere0.857,0.908,0.925,and1.050forPadre,
Carmel,Butte,andNonpareilcultivars,respectively.Diversityindicesfluctuatedamongthe
examinedcultivarsacrossthegrowingseasonbutnoneofthecultivarsexhibitedconsistently
lowerorhigherdiversityonanygivendate(Fig2).Overall,fungaldiversitywashighestfrom
July1stto15thforallstudiedcultivarsexceptforButte,inwhichthehighestvaluewasdetected
onJune1st.
Developingpistachios
Over50,000isolateswereenumeratedduringthecourseofthestudy.ThegeneraAureobasi-
dium(25.4%),Cladosporium(24.6%),Alternaria(19.2%),Penicillium(7.6%),Aspergillus
(2.3%),Botrytis(1.7%)andblackandwhiteyeasts(15.2%)wererecoveredatrelativelyhighfre-
quenciesintheexaminedpistachiofruitsduringcertainperiodsorthroughoutthewholesea-
son.OtherlessfrequentlyrecoveredgeneraincludedEpicoccum,Fusarium,Mucor,Rhizopus,
andPaecilomyceswithlessthan1%ofthepopulation.Throughoutthecourseofthestudy,
densitiesofAlternariaandyeastschangedmoderatelywhiledensitiesofBotrytis,Penicillium,
andAspergilluschangedconsiderably.PropagulesofAspergillusspp.increasedtosignificant
proportionsduringthefirstweekofAugust(average200CFU/g)andcontinuedtoincrease
throughoutthatmonth(average1,500CFU/g;Fig1).
Fig2.Shannon-Weinerdiversityindicesoffungalspeciesdetectedinfouralmondcultivarsduringthecourseofthestudyinanexperimentalorchardatthe
KearneyAgriculturalResearchandExtensionCenterinParlier,CA.
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Fungiinfectingalmondthroughoutcropdevelopment
AbilityofAF36toreduceaflatoxincontaminationwhenchallengedwith
toxigenicstrainsonalmondsubstrate
Almondsco-inoculatedwithAF36andhighlytoxigenicstrainsofeitherA.flavusorA.parasi-
ticushad>95%lessaflatoxinsthanalmondsinoculatedwithtoxigenicstrainsalone(Fig3).
AF36washighlyefficientinreducingbothaflatoxinB (bybothA.flavusandA.parasiticus)
1
andG (byA.parasiticus).
1
Discussion
ThisstudydescribesfrequenciesofthefungusA.flavusthroughoutthealmondgrowingsea-
son.Inaddition,weinvestigatedfrequenciesofmembersofvegetativecompatibilitygroup
(VCG)YV36inalmondblossomsandhoneybeespollinatingtheblossomsofthiscrop.YV36
istheVCGtowhichtheaflatoxinbiocontrolagentAspergillusflavusAF36belongs[44].Thisis
thefirststudyinwhichfrequenciesofanA.flavusgenotypearemonitoredthroughoutthis
criticalperiodforalmondproduction.Finally,theabilityofAF36’sactiveingredienttolimit
aflatoxincontaminationinalmondsubstratewasinvestigated.Ourresultsindicatethatfre-
quenciesofYV36,andA.flavusingeneral,arelowandinfrequentduringalmondblossoming-
pollinationperiodandthatdensitiesofAspergillusspp.remainrelativelylowindeveloping
almondfruitsuntilthelatterstagesofnutmaturation,inAugust.Inaddition,AF36wasfound
toeffectivelylimitaflatoxincontaminationwhenchallengedwithhighlytoxigenicAspergillus
strainsduringlaboratorycompetitionassaysinalmondkernels(Fig3).
Fig3.AbilityofAspergillusflavusAF36’sactiveingredienttoreduceaflatoxinaccumulationinviablealmonds
whenco-inoculatedwithtoxigenicA.flavus2A1L-11orA.parasiticus4C1P-11isolates.Bothtoxigenicisolatesare
nativetoCaliforniaalmondagroecosystemandproducelargeaflatoxinquantitiesinseveralsubstrates.Asterisks
indicatesignificantdifferences(Welch’st-test;α=0.05)inaflatoxinconcentrationsbetweentoxigenicisolates
inoculatedaloneandthatisolateco-inoculatedwithAF36.Almondfermentationsco-inoculatedwithAF36
accumulatedover95%lessaflatoxinsincomparisontoalmondsinoculatedwithatoxigenicisolatealone.
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Fungiinfectingalmondthroughoutcropdevelopment
ThespeciesA.flavusisapathogenofplants,animals,andinsects[45].Itsroleasaplant
pathogenhasreceivedconsiderableattentionbecauseitcontaminatesawiderangeofcrops
withaflatoxins,whichposeserioushealththreatstobothhumansandanimalsevenatverylow
concentrations[10].Aflatoxincontaminationofsusceptiblecropsiscommoninregionswith
tropicalandsubtropicalclimates[46].Inaddition,susceptiblecropsthatareirrigatedcanbe
subjectedtohotandhumidenvironments,favoringaflatoxincontamination.Furthermore,
duringthelastdecade,susceptiblecropshavesufferedfromaflatoxincontaminationeventsin
areastraditionallyfreeofcontaminationbecauseofdroughtconditionsdrivenbyclimate
changewhichpredisposecropstoinfectionbyaflatoxigenicfungi[47,48].
Almond,acropofgreateconomicimportanceforbothCaliforniaandtheUS,poses
increasedriskofaflatoxincontaminationduetorecentdroughtconditions[5,49].Inthepast,
theonlyeffectivepreventivemeasuretolimitaflatoxincontaminationinalmondwastocontrol
thenavelorangeworm,whichcreateswoundsforinfectionwhenlarvaeboreintothenutmeat
andcarriespropagulesofaflatoxin-producingspecies.Forothercommercialcropsaveryeffec-
tiveapproachtoreduceaflatoxincontaminationistheuseofatoxigenicA.flavusstrainsasbio-
controlagentstocompetitivelydisplaceaflatoxin-producersduringcropdevelopment[6,18].
ThebiocontrolagentAspergillusflavusAF36isoneofthetwoatoxigenicbiopesticides
registeredwiththeUSEPAthatuntilearly2017,wasapprovedforuseincotton,maize,and
pistachio.ThesecropscanbetreatedyearlyatlabelratesperhectarewithAF36inArizona,
California,NewMexico,andTexas[27,28].Fortunately,thisbiopesticidewasapprovedby
USEPAinMarch2017foruseinalmondandfigcrops[21].Theactiveingredientfungusof
AF36belongstoVCGYV36.ThisVCGishighlyassociatedwithalmondthroughoutCalifor-
nia[22].ThehighnumberofrejectedalmondloadsexportedtoEuropeduetoexceedingafla-
toxintolerancelevels(particularlyin2007),ahighassociationofYV36withalmond,and
successfulaflatoxinreductionsbyAF36inothercropssparkedaninterestinthealmond
industrytouseAF36toreduceaflatoxincontaminationofalmonds.InordertoobtainUSEPA
approvaltouseAF36inalmond,severalrequirementsneededtobeaddressedandsatisfied,
includingprovidingevidencethattheuseofAF36inalmondposesnothreattothecritically
endangeredhoneybeepopulations.Certainacademicandgovernmentalsectorsfearedthat
fieldreleaseofanorganismwithpotentialtonegativelyaffecthoneybeeswillaccentuate
honeybeedecline[29,30]inCalifornia,whereseveralcrops,includingalmond,relyonhoney
beesforpollinationOntheotherhand,A.flavusisnotamajorpathogenofhoneybeespolli-
natingalmondsinCalifornia[31,32].TheapplicationofAspergillusflavusAF36inthefieldis
plannedforlateJuneorduringJuly,longafterthepollinationofalmondandtheflightsof
honeybeesinalmondorchardshavebeencompleted.ByJune–July,mostcommercially-man-
agedhoneybees(whichvirtuallypollinateallalmondcropsinCalifornia)havebeentrans-
portedtootherstatestoprovidetheirpollinationservicestoothercrops[31].
Only2.1%ofexaminedblossomsofthefouralmondcultivarswerecontaminatedwithA.
flavus.Thisoccurredregardlessofcontinuousexperimentalreleaseofbothatoxigenicand
toxigenicAspergillusfungiduringpreviouscroppingseasons[22,33]andrelativelyhighnatu-
ralAspergillusdensitiesthroughoutCalifornia’sCentralValley[5,6,16].Low(<3%)frequen-
ciesofAspergillusspp.inalmondblossomswerepreviouslydetectedinseveralunrelated
studiesinwhichthousandsofblossomswerescreenedforfungalcontaminants(Michailides,
unpublishedresults).ResultsfrompreviousandthecurrentstudysuggestthatAspergillusspp.
remainpredominantlyinactiveduringalmondblossomingduetothelowtemperaturespreva-
lentduringthatperiod(lateFebruaryandduringMarchinthenorthernhemisphere).Fur-
thermore,A.flavuswasdetectedonlyinblossomscollectedin2016withonlyoneisolate
belongingtoVCGYV36.Also,wedidnotobservesporulatingAspergillusspp.onblossoms
underthefieldconditions.
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