Table Of ContentEVALUATIONOFTHEPARASITICRELATIONSHIPOFMELANOSPORAAND
OTHERALLIEDGENERAWITHFUSARIUMOXYSPORUM
By
ROBERTMARTINHARVESON
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOLOFTHE
UNIVERSITYOFFLORIDAINPARTIALFULFILLMENTOFTHE
REQUIREMENTSFORTHEDEGREEOFDOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
1999
ACKNOWLEDGMENTS
IwouldfirstliketoextendmythankstoDr.G.N.Agriosandthecommitteein
chargeofawardingplantpathologydepartmentalassistantships.Withoutthispossibility,I
wouldnothavebeenabletocometoFloridatocompletemyeducation.Most
importantly,Iwanttothankmymajorprofessor,Dr.JimKimbroughforlisteningtomy
unorthodoxresearchideasandbeingwillingtosupportthemataverydifficulttimeinmy
career.Hisconstantsenseofhumor,enthusiasm,andguidancewillalwaysbeappreciated
andneverforgotten.Iwouldalsoliketothankmyothercommitteemembers-Dr.Don
Hopkins,Dr.GarySimone,Dr.AndyOgram,andDr.BillZettler-foragreeingtoserveon
mycommitteeatthelastminuteandtheirinputthroughoutthecourseofthisproject.Dr.
Hopkinsalsoagreedtosupportperipheralareasrelatedtothisworkbyfurnishinghis
researchplotsandlaboratoryinLeesburg.Unfortunately,noneofthisresearchwill
appearinthisdissertation.Dr.Simonewasinstrumentalinalsosharinghislabfacilities
andbeingtheretolistentomeventandadvisemewhenmycourseofstudychanged.Dr.
Ogramhasbeenhelpfulinfindingflawsinthiswork,whichsubsequentlyledtoan
improvedfinalproduct.Dr.Zettler'scontributionisparticularlyimportant.Heservedas
proxyforboththeoralexamandthefinaldefense,hencehehasbecomeaformal
committeememberinliterallythetwelfthhour.Withouttheencouragementand
assistancefromDr.GerryBennyandUllaBennyoverthelastthreeyears,thisprojectand
mysanitywouldprobablynotbewheretheyarenow.IwanttoalsothankDrs.Dave
Mitchell,DanPurcifiill,andTomKucharekforadviceandacademicdiscussionsoverthe
years.TheinfluenceandmentoringabilityofDr.CharlieRushonmycareeris
incalculable.Iowehimadebtofgratitudethatcanneverfullybeexpressedorrepaid.I
amalsogratefultoDr.CorbyKistlerforsosuccinctlyillustratingtomethatmyultimate
futureleddownadifferentpaththanIfirstbeganwhenstartingmydoctoralwork.Iwant
toalsoexpressmyappreciationtoGalenJonesandJackBishkofortheirconsultationand
recommendationsforthevariousstatisticalanalysesusedinthisstudy.Ineedtoalso
acknowledgemyparents,WayneandMaralynHarveson,andmygrandmother,Marietta
Martin,foralwaysbelievinginmeandgivingunconditionalsupportandencouragement
foranyendeavorthatIhavechosentoundertake.Finally,Iwishtothankmywife,
Tammy,forunderstandingmysometimesunreasonabledrivetocompletethisworkand
forreluctantlytoleratingthe12-14hourworkdaysspentoverthelastyearandahalf.
Herwillingnesstoconsidermovingtoanewandtotallyforeignlocationhasbeena
tremendouscomforttomewhilefinishingmywork.Thecompletionofthisprojectwould
nothavebeenpossiblewithouther.
TABLEOFCONTENTS
ACKNOWLEDGMENTS u
ABSTRACT vi
CHAPTERS
1 INTRODUCTION 1
2 ATAXONOMICSTUDYOFMELANOSPORAANDITSALLIES 17
17
22
25
Discussion 38
3 ENHANCEMENTOFGROWTHANDSPORULATIONOF
MELANOSPORAANDITSALLIESBYFUSARIUM 40
OXYSPORUM
Introduction 40
MaterialsandMethods 43
Results 51
Discussion 63
4 PARASITISMANDMEASUREMENTOFDAMAGETOFUSARIUM
OXYSPORUMBYSPECIESOFMELANSOPORA,
SPHAERODESANDPERSICIOSPORA 70
Introduction 70
MaterialsandMethods 72
Results 79
Discussion 90
5 ECOLOGYANDUSEOFSPHAERODESRETISPORAVAR.
RETISPORAFORTHECONTROLOFFUSARIUMWILT
OFWATERMELON 93
Introduction 93
MaterialsandMethods 95
Results 100
Discussion 110
6 SUMMARYANDCONCLUSIONS 116
REFERENCES 120
BIOGRAPfflCALSKETCH 129
AbstractofDissertationPresentedtotheGraduateSchooloftheUniversityofFlorida
inPartialFulfillmentoftheRequirementsfortheDegreeofDoctorofPhilosophy
EVALUATIONOFTHEPARASITICRELATIONSHIPOFMELANOSPORAAND
OTHERALLIEDGENERAWITHFUSARIUMOXYSPORUM
By
RobertMartinHarveson
August1999
Chairman: J.W.Kimbrough
MajorDepartment: PlantPathology
Fusariumwilt,causedbyFusariumoxysporum,hasbeenaproblematicdiseasefor
manydifferentcropsforover100years.Thediscoveryofagroupofpyrenomycetous
ascomycetesfoundnaturallyinassociationwithseveralformaespecialesofF.oxysporum
fromfieldsamples,hasgeneratedtheinteresttoinvestigatetheuseofthesefungias
biocontrolagentsforwatermelonwiltcausedbyF.oxysporumfsp.niveum(FON).The
firstofthesefungiwasisolatedfromthesugarbeetpathogen,F.oxysporumfsp.betae,
andwasidentifiedasMelanosporazamiae.Laterfindingsfromotherwilt-infectedcrops
yieldedanotherfiveisolatesthatverycloselyresembledtheoriginalM.zamiae.However,
baseduponcurrenttaxonomiccriteria,thesefiveisolatesweredeterminedtoconsistof
twodistinctlydifferentgenera,Sphaerodesrelisporavar.retispora,andPersiciospora
moreaui.
AllsixisolateswerefoundtobestronglydependentuponF.oxysporumfor
growthandsporulation.Lineargrowthandproductionofperitheciaweresignificantly
improvedforallisolatesindualculturewithF.oxysporumorgrowinginfiltratefroma
liquidcultureofFusarium.Significantdifferencesingrowthwereobservedamongand
betweenthedifferentpyrenomycetes.WithoutthepresenceofF.oxysporumorits
metabolicgrowthproducts,growthandreproductionweregenerallyverypoorforall
isolates.ThebettermediawithoutFusariumtendedtobethosecomposedofsimple
sugarslikedextroseormaltose,whilethoseproducingtheworstresultscontainedstarch
orcellulose.AllisolatesweredeterminedtobeparasiticonF.oxysporumthroughtheuse
ofseveralculturalassays,butnotallwerepathogenictoFON. Persiciosporacausedno
detectablereductioninhyphalgrowth,whereastheSphaerodesisolateswerethemost
harmful.
IsolateLE{S.relispora)wasselectedfordiseasereductionevaluationsinthe
greenhouse.Itwasincorporatedintosodiumalginatepelletsanddeliveredtotheinfection
courtbyeitherroottreatmentoramendmenttoinfestedsoilsbeforetransplantation.Yield
datawerecollectedaftereightweeks,andconsistedofmortalitycountsandtotalplantdry
weights.Thistechniquedidsignificantlyimproveplantdryweightsoftreatedplants
comparedtotheFON-inoculatedcontrols.Thesearepromisingresultsforanalternative
methodformanagingFusariumwiltofwatermelon.However,itisnotknownwhether
thisprocedureisefficaciousorevenfeasibleunderfieldconditions.Ifproventobe
successfulinthefield,thisprocedurecouldalsopresumablybeutilizedfordiseasecontrol
withothervegetablecropsinFloridathataresimilarlygrownastransplants.However,
caremustbetakenfortheselectionofthepotentialbiocontrolisolates,becausenotall
includedinthisstudywerefoundtoequallycolonizeorinducedeleteriouseffectsuponall
formaespecialesofF.oxysporum.
vii
INCTHRAOPDTUECRTI1ON
ThegenusFusariumwasfirsterectedin1809byLinkwhenhedescribedF.
roseumasthefirstspecies(Booth1984).Fusariumspeciesareubiquitoussoilfungi
foundinmanyclimaticextremessuchasdesertsandthearctic,inadditiontotemperate
andtropicalregionsoftheworld.Theycanbesaprophytesandpathogensinfectingboth
animalsandplants(Nelson1981).Becauseofvariationinculture,ithasbeendifficultfor
workerstocorrectlyclassifythisgenusintospecies.ThebasisforFusariumclassification
wasestablishedbyAppelandWollenwebber(1910).Theyplacedspeciesstudiedinto
sectionsbasedonvariousmorphologicalcharacteristics(Coons1928).Thesesections
werelaternamedandexpandedtoincludeatypespeciesforeachsection(Wollenwebber
1913).
Ofthe43speciesofFusariumlistedbyBooth(1971),27arepathogenictoplants
andcausesomeofthemostimportantdiseasesinagriculture.Thepathogensmaybe
dividedintothreemaincategories:thewilts,therootrotscausedprimarilybyF.solani,
andthosefungiattackinggraminaceousplants,suchasF.moniliforme,F.graminearum,
F.culmorum,andF.avenaceum(Price1984).Thevascularwilts,causedbyformae
specialesofF.oxysporum,areconsideredtobethemosteconomicallyimportantdueto
thediversityofplantsattackedandthedestructivenessofthediseases(Nelson1981).
Fusariumoxysporumwasthefirstpathogenicspeciesdescribed,andwasincluded
inthesectionElegansbyWollenwebberandReinking(1935).Theydividedthissection
intothreesubsectionsandfurthersubdividedthesesubsectionsinto10species,18
varieties,and12forms(Nelson1981).Theirtaxonomicsystemwasdifficulttouseby
othersbecausetheyplacedsuchgreatemphasisonsporemeasurementsandseptations,
whicharehighlyvariable.
SnyderandHansen(1940)simplifiedWollenwebberandReinking'ssystemby
combiningallmembersofthesectionElegansintoonespecies,F.oxysporum,bythe
utilizationofstandardculturemediaandincubationtemperatures.Thedifferentparasitic
formswererecognizedbytheirselectivepathogenicitytospecificcropsandwere
designatednsformaespeciales.Formaespecialesarealsofurthersubdividedinto
biologicalracesbasedonpathogenicitytodifferentialcultivarscontainingdifferent
resistancegenes.
RecognitionofthelimitingeffectoftheFusariumwiltsoncropproductiongoes
backtothe1890's.Atkinson(1892)describedthefirstFusariumwiltdisease,and
referredtoitas"Frenching."IthadbeencausingseriouslossestoAlabamacotton
growers.Hedemonstratedthepresenceofgummysubstancesblockingthevascular
elementsandillustratedthephialidsproducingthemicroconidia,whichhasproventobe
themaindiagnosticcriterionforidentificationof/7,oxysporum.Healsonamedthecausal
agentasF.vasinfectum(Atkinson1892),whichisnowknowntobetheformnamefor
Fusariumwiltofcotton.Therearemanyadditionalearlyexamplesofcropsthreatenedby
Fusariumwiltsincludingpotatoes,celery,tomatoes,flax,bananas,cabbage,cowpea,and
watermelon.
Thecultivatedwatermelon,Citrulluslanatus(Thunb.)MatsumandNakai,isthe
mosteconomicallyimportantmemberofthegenusCitrullus,andisgrownforfresh
consumptionasadesertvegetable.Thecitron,C.lanatusvar.citroides(L.H.Bailey)
Mansf,isnoteatenfresh,butiscandied.Ithasalsobeenimportantinbreedingfor
diseaseresistancebecauseitreadilycrosseswiththewatermelon,andisresistantto
Fusariumwilt.WatermelonsareanimportanttruckcropintheSouth.Commercial
productionisapproximately184,000acresnationally,concentratedinFlorida,Texas,
Georgia,andCalifornia(Peirce1987).Ithasanestimatedgrossmarketvalueof$150
million(Martyn1986).
ThewatermelonisconsideredtobenativetothesoutherndesertsofAfrica.
DavidLivingstonereportedthatwildpopulationscoveredvastareasoftheKalahari
Desertafterunusuallyheavyrains.Bothnativesandanimalswereobservedconsuming
thefruit(Livingstone,1859).Carrier,however,concludedwronglythatitwasindigenous
toNorthAmericabasedondescriptionsbyFrenchexplorersofIndiansgrowingmelonsin
theMississippiValley(Carrier1924).
Theactualtimeandplaceofdomesticationofthewatermelonisunknown,butby
2000B.C.itwasbeingcultivatedalongtheNilebyEgyptiansandwidelygrownby
prehistoricagriculturalpeoplesofsub-SaharaAfrica(Peirce1987,Sauer1994).
WatermelonswereintroducedtoIndiaandChinabyAD.1100,andbroughttoEuropeby
theMoors.PopularityinEuropespreadslowly,presumablybecauseofthelong,hot
growingseasonrequiredforgoodyields(Sauer1994).InNorthAmerica,Spanish
colonistsweregrowingtheminFloridaduringthe1500's,andby1650,DutchandBritish
coloniesintheNortheastwerecultivatingthemabundantly(Peirce1987).Watermelons
