fungal control - british society for plant pathology abstract booklet 22.9...some of them, but we...
Post on 29-Jun-2018
214 Views
Preview:
TRANSCRIPT
1
Contents
Page 2………………………………………………..President’s welcome 4………………………………………………..Programme 10………………………………………………..Awards 12…………………………………………….. Opening plenary talk abstracts 15…………………………………………….. BMS talk abstracts – Exploitation 1 (Tuesday 12th) 19……………………………………………...BMS talk abstracts – Control 1 (Tuesday 12th) 23………………………………………………BMS talk abstracts – Exploitation 2 (Wednesday 13th) 26………………………………………………BMS talk abstracts – Control 2 (Wednesday 13th) 28………………………………………………BSPP talk abstracts (Tuesday 12th) 38………………………………………………BSPP talk abstracts (Wednesday 13th) 44………………………………………………Closing plenary talk abstracts 46………………………………………………Poster abstracts 82………………………………………………Date of next BSPP presidential meeting
2
BritishMycologicalSocietyandBritishSocietyforPlantPathologyJointPresidentialMeeting2017
FungalExploitationandFungalControl
UniversityofNottingham
11th–13thSeptember2017
Presidents’WelcomeOnbehalfoftheBritishSocietyforPlantPathologyandtheBritishMycologicalSociety,itisourpleasuretowelcomeyoutothisjointannualmeetinginNottinghamUniversity.Despitethefactthatthetwosocietiessharealonghistoryandhavemuchincommon,asfarastheOrganisingCommitteeisaware,thisisthefirsttimeajointmeetinghasbeenheld.Thethemesofthisconferencearethecontrolofplantpathogens(mainlyfungi)andtheexploitationoffungalspecies.Plantpathogenscontinuetocausesubstantiallossestofarmersinallpartsoftheworld.Themainmethodsusedtocontroldiseasesaregeneticdiseaseresistanceincropcultivars,fungicides,biologicalcontrolagentsandculturalpractices.Thisconferencewillhighlightsignificantadvancesinthefirsttwomethods.Cultivarswithimproveddiseaseresistance,whichcombineseffectivenesswithlongevity,arebeingreleasedthroughpartnershipsbetweenacademicscientistsandplantbreedingentities.Newfungicidesarebeingdevelopedandbettermethodstodelayandreducetheimpactoffungicideresistancearebeingdeployed.Aparticularlypleasingaspectofthisworkisthatitisnowbecomingclearthatthelongevityoffungicidesisextendedbygoodgeneticdiseaseresistanceandviceversa;fungicidesprotectthelongevityofgeneticdiseasegenes.AremainingfrustrationisthatGMdiseaseresistancehasnotbeenwidelydeployedevenaswepass20yearssincethefirstcloningofR-genes.Somefungiandoomycetescanalsoinfectanimalsandcreateseriousproblems,oftenresultinginfatalities.Newmethodstodetectsuchpathogensatanearlystagewouldresultinbettertreatmentpractisesandbettersurvivalrates.Inmanyanimal–fungalinteractions,basicbiologyquestionssuchas“howdoesthepathogeninfect?”and“whatdoesthehosttodefenditself?”cannotbeansweredatpresent.Forsomeanimaldiseases,controloptionsareverylimitedandalternativemethodsareurgentlyneeded.Besidesbeingpests,fungicanalsobeverybeneficialtous.Obviously,weliketoeatsomeofthem,butwecanalsomanipulatethemandusethemforbiotechnologicalpurposestodevelopmedicines,forexample;oralternatively,usethemforbiomassdegradation,insectorfungalbiocontrolandenhancingproductivitythroughbetterunderstandingofsymbiosisinmycorrhizalinteractions.
3
Itpromisestobeanexcitingannualmeetingandwewouldliketothanktheorganisingteamfrombothsocieties:PaulDyer,SimonAvery,MatthiasBrockandElizabethOrton,forputtingtogethersuchastimulatingprogramme.WealsothankAlisonTorfortheabstractbookletandMoiraHartwhoassistedwiththeorganisation.WewishyouafruitfulandenjoyabletimeinNottingham.RichardOliver,PietervanWestandNickRead(formerBMSpresident)TheBSPPandBMSaregratefulforthesupportfromApolloScientific,CABI,ElsevierandWiley.
4
Monday 11th September
13.00 Lunch and registration 14.00 Welcome 14.15 Plenary -‐ BSPP President Richard Oliver Translating understanding of plant pathogen control genetics into
grower profits 15.00 Plenary -‐ BMS President Pieter van West Invasive oomycetes are a serious threat to our natural and man-‐made
aquatic ecosystems 15.45 Coffee 16.15 Plenary -‐ Richard Michelmore The impact of new technologies on strategies to increase the
durability of disease resistance 17.00 Plenary -‐ Mark Lynas Anti-‐science and post-‐truth: why we got it so wrong about GMOs 17.45 Poster session and drinks reception (sponsored by Elsevier) 18.15 BSPP AGM Small lecture theatre 19.30 Dinner
5
Tuesday12thSeptember
ConcurrentBMS(Exploitation1) ConcurrentBSPP
9.00 BMSInvited-VeraMeyer BSPPInvited-BruceMcDonald Movingfromdescriptivetopredictivebiology:Rationalrewiringof
thecellfactoryAspergillusnigerUsingpopulationgenomicstodeterminethegeneticbasisofpathogenadaptationinagro-ecosystems
9.30 BMSInvited-ScottBaker BSPPInvited-LiseNistrupJorgensen Fungiforbiotechnology WhydoesIPMnotleadtomorereductionsinfungicides? 10.00 MuhammadAkbar:Anovel inoculationmethodofmycorrhizae in
wheat fields has remarkable effects on crop yield and soilproperties
VanessaMcMillan:Identifyingrootresistanceintake-alldisease
10.20 ElenaGeib:AspergillusnigerversusAspergillusoryzae:Expressionplatformsforheterologoussecondarymetaboliteproduction
YongjuHuang:Ignoreduntilrecently-thestoryofthefungalpathogenLeptosphaeriabiglobosa
10.40 Coffee Coffee11.10 BMSInvited-EckhardThines PHGregoryStudentCompetitionTalks
UnravellingpyriculolbiosynthesisanditsregulationinthericeblastfungusMagnaportheoryzae
1)CorinneArnold(JIC):PowderyMildew:ataleoftwofungicides
2)ShaoliDasGupta(RHUL):Blumeriametallo-proteaselikeeffector;Azincophoreactingasauniversalvirulencefactorinfungi?
11.40 KristiinaHildén:Biochemicalcharacterizationofmannan-acting 3)KathrynHales(Warwick):Understandingtheecologyand
6
enzymesfromthewhite-rotfungusDichomitussqualens epidemiologyofPythiumviolaetoenablediseasemanagementincarrotcrops.
4)SaschaJenkins(Warwick):Examiningidentity,phylogenyandpathogenicityfactorsinFusariumspeciesaffectingpea.
12.00 MichaelaNovodvorska:PigmentbiosynthesisinPenicilliumroqueforti,whybeblue?
5)ChinthaniKarandeniDewage(Hertfordshire):FinemappingofamajorgenelocusforresistanceagainstPyrenopezizabrassicae(lightleafspot)inBrassicanapusandidentificationofcandidateresistancegenes
12.20
Lunch
6)JosephMoughan(Rothamsted):Fromthefieldtothelabandbackagain:exploringlegacyeffectsofrootgeneticsandfungicidefortake-allrootdiseasecontrolinwheatrotations.
13.30 Elsevierauthorworkshop ConcurrentBMS(Control1) ConcurrentBSPP
14.30
BMSInvited-AxelBrakhage BSPPInvited-VivianneVleeshouwers
Melanin-dependentpathogenicityofAspergillusfumigatus ExploitingeffectorsinbreedinganddeploymentofRgenesinpotato
15.00 JanetQuinn:FungalSAPKpathwaysasmediatorsofvirulence FranziskaTrusch:TheRxLRMotifoftheHostTargetingeffector
AVR3aofPhytophthorainfestansiscleavedbeforesecretion
15.20 CindyVallieres:Synergisticcombinationstargetingtranslationfidelityanddiversefungalpathogens.
SarahSchmidt:Knowyourenemy-engineeringresistanceagainstFusariumwiltinbanana.
7
15.40 Coffee Coffee 16.10
BMSInvited-AlBrown
BSPPInvited-Karl-HeinzKogel
Candidaalbicansisamovingtargetfortheimmunesystemasitadaptstohostsignals
TheAgronomicPotentialofGeneSilencingApplication
16.40 Rhys Farrer: Targeting infection mechanisms for two amphibian-
infectingchytridspecies.LaurenceBindschedler:Combiningproteomicsofhaustoriaandgenesilencingtodiscoverkeyplayersofvirulenceduringbarleypowderymildewinfection
17.00 Posterssessionanddrinksreception(sponsoredbyWiley)
BMSAGMSmalllecturetheatre
19.30 Conferencedinner
Wednesday13thSeptember
ConcurrentBMS(Exploitation2) ConcurrentBSPP9.00 BMSInvited-RonalddeVries BSPPInvited-SanderSchouten Diversityinplantbiomassdegradationapproachesoffungi Directandindirecteffectsoffungalendophytesonplant-parasitic
nematodes 9.30 IolyKotta-Loizou:Mycovirusesasprospectiveenhancersof
biocontrolagentsChristianVoigt:Improvingplantresistancetofungalpathogensbycallosemodification
9.50
Stefany Solano:Genomic and genetic analysis of a basidiomyceteyeastfocusedonthecharacterisationofMELbiosynthesis
AndrewTaylor:UnderstandingthegeneticcontrolofpathogenicityandresistanceforFusariumoxysporuminOnion
8
10.10 Sara Casado López: Intraspecies diversity and induction of thewooddecaybythewhiterotfungusDichomitussqualens
RyanAmes:UnderstandingappressoriumdevelopmentinMagnaportheoryzae
10.30 Coffee Coffee Concurrent.BMS(Control2) ConcurrentBSPP
11.00 BMSinvited-AlanGange BSPPInvited-FrankVandenBosch Exploitingunspecialisedendophytesforbiologicalcontrolofinsects
andplantsIntegrateddeploymentofhostresistanceandfungicides:managingevolutionofvirulenceandinsensitivity
11.30 BMSInvited-MikeBromley BSPPInvited-NeilPaveley RegulationofDrugResistanceinAspergillusfumigatus Integrateddeploymentofhostresistanceandfungicides:
economics,efficacyandevolution
12.00 TakanoriFurukawa:SystematicfunctionalannotationoftranscriptionfactorsinthemajormouldpathogenAspergillusfumigatus
AndrewLeader:Fenpicoxamid(InateqTM,activefungicide),anewfungicidewithutilityincerealsforcontrolofZymoseptoriatritici(Septorialeafblotch)andotherdiseases
12.20
Endofsession
AviceHall:ControllingStrawberrypowderymildewsrequiresallyear-roundvigilance
12.40 Lunch
9
PlenarySessionandClose
13.30 Plenary-RobJohnson TheScienceofQuorn
14.00 Plenary-NataliaRequena Fungalweaponstomanipulateplants
14.45 PrizesandClose
10
Awards The Boards of the BSPP and BMS wish to encourage young plant pathologists and mycologists to present their work. There are two BSPP prizes and one BMS prize awarded at the presidential meeting: The P H Gregory prize, awarded for the presentation of an oral paper. The competition is open to (a) members of BSPP who have not previously presented a paper to a meeting of a learned society and (b) all registered postgraduate students, whether or not they are members of the society, or have presented a paper before to a meeting of a learned society. Contestants should not have entered the P H Gregory competition previously. The winner receives a certificate and a cheque for £250. Philip Gregory (1907–1986) pioneered aerobiology as a topic for research, combining many disciplines to contribute to better understanding of fungal spore dispersal and plant disease epidemiology. He developed theories of spore dispersal, published in his classic paper on the dispersion of airborne spores (Gregory PH. (1945) Trans. Br. Mycol. Soc. 28: 26–72). He became Head of the Plant Pathology Department at Rothamsted in 1958 where he further developed his research on spore dispersal and sedimentation velocities. In his retirement, Philip Gregory continued work on elucidating the epidemiology of black pod disease of cocoa in Nigeria, and maintained enthusiasm for his wide range of scientific interests. He proudly and enthusiastically showed his garden to visitors and they often regarded this as the highlight of their visit to Harpenden (Source: Lacey et al. (1997) Ann. Rev. Phytopathol. 35: 1—14). The John Colhoun Prize, awarded for a poster. The work presented in the poster must form part of a research project conducted by the entrant in support of a PhD or Masters degree, and the entrant must not have been awarded the degree prior to the deadline for abstracts date. Students need not be members of BSPP. The winner receives a certificate and a cheque for £100. John Colhoun (1913–2002): cryptogamist and plant pathologist. John Colhoun was awarded a MAgr in 1937 at Queens University, Belfast, and then moved to Imperial College, London, working on fungal pathogens of apples for his PhD, awarded in 1940. He returned to Northern Ireland where he worked on flax, leading to a definitive text (Muskett A. E. & Colhoun J. (1947): The Diseases of the Flax Plant). He became reader at Queen’s University in 1954. Subsequently, he took up the Chair of Cryptogamic Botany at the University of Manchester in 1960, where he worked on Fusaria, Phytophthora, Septoria and Phoma, with hosts ranging from cereals to chrysanthemum, yam, oil palm, and banana. In 1968, he was elected Chairman of the Federation of British Plant Pathologists, forerunner of the British Society for Plant Pathology. He retired from Manchester University in 1980 as Professor Emeritus, having occupied the Barker Chair of Cryptogamic Botany for 20 years. (Source: Epton H. (2003) Mycol. Res. 107: 377–381). The BMS Student Poster Prize: Awarded to the best mycology student poster at the meeting.
12
OpeningPlenaryTalksTranslatingunderstandingofplantpathogencontrolgeneticsintogrowerprofitsRichardP.OliverCentreforCropandDiseaseManagement,CurtinUniversity,Perth,WA,AustraliaEmail:Richard.oliver@curtin.edu.auPlantfungalpathogenscausemajoravoidablelossestocropinallpartsoftheworld.Reliabledataonthedegreeoflossesisrare.ThatAustralia,withitslowinputsandfrequentdroughts,isthecountrywiththehighestdocumentedoverallpercentagelossesinbroadacreagricultureiscertainlyduetothepaucityofdatafromotherregions.ThemajordiseasesofgraincropsinAustraliathattheCCDMworksonarecausedbyarangeofnon-obligatefungi;thewheatdiseasestanspotcausedbyPyrenophoratritici-repentis,andseptorianodorumblotch(SNB)causedbyParastagonosporanodorum,thebarleynetblotchdiseasescausedbyPyrenophorateres,thelegumeleafblightdiseasescausedbyvariousAscochytaspp.andthecanoladiseasecausedSclerotiniasclerotiorum.DiseasescausedbyrustshavebeenreasonablywellcontrolledbythedeploymentofmajorR-genes.Suchmajorgeneswerenotavailablefortheseblightdiseases.Thestrategydeployedtocontrolthemhadtoawaitthedevelopmentofsomeunderstandingabouthowthepathogensinteractwiththehost.AcollaborativeefforthasshownthatatleastfortanspotandSNB,thekeywastodeterminethatthediseaseswerecontrolledbytheinteractionofpathogengeneproductsnamednecrotrophiceffectors(NEs).NEsinteractwithcorrespondinghostsensitivitygenes.ThekeytoimprovingdiseaseresistancewastotransferthisknowledgetobreederssothattheycouldeliminategermplasmcarryingrelevantNEsensitivitygenes.FungicidesremainthebackstopoffungaldiseasecontroleveninlowintensitycountrieslikeAustraliaandespeciallysince2005whenpricesdropped.FungicideresistancewassoondiscoveredinmanypathogensinAustralia.Thepatternoffungicideresistanceconfirmedthedevelopingunderstandingthatfungicideresistancecanbedelayedbyapplicationofcoreevolutionaryprinciplesthatguideustouseminimumdoses,minimumnumbersofapplicationsandmixturesoralternationsofmodesofaction.Itisalsobecomingclearprudentuseoffungicidesextendsthelifeofgeneticdiseaseresistanceandviceversa.
13
Invasive oomycetes are a serious threat to our natural and man-made aquaticecosystemsPietervanWestAberdeen Oomycete Laboratory, International Centre for Aquaculture Research andDevelopment at the University of Aberdeen, College of Life Sciences and Medicine,InstituteofMedicalSciences,Foresterhill,Aberdeen,AB252ZD,Scotland,UK.Email:p.vanwest@abdn.ac.ukOomycetesarefungal-likeorganismsthatareclassifiedasChromalveolatesandphylogeneticallygroupedwithdiatomsandbrownalgae.Theyareamongthemostimportantgroupsofdisease-causingorganismsinbothagricultureandaquacultureandthusrepresentahugethreatforglobalfoodsecurity.Someaquaticspeciescancauseseriousenvironmentaldisasters,wipingoutournativeaquaticanimals,forexampleEuropeancrayfishandalsoseveralamphibianshavebeenseverelyaffectedorhavebecomeextinct.Oneparticularpathogen,Aphanomycesinvadans,isnowapproachingEuropeanwatersandrepresentsadevastatingpathogen.Itisatremendousproblemincountrieswhereithasalreadyarrivedandhasestablished(e.g.Bangladesh,IndiaandpartsofAfrica).TherearealsootheraquaticoomycetepathogensknownincludingHaliphthoros,Halioticida,Lagenidium,Atkinsiellaspp.thatinfectmarineandbrackishanimalsincludinglobsters,langoustines,abaloneandprawns.Saprolegniaparasiticaisoneofthemostdestructivefishpathogensandisfoundinmostfreshwaterenvironmentsaroundtheworld.Remarkably,verylittleisactuallyknownaboutthebiologyoftheseaquaticpathogens.However,whatwedoknowisthattheyare:1)potentialinvadersofmarine&freshwaterhabitats,2)theycancauseseriouseconomicorenvironmentaldamageandmostimportantly,3)theyarealluncontrollableatpresent.Anoverviewofthesevariousanimalpathogenicoomycetesispresented.Furthermore,ourcurrentknowledgeaboutthecellularandmolecularinfectionstrategiesofS.parasiticaandA.invadansisbeingdiscussed.Inordertosuccessfullyinfecttheirhoststheseparasitesrelyontheireffectorrepertoires.Effectorsaresecretedproteinsofthepathogenthatcanalterthehostandwhichadvancetheinfectionprocess.Alargenumberofeffectorsislocatedattheinterfacebetweenpathogenanditshostandfulfilafunctionontheoutsideofthehostcell.Sucheffectorsareoftenclassifiedasextracellulareffectors.Whileseveralotheroomyceteeffectorsareabletotranslocateintotheirhostcellswheretheycan,forexample,interferewithdefenceresponsesofthehost.Theseeffectorsarecommonlyreferredtoasintracellulareffectors.Themechanismoftranslocationordeliveryoftheintracellulareffectorsisunderintenseinvestigationandseveralroutesofentryhavebeenproposed.Herewewilldiscussourrecentadvancesinunderstandingsecretion,deliveryandfunctionsofeffectorsfromanimalpathogenicoomycetes.
14
The impact of new technologies on strategies to increase the durability of diseaseresistance.RichardW.MichelmoreThe Genome Center & Department of Plant Sciences, University of California, Davis,Californa,95616,USA.Email:rwmichelmore@ucdavis.eduGreatadvanceshaverecentlybeenmadeintheunderstandingofthemolecularandgeneticbasisofdiseaseresistanceinplants.Itisnowtimetodeploythisknowledgetoprovidemoredurablediseaseresistance.Manyadvanceshavebeenenabledbytechnologicalimprovements.Inparticular,highthroughputDNAsequencingenablesdetailedanalysisofcropsandtheirpathogens.Itisnowpossibletocharacterizevariablepathogenpopulationsandusethisinformationfortherationaldeploymentofresistancegenessoastomaximizetheevolutionaryhurdleforthepathogentobecomevirulent.Muchofourworkoverthepastthirtyyearshasfocusedonresistancetodownymildewinlettuce.Lettuce(Lactucasativa)isoneofthemostvaluablevegetablecropsanddownymildew,causedbyBremialactucae,isitsmostimportantfoliardiseaseworldwide.Theuseofresistantvarietiesisthemosteffectivemethodforcontrollingthisdisease;however,pathogenvariabilityhasledtotherapiddefeatofindividualresistancegenes.Over50resistancegeneshavebeenidentifiedandlettucedownymildewisoneofthebestgeneticallycharacterizedplantdiseases.Wholegenomesequencingofmultiplegenotypeshasallowedtheidentificationofcandidateresistancegenesinthehostandvirulencefactorsinthepathogen.Knowledge-drivendeploymentofeffectiveresistancegenesasgenepyramidsprovidestheopportunityformoredurableresistance.Genestackingusinggenomeeditinghasthepotentialmakingthisprocessmoreefficient.Inaddition,host-inducedgenesilencingofvitalpathogengenespresentspotentiallyinsurmountableevolutionaryhurdlesforthepathogentoovercome.Anti-scienceandpost-truth:whywegotitsowrongwithGMOsMarkLynas@mark_lynasEmail:marklynas36@gmail.comWith“post-truth”theOxfordEnglishDictionarywordoftheyearfor2016,itseemsasifanewzeitgeistischallengingthewholebasisofevidence-basedthinkingandthescientificmethod.Buthownewisthisreally?MarkLynas,areformedformeranti-GMOactivist,tracessomeofthedeeperrootsoftoday’s“post-truth”attitudesintheenvironmentalmovement’srejectionofgeneticallymodifiedcropsandothertechnologies-likenuclearpower-thatwereseenasbeing‘unnatural’.
15
BMSTalksMoving from descriptive to predictive biology: Rational rewiring of the cell factoryAspergillusnigerVeraMeyerDepartmentAppliedandMolecularMicrobiology,TechnischeUniversitätBerlin,Berlin,13355,GermanyEmail:vera.meyer@tu-berlin.deFungalbiotechnologyiscurrentlyundergoingarenaissancewithimportantimplicationsforitsroleasplatformtechnologyforthesustainableproductionofproducts,goodsanddrugs.AlliedtothisaretherecentadvancesinfungalSystemsandSyntheticBiologywhichcanbeseenastwocomplementaryapproachestoinvestigatethecomplexityofbiologicalsystemsincludingfungi.WhereasSystemsBiologyanalyzescellularsystemsinaniterativecycleofhigh-throughputgenerationof‘omics’dataandmodeling,SyntheticBiologytakesaconstructiveapproachtoreengineerbiologicalnetworksandtodesignnovelbiologicalpartsandcircuitswithnon-naturalfunction.Bothdisciplines–systemsanalysisanddesign–ideallycomplementeachotherand,notsurprisingly,aimtodiscoverthedesignprinciplesof(fungal)Life.OurgoalistounderstandandrationallyrewiretheindustrialcellfactoryAspergillusniger.Ontheonehand,wegenerateandevaluatedifferentomicsdatatypesforA.nigerandusetheseBigDatatopredictgenefunctionaswellasgeneregulatorynetworks.Ontheotherhand,weengineertitratableandtightlyregulatedconditionalmono-andpolycistronicgeneswitchesandtargetthemtospecificgenomicloci.Bycombiningthesetwoactivities,wenotonlysuccessfullyimproveA.nigerasproducerforproteins,newnaturalproductsorevennew-to-naturecompounds,wearealsoenteringanewerainAspergillusnigerbiology:wearemovingfromdescriptivetopredictivebiology.FungiforbiotechnologyScottE.Baker1,21Joint BioEnergy Institute, Emeryville, California; 2Environmental Molecular SciencesLaboratory,PacificNorthwestNationalLaboratory,Richland,Washington,USAEmail:scott.baker@pnnl.govFungihavebeenlongbeenexploitedforproductionofsmallmoleculesandenzymesforcenturies.Theunderlyingbiologyoffungalbioprocesses,however,isnotwellcharacterized.Usingavarietyofcellbiological,geneticandgenomicmethodswehaveassessedthebiologicalpathwaysunderlyingbothenzymeandsmallmoleculeproduction.Wehaveexploredenzymeproductioninthreephylogeneticallydiverse
16
filamentousascomycetesfindingsharedgeneticdeterminantsofhighenzymeproduction.Inaddition,wehaveusedavarietyofcellbiologicalandgeneticmethodstobetterunderstandfactorsimportantforlipidproductionintheoleaginousyeast,Yarrowialipolytica.AnovelinoculationmethodofmycorrhizaeinwheatfieldshasremarkableeffectsoncropyieldandsoilpropertiesMuhammadAkbar1,SafeerA.Chohan1,MuhammadS.Iqbal1,NazirAslam1,AqeelAhmad2andTayyabaKhalil11DepartmentofBotany,UniversityofGujrat,Gujrat,50700,Pakistan;2InstituteofAgriculturalSciences,UniversityofthePunjab,Lahore,54000,Pakistanemail:makbarpu@gmail.comIn recent years there is growing trend towardsorganic farming tomeet food securityand food safety. Inorganic fertilizers are available to boost yield but there areenvironmentalandpublichealthconcerns.Amongstvariousorganicalternatives,usingmycorrhizal species is one of the most promising options. Therefore, the presentresearchwasconductedtoevaluatetheinfluenceofindigenousmycorrhizalspeciesonthe growthofwheat and soil properties under field conditions. In total 11 species ofmycorrhizaewere identified fromtheexperimentalareas, themostprominentgenerabeing,Claroideoglomus,RhizophagusandFunneliformis.Forinoculationofmycorrhizae,theirnativedensitywasmaintainedwithanovelideathatthesespeciesworkbetterinconsortia when their native population density was maintained. There were eightdifferent treatments having plot sizes as 6meters x 2meters. Thewhole experimentwasrepeatedattwodifferentsites.Bio-inoculationofconsortiaofdifferentmycorrhizalspeciesshowedasignificant increaseinallgrowthparametersstudiede.g.,numberoftillerperplant(upto39.53%),plantheight(upto13.66%),drybiomass(upto15.84%),grainyield(upto18.69%)andhayweight(upto15.69%).Moreover,remarkableeffectswereencounteredonsoilfertilitysuchassoilorganicmatter,availablephosphorusandpotassiumwere increasedup to92.30%,12.24%and8.83%, respectively.Thepresentstudyconcludesthatincreasingthemycorrhizalinoculumincropfieldsbykeepingtheirdensityasnativehastremendouseffectsoncropproductivityandsoilfertilitystatus.Aspergillus niger versus Aspergillus oryzae: Expression platforms for heterologoussecondarymetaboliteproductionElenaGeibandMatthiasBrockFungalGeneticsandBiology,UniversityofNottingham,Nottingham,NG72RD,UKEmail:mbxeg3@nottingham.ac.ukTo exploit fungal secondary metabolite production a strong and specific expressionsystemisrequired.WedevelopedsuchanovelexpressionsysteminAspergillusniger,
17
which bases on regulatory elements from theAspergillus terreus terrein biosyntheticgenecluster.Weconfirmeditssuitabilitybyheterologousproductionofpolyketides(e.g.lecanoricacid),non-ribosomalpeptidesynthetase-likeproducts(e.g.aspulvinoneE)andusedittocharacterisetheAsp-melaninbiosynthesispathwayfromA.terreus.ThelatterstudiesledtoaninterestintheunderstandingofthechemistryofNRPS-likeenzymesthatproducemetaboliteswithantifungal,cytotoxic,antitumorigenicandantiviralactivity.WhiledifferentNRPS-likeenzymesmayacceptthesamesubstratetheresultingproductdependsonthebiochemistryofthethioesterasedomain.Tostudythesedomains,wecomparedtheaspulvinoneEsynthetaseMelAfromA.terreuswiththeatromentinsynthetaseInvA5fromPaxillusinvolutus.WhilerecombinantexpressionofmelAinA.nigerresultedinaspulvinoneEproduction,expressionofinvA5ledtoarangeofproductsthatwerealldistinctfromatromentin.WethereforespeculatedthatthephysiologyofA.nigermightleadtoamodificationoftheInvA5-derivedmetabolite.Consequently,invA5expressionwasstudiedinthealternativehostAspergillusoryzaeand,indeed,atromentinwasproduced.Inconclusion,ourrecombinantexpressionsystemisperfectlysuitedforheterologousproductionofawiderangeofdifferentsecondarymetabolites.However,asthemetabolicphysiologyoffungidiffers,atleasttwodifferentexpressionplatformsshouldbeselectedwhenaiminginthecharacterisationofnovelsecondarymetabolitebiosynthesisgenes.Unravelling pyriculol biosynthesis and its regulation in the rice blast fungusMagnaportheoryzaeEckhardThines1,2andStefanJacob21Institute of Molecular Physiology, University of Mainz, D55128 Mainz, Germany;2InstítuteofBiotechnologyandDrugResearch(IBWF),D-67663Kaiserslautern,GermanyEmail:thines@uni-mainz.deFungalsecondarymetaboliteshaveinthepastbeenverysuccessfulleadstructuresforpharmaceuticaloragrochemicalapplications.Manyofthosecompoundshavebeendiscoveredinaxenicculturesinartificialmedia,whicharepoorsurrogatesformimickinganorganism’snaturalhabitat.Asaconsequenceonlyasmallnumberofgenespotentiallyinvolvedinthebiosynthesisofsecondarymetabolitesareexpressedundertheseconditions.Magnaportheoryzaeisthecausalagentofblast,themostdevastatingfungaldiseaseoncultivatedrice.Thefungusisknowntoproducesecondarymetabolitesinvolvedinplant/pathogeninteraction,suchaspyriculol/pyriculariol.Withinthegenome20genesencodingpolyketidesynthaseshavebeenidentified,whereasqPCRexperimentsrevealedthatonlyfourofthemaretranscribedinaxenicculture.Apartfromcultivationvariationapproachesmolecularbasedtechniqueshavebecomesuitablemethodsinordertoexploretheproductsofsilentsecondarymetabolitepathways.
18
Inordertoinvestigateregulatoryelementsofsecondarymetabolismandtoidentifynewnaturalproductsmanytechnologiescanbeapplied.Forthosestudiesgenedeletion/genedisruptionstrainsweregenerated,promoterswerechangedandtranscriptionfactorsorotherpleiotropicregulatorswereover-expressed.Furthermoreepigeneticstrategiesweresuccessfullyappliedinordertoelucidatenewsecondarymetabolites.TheapplicationofdemethylaseinhibitorsledtotheidentificationofnovelpolyketidesfromM.oryzaethathavenotbeenisolatedfromextractsofaxenicculturesbefore.Apartfromtheidentificationofnewsecondarymetabolitesandtheirpotentialroleinpathogenicitythestudieswillprovideinsightsintotheregulationofphytotoxinbiosynthesisinplant/pathogen-interactions.Biochemicalcharacterizationofmannan-actingenzymesfromthewhite-rotfungusDichomitussqualensAngelZhang1,JaanaKuuskeri1,JohannaRytioja2,RonalddeVries3,MiiaR.Mäkelä1andKristiinaHildén11DivisionofMicrobiologyandBiotechnology,DepartmentofFoodandEnvironmentalSciences,UniversityofHelsinki,Helsinki,Finland;2DepartmentofChemicalTechnologyandBiotechnology,AaltoUniversity,Espoo,Finland;3FungalPhysiology,WesterdijkFungalBiodiversityInstitute&UtrechtUniversity,Utrecht,TheNetherlandsEmail:kristiina.s.hilden@helsinki.fiWhite-rotfungihaveabilitytodecomposeallcomponentsofplantcellwallduetotheirwideenzymerepertoire.Theseenzymescouldbeusedforbioconversionofvariouslignocellulosesinindustrialapplications.Asatypicalwhite-rotfungus,Dichomitussqualenshasanextensivesetofgenesencodingenzymesforplantpolysaccharidedegradation.Thepreviousstudy[1]indicatedthatD.squalensexpressesandsecretesvarietyofmannanolyticgenes/enzymeswhengrowingondifferentplantbiomasses.Inthecurrentstudy,wecharacterizedfourmannan-actingenzymesderivedfromD.squalens.Inparticular,twoβ-1,4-endomannanases(GH5_7),oneβ-1,4-mannosidase(GH2)andoneα-1,4-galactosidase(GH27)wereclonedandexpressedinPichiapastoris.Theoptimaltemperaturesoftheexpressedrecombinantenzymeswereintherangeof50to60°CandoptimalpHfrom3to5.Themolecularmassofthecharacterizedproteinsrangedfrom47to110kDa.Thehighestthermaltolerancewasobservedfromβ-1,4-endomannanase(MAN1).MAN1retainedover50%ofitsenzymeactivityafter30minutesofincubationat70°C.Themeasuredenzymeactivitiesfromtwoofthecharacterizedenzymes,namelyMAN1andα-1,4-galactosidase,exhibitedhighenzymaticactivitiestowardsthetestedsubstrates.Overall,theresultssuggestthehighpotentialofD.squalensasasourceofindustrialenzymes.
19
[1]J.Rytioja,K.Hildén,M.DiFalco,M.Zhou,M.V.Aguilar-Pontes,O.-M.Sietiö,A.Tsang,R.P.deVries,M.R.Mäkelä,Environ.Microbiol.(2017)doi:10.1111/1462-2920.13652PigmentbiosynthesisinPenicilliumroqueforti,whybeblue?MichaelaNovodvorska,MatthewCleere,JackWhittaker,PaulS.DyerSchoolofLifeSciences,NottinghamUniversity,UniversityPark,Nottingham,NG72RD,UKEmail:michaela.novodvorska@nottingham.ac.ukPenicilliumroquefortiisanindustriallyimportantfungusthatisusedasastarterculturefortheproductionofblue-veinedcheese.Thefungusproducesvolatilecompoundsthatare responsible for theuniquebluecheesearoma, thesecompoundsbeinggeneratedfromtheextensivecatabolismofmilk lipidsandproteins.Thebluecolourcomesfrommelanin, and possibly other, pigments covering the surface of spores formed duringgrowth in the cheese. It is known that a DHN-melanin biosynthetic pathway isresponsibleforconidialmelaninpigmentbiosynthesisincloselyrelatedspeciessuchasAspergillus fumigatusandPenicilliummarneffei. In thisstudywe investigatedwhetherthesamepathwayispresentinP.roquefortiandassessedtheimpactofthepathwayonresistanceagainstenvironmentalfactors,mycotoxinproductionandvolatileprofile.Weidentified six homologous genes of a canonical DHN-melanin biosynthetic pathway,developed a novel transformation protocol for P. roqueforti, and generated deletionstrains.Deletionof eachof these geneshadan impactonpigmentbiosynthesis,withdeletion mutants producing conidia exhibiting colour differences to the wild-typeparentalstrain.DeletionmutantsshoweddecreasedviabilitywhenexposedtoUVlightandelevated temperatures, indicating the importanceofpigmentproduction in sporesurvival under varying environmental conditions. Production of mycotoxins such asmycophenolic acid and roqueforine C was greatly reduced when the pigmentbiosynthesispathwaywasnot fully functional.Deletionofeachof thepathwaygenesalso resulted in a change in production of flavour volatiles. These findings are ofrelevanceforcheesemanufacture.Melanin-dependentpathogenicityofAspergillusfumigatusAxelA.Brakhage1,21Department,MolecularandAppliedMicrobiology,LeibnizInstituteforNaturalProductResearch and Infection Biology (HKI), Jena, Germany and 2Institute for Microbiology,UniversityofJena,GermanyEmail:axel.brakhage@leibniz-hki.deThefilamentousfungusAspergillusfumigatusisthemostimportantair-bornefungalpathogencausing90%ofallsystemicAspergillusinfectionsinhumans.Previously,we
20
showedthattheconidialpigmentconsistingofdihydroxynaphthalene(DHN)melaninrepresentsavirulencedeterminant.ConidialackingDHN-melaninshowreducedvirulenceinamouseinfectionmodel.WefoundthatthepresenceofDHN-melaninontheconidialsurfaceinfluencestheresponseofimmunecellsatvariouslevels.Forexample,DHN-melanininhibitsphagolysosomalacidificationandcausesincreaseddamagetomacrophagescomparedtonon-pigmentedpksPmutantconidia.WediscoveredanovelmolecularmechanismbywhichDHN-melaninleadstothedisturbanceoflipidraftmicrodomainsinthemembraneofphagolysosomes,therebypreventingtheassemblyandactivityofvATPaseandthusinhibitingtheacidificationofthephagolysosome.Also,assemblyofNADPHoxidasecomplexwaslipidraft-dependentandreducedbymelanizedconidia.Furtherclassificationofthelipidraftmicrodomainsshowedthatflotillin-enrichedlipidraftsco-localizedwithsphingolipidsonphagolysosomalmembranescontainingpksPconidiabutnotwild-typeconidia.FungalSAPKpathwaysasmediatorsofvirulenceAlisonM.Day1,CarmenM.Herrero-de-Dios2,AlistairJ.P.Brown2andJanetQuinn11Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, NewcastleUniversity,Newcastle upon Tyne,NE2 4HH,UK; 2Aberdeen FungalGroup, Institute ofMedicalSciences,UniversityofAberdeen,AberdeenAB252ZD,UK.Email:janet.quinn@ncl.ac.ukRobuststressresponsesarecommonvirulenceattributeswithinabroadrangeoffungalpathogens,includingthehumanfungalpathogenCandidaalbicans.Centraltosuchstressresponsesarethestressactivatedproteinkinases(SAPK),whichareevolutionarilyconservedeukaryoticsignallingmodulesthatareessentialforthevirulenceofhumanpathogenicfungi.TheHog1SAPKisessentialforC.albicansvirulence,howeveritsstructuralandfunctionalconservationwithanalogoushumanSAPKs(p38/JNK)makesthedevelopmentofantifungaldrugsthatwillspecificallytargetfungal,andnothuman,SAPKschallenging.Becauseofthis,thereismuchinterestinidentifyingfungal-specificregulatorsofSAPKsaspotentialdrugtargets.Suchtargetsincludetwo-componentrelatedphosphorelaysystems,whichareusedbyfungi,butnotmammals,tosenseandrelaystresssignalstoSAPKmodules.Suchphosphorelaysystemscompriseofastress-sensinghistidinekinase,anintermediaryphosphorelayprotein,andaterminalresponseregulator.HereIwillpresentrecentfindingsfrommygroup,whichhavedissectedtheroleofkeyphosphorelayproteinsinregulatingHog1,andexploredtheirpotentialasantifungaldrugtargets.
21
SynergisticcombinationstargetingtranslationfidelityanddiversefungalpathogensCindyVallieres,ElenaMoreno-Martinez,AshleighGilliot,SaraL.HollandandSimonV.Avery.SchoolofLifeSciences,UniversityofNottingham,UniversityPark,NottinghamNG72RD,UK.Email:C.Vallieres@nottingham.ac.ukAwiderangeoffungicides(orantifungals)areusedinagricultureandmedicine.Unfortunately,thegrowingresistancetocurrentantifungalshasbecomeamajorissue,andthereforenewagentswithnovelmodesofactionareurgentlyneeded.Combinationtreatmentsofferanefficientmethodformanagingfungalgrowthbyenhancingefficacycomparedtoindividualcompounds,decreasingthedoseofsingleagentusage,andsubsequentlyloweringthedrugtoxicityandcost.Moreover,combinatorialinhibitioncanslowtheevolutionofresistance.Astudyconductedinourgroupshowedthatdifferentaminoglycosideantibioticscombinedwithdifferentsulphate-transportinhibitorsproducedstrong,synergisticgrowth-inhibitionofseveralhumanandplantpathogenicfungimainlybycausing,viadifferentmechanisms,errorsinmRNAtranslation.Theresultsindicatethattranslationfidelityisapromisingnewtargetforcombinatorialtreatmentofundesirablefungi.Inlinewiththisstudy,wescreenedawiderrangeofcompoundsknowntoaffecttheavailabilityoffunctionalaminoacidsincombinationwitheitheri)anotheragentthataffectstheavailabilityoffunctionalaminoacidsorii)anaminoglycosideantibiotictouncovernewsynergisticdrugcombinationstargetingtranslationfidelity.Screenswereperformedinthreefungalphytopathogens(i.e.,Rhizoctoniasolani,SeptoriatriticiandBotrytiscinerea)andthreefungalpathogensofhumans(i.e.,Cryptococcusneoformans,CandidaalbicansandAspergillusfumigatus).43combinationsoutof172wereabletoabrogatefungalgrowthwith9combinationsbeingrepresentedinatleastthreepathogenicfungi.Thus,thisstudyunveilsatargetprocessandchemicalcombinationswiththerapeuticpotentialagainstdiversefungalpathogens.CandidaalbicansisamovingtargetfortheimmunesystemasitadaptstohostsignalsGabrielaM. Avelar, Delma S. Childers, Judith Bain, Arnab Pradhan, Daniel Larcombe,LarsErwig,ElizabethR.Ballou,NeilA.R.GowandAlistairJ.P.BrownAberdeenFungalGroup,MRCCentreforMedicalMycology,InstituteofMedicalSciences,UniversityofAberdeen,AberdeenAB252ZD,UKEmail:al.brown@abdn.ac.ukThepotencyoftheimmunesystemplaysakeyroleinfungalcolonisationandinfection.MoststudiesofCandidaimmunologyhavebeenperformedonfungalcellsgrownunderstandardisedconditionsinvitro.ByexaminingtheimpactofkeyhostsignalsuponCandidaalbicansinvitro(suchaschangesincarbonsources,pH,temperature,oxygenation,stressesandmicronutrients),wefindthat,inadditiontoaltering
22
resistancetohoststressesandantifungaldrugs,somehostsignalstriggerchangesinthefungalcellwall.ThisleadstosignificantmodulationofkeyPathogenAssociatedMolecularPatterns(PAMPs)onthefungalcellsurface,turningC.albicansintoamovingtargetfortheimmunesystem.InparticularwehavefoundthatphysiologicallevelsofL-lactatetriggerβ-glucanmaskingviaaspecificlactatesignallingpathwaythatincludesthereceptorGpr1andthetranscriptionfactorCrz1.Lactate-inducedβ-glucanmaskingleadstoareductioninphagocyticrecruitment.ThiscorrelateswithanincreaseinthevirulenceofC.albicans.Therefore,wearguethattheadaptationofC.albicanstohostmicroenvironmentscanaffectitssusceptibilitytoantifungaltherapy,itsdetectionbytheimmunesystem,anditspathogenicity.ThesefindingsmayberelevanttootherpathogenicCandidaspecies.Targetinginfectionmechanismsfortwoamphibian-infectingchytridspeciesRhysA.Farrer1,2,AnthonyNash3,AnMartel4,ElinVerbrugghe4,AmrAbouelleil1,RichardDucatelle4,JoyceE.Longcore5,TimothyY.James6,FrankPasmans4,MatthewC.Fisher2andChristinaA.Cuomo11 Genome Sequencing and Analysis Program, Broad Institute of MIT and Harvard,Cambridge, Massachusetts 02142, USA; 2 Department of Infectious DiseaseEpidemiology,SchoolofPublicHealth,ImperialCollegeLondon,LondonW21PG,UK;3Department of Chemistry, University College London, London WC1H 0AJ, UK; 4
Department of Pathology, Bacteriology and Avian Diseases, Faculty of VeterinaryMedicine,GhentUniversity,Salisburylaan133,B-9820Merelbeke,Belgium;5SchoolofBiologyandEcology,UniversityofMaine,Orono,Maine04469,USA; 6DepartmentofEcologyandEvolutionaryBiology,UniversityofMichigan,AnnArbor,Michigan48109,USAEmail:rfarrer@broadinstitute.orgNearlyhalfofallamphibianspeciesaredeclininggloballyduetofactorssuchashabitatlossanddisease.Tworelatedfungalpathogens(Batrachochytriumdendrobatidis;BdandtherecentlyidentifiedB.salamandrivorans;Bsal)havebeenattributedtotheseglobaldeclinesandextinctions.Bycomparingthegenomecontentandgeneexpressionduringinfection,weidentifiedfundamentaldifferencesinthemolecularbasisofpathogenesis.Notably,weidentifiedanewgroupofsecretedgenesinBsalthathavenosequencesimilaritytopreviouslycharacterizedgenes,butarehighlyexpressedinthehost.Usingstructuralpredictiontools,wearestartingtogaincluestoitspossiblefunctions.Additionally,weidentifiedalargeexpansionofmetalloproteasesinBsalcomparedtoBd,geneswhichhavebeenalsobeenreportedinavarietyofdermatophytes.PreliminaryworktosilencethesegenesusingRNAiisbeinginvestigated.Wefoundthatbothofthesecandidatepathogenicityfactorsweredifferentiallyexpressedinthepresenceofthehost,andthatthehostresponseishighlydistincttothetwopathogens.Together,theseanalysesdemonstratethedivergent
23
infectionstrategiesandimmuneresponsetowithinthisamphibian-killinggenus,andtwopossibleroutestofurtherunderstandtheirfunctionalmechanisms.DiversityinplantbiomassdegradationapproachesoffungiRonaldP.deVriesFungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal MolecularPhysiology,UtrechtUniversity,Uppsalalaan8,3584CTUtrecht,TheNetherlandsEmail:r.devries@westerdijkinstitute.nlTherearefewaspectsoffungalbiologythatareasdiverseastheirstrategiestoobtaincarbonandenergyfromtheirnaturalsubstrates.Plantbiomass,andinparticularlignocellulose,isamajorsubstrateformanyfungiduetoitsabundanceinnatureanditshighcontentofmonosaccharides.Theavailabilityof(post-)genomicdatasetshaveprovidedanunprecedentedviewonthestrategiesoffungitodepolymerizeandconvertplant-basedpolysaccharides.Comparativegenomicsrevealedahugevariationingenome(CAZome)contentwithrespecttolignocelluloseactiveenzymesandinmanycasesthesevariationscouldbelinkedtothenaturalbiotopesofthespecies.However,thedifferenceinstrategygoesbeyondthegenomicvariations.RecentstudiesinourlabrevealedthatevencloselyrelatedspecieswithsimilarCAZomesproducesignificantlydifferentenzymesetswhenexposedtothesamesubstrate.Thissuggestssignificantdifferencesintheregulationofthegenesencodingtheseenzymesaswellasapreferencefordifferentcomponentsoflignocellulosebythedifferentspecies.Inthispresentation,majordifferencesobservedacrossthefungalkingdomwillbepresentedandplacedintocontextofbiotopeandlifestyle.Secondly,differencesbetweenspeciesofthegenusAspergilluswillbepresentedasashowcaseforpost-genomicvariation.Finally,Iwilladdresshowtheseinsightscanbeusedtoimprovecommercialenzymecocktails.MycovirusesasprospectiveenhancersofbiocontrolagentsIolyKotta-Loizou1andRobertH.A.Coutts21Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK;2Department of Biological and Environmental Sciences, University of Hertfordshire,Hatfield,AL109AB,UK.Email:i.kotta-loizou13@imperial.ac.ukThecurrentworldpopulationisca.7.5billionandapproximately2billiontons/yearofcropsareneededtocoverournutritionalrequirements.Pesticidesareusedtoprotectthecropsfromdamageandincreaseagriculturaloutput;however,theyareconsideredoneofthetopsixglobalthreatsgreatlyimpactingthebiodiversityoftheenvironmentandputting7millionpeopleatrisk.Environmentallyfriendlyalternativestopesticides
24
arebiologicalcontrolagents,includingcommerciallyavailableentomopathogenicfungiwhosegrowthandpathogenicitymaybeenhancedbymycoviruses.Theultimateaimoftheprojectistoinvestigatemycovirus-inducedhypervirulenceinentomopathogenicfungi.TothisendpanelsofBeauveriabassiana,MetarizhiumanisopliaeandLecanicilliummuscariumwerescreenedforthepresenceofdouble-stranded(ds)RNAelements.Amongothermycovirusesdiscovered,membersofthenovelvirusfamilyPolymycoviridaewereidentifiedasthemostpromisingenhancers,sincetheynaturallyconfermildhypervirulencetotheirhosts,arenotconventionallyencapsidatedandareinfectiousasnakeddsRNAfacilitatinggeneticmanipulations.Themechanismscontrollingmycovirus-inducedhypervirulencearestillunclearbutsuchknowledgewouldfacilitategeneticengineeringofknownmycovirusestoincreasehypervirulence.Off-targetRNAsilencingisexpectedtoplayarolegiventhedsRNAnatureofthesevirusesthatactasbothtriggersandtargetsoftheantiviraldefencesystem.Additionally,host-virusprotein-proteininteractionsviaEukaryoticLinearMotifsleadingtodisruptionofhostpathwaysmayalsobeimplicatedinaccordancewithotherhost-pathogensystems.Inconclusion,thisstudydiscussesthepotentialofmycovirusesasenhancersofbiocontrolagents.Genomic and genetic analysis of a basidiomycete yeast focused on thecharacterisationofMELbiosynthesisStefanySolano1,2,AnnaSobolewska3,DougCossar3,MarkCaddick1&AlistairDarby11Institute of Integrative Biology, University of Liverpool, UK; 2Escuela de CienciasBiologicas,UniversidadNacional,CostaRica;3CrodaEurope,Ditton,Widnes,UKEmail:S.Solano-Gonzalez@liverpool.ac.ukThisworkfocusesonabasidiomyceteyeastknowntobeagrassendophyte.Ofspecificinterestisthestrain’sabilitytoproduceMannosylerithritollipids(MELs).Theseareofinteresttoindustryasbiosurfactantsandareclassifiedintofourgroups(A–D)accordingtotheirstructure.ThebasidiomyceteinthisstudyisprimarilyaMELCproducer.Theregulationandproductionofthesesecondarymetabolitesandtheunderpinningbiologyremainunclear,despitethemanystudiescarriedoutwithbasidiomycota,Ustilagomaydis,andPseudozymaaphidis.CurrentlytherearefewannotatedgenomesforMELproducingfungi.Wethereforeundertookthegenomesequencing,assemblyandannotationofourisolate,asafirststeptoinvestigatingitspotentialforindustrialexploitation.UsingthePacBiosequencingplatformwehaveproducedagenomeassembly,composedof34contigs.Transcriptomesequencinghasbeenutilisedtofacilitategenecalling,withatotalof6603genesbeingidentified,ofwhich4877havebeenautomaticallyannotatedusingprimarydatabases.AmongstthesearethefivegeneswhichformtheputativeMELbiosyntheticcluster.Phylogeneticanalysisshowsthatamongstsequencedfungi,ourstrainismostcloselyrelatedtoS.reilianumandU.maydis(bothplantpathogens)andsurprisinglynotPseudozymaspecies,ashadbeenexpected.Wehaveundertakentranscriptomeanalysisto
25
investigategeneexpressionassociatedwithMELproductionoverafivedaytimecourseandwearecurrentlyusingdirectedgenereplacementtoinvestigatethefunctionofspecificgenesinthebiosyntheticclusterandtheroleofputativeregulatoryproteinsinMELproduction.IntraspeciesdiversityandinductionofthewooddecaybythewhiterotfungusDichomitussqualensSaraCasadoLópez1,WilliamB.Chrisler2,SamuelPurvino2,AliceDohnalkova2,GalyaOrr2,ErrolRobinson2,ErikaZink2,ScottBaker2,KristiinaHildén3,MiiaR.Mäkelä1,3andRonaldP.deVries1,31 Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal MolecularPhysiology, Utrecht University, Utrecht, 3584CT, The Netherlands; 2 EnvironmentalMolecular Sciences Laboratory, Department of Energy, Richland, WA 99354, USA;Department of Food and Environmental Sciences, University of Helsinki, Helsinki, FI-00014,Finland.Email:s.casado@westerdijkinstitute.nlDichomitussqualensisawhiterotfungusthatpreferablygrowsandcolonizesdeadsoftwood,butisalsoabletoutilizehardwood.ItiscommonlyfoundinthenorthernpartsofEurope,NorthAmericaandAsia.Asaresultofsexualcrossingoftwocompatiblemonokaryons,afertiledikaryonisformedandfruitingbodiescanbeproduced.Inthisstudyweaimedtodissectthedifferencesinwoodcolonizationbyasetofmono-anddikaryoticstrainsofD.squalens,toanalyzehowvariedthisprocesscanbewithinonespecies.Forthispurpose,weintegratedacombinationoftranscriptomics,proteomics,metabolomicsandhighresolutionmicroscopyenabledbythecombinedfacilitiesoftheJGIandEMSLinstitutes.Allstrainsweregrownonsolidsprucesticks,andsamplingwasperformedaftertwoandfourweekstoevaluatethevariationbetweenthestrainsduringthewooddecayprocess.Moreover,weperformedtranscriptomicsoftwoofthestrains(mono-anddikaryon)onplantpolysaccharidederivedsugarstostarttounraveltheinductionmechanismofcarbohydrateactiveenzymes(CAZymes)encodinggenesinD.squalens.
26
ExploitingunspecialisedendophytesforbiologicalcontrolofinsectsandplantsAlanC.Gange,AmandaF.Currie,NadiaAbRazakandRuthChittySchool of Biological Sciences, Royal Holloway, University of London, Egham, SurreyTW200EXUKEmail:a.gange@rhul.ac.ukHerbaceousplantsharbourdiversecommunitiesofendophyticfungiintheirrootsandshoots.However,comparedtoendophytesingrassesorwoodyplants,almostnothingisknownabouttheecologyofthesefungi.Ithasbeensuggestedthatastheseendophyteareunspecialisedandubiquitous,thentherelationwiththeirhostsmaybealooseone,withlittleeffectsontheplantitself,orotherorganismsthatattackthehost,suchasinsects.Oneparticulargroupoffungithatcanoccurasendophytesinplantsaretheentomopathogensandthereisincreasinginterestintheuseofthesefungiasinternalplantprotectants.Herewereporttheresultsofameta-analysis,toexaminetheeffectsofunspecialisedendophytes,includingentomopathogens,oninsectherbivores.Wewillcombinethiswithsomeofourownexperimentaldatatoshowthatthesefungimayhavegreatpotentialinbiologicalcontrolofinsectpests.Contrarytoexpectations,someunspecialisedendophytescanalsohavedirecteffectsonthegrowthoftheirhostsintheabsenceofantagonists,andwewillexplorehowsucheffectsmaybeexploitedinthebiologicalcontrolofweeds.RegulationofDrugResistanceinAspergillusfumigatusMichaelJ.BromleyManchester Fungal Infection Group, School of Biological Sciences, University ofManchester,Manchester,UnitedKingdom.Email:Mike.bromley@manchester.ac.ukAspergillusfumigatusisthemostimportantairbornemouldpathogenandallergenworldwide.Estimatessuggestthatover3millionpeoplehaveinvasiveorchronicinfectionsthatleadtoinexcessof600,000deathseveryyear.Veryfewdrugsareavailabletotreatthevariousformsofaspergillosisandwerelypredominantlyontheazoleclassofagents(Itraconazole,Voriconazole,PosoconazoleandtherecentlylicensedIsavuconazole).Resistancetotheazolesisemerging.Forindividualsthatareinfectedwitharesistantisolatethemortalityrateexceeds88%.Therapyfailureisinpartattributedtodelaysinadministeringalternativetherapies,somethodstorapidlydetectresistancearecritical.Whileresistanceinaround50%ofclinicalisolateshasbeenlinkedtomodificationofthegeneencodingthetargetoftheazoles,cyp51A,ourunderstandingofwhatleadstoresistanceintheremainingstrainsislacking.Wehaveundertakenaprogrammeofworktoidentifytranscriptionalandpost-transcriptionalregulatorsofazoleresistance.Wehavediscoveredacohortoftranscriptionfactors,includingtheCCAAT-bindingcomplexandNegativeCofactor2,
27
whichmodulateazoletolerability.Withaviewtounderstandingwhichgenesaredirectlyregulatedbythesetranscriptionfactors,wehaveperformedgenome-wideprotein-DNAinteractionanalysisusingChIP-seq.Wehaveidentifiedthatthesetranscriptionfactorsbindthepromotersofgenesknowntobeassociatedwithazoletoleranceincludingcyp51Athedrugtransportercdr1B.OurChip-seqdataprovidesevidencetosuggestthattheseregulatorsbindthepromotersofmanygeneshoweverdonotalwaysmodulatetheirexpression.SystematicfunctionalannotationoftranscriptionfactorsinthemajormouldpathogenAspergillusfumigatusTakanoriFurukawa,NormanvanRhijn,PanosPapastamoulis,SayemaKhan,LeaGregson,PaulBowyer,MagnusRattray,MichaelBromley,ElaineBignellManchester Fungal Infection Group, Division of Infection, Immunity, and RespiratoryMedicine,UniversityofManchester,Manchester,M139NT,UKEmail:takanori.furukawa@manchester.ac.ukAspergillusfumigatusis,globally,themostimportantairbornefungalpathogenofhumansthatcausesover90%ofallAspergillus-relatedinfections.Ithasbeendemonstratedthatthevirulenceofthisfungusismultifactorialandseveralvirulence-determiningfactorshavebeenidentified.Inparticular,theabilityofthefungustorespondtovarioushost-imposedstresseshasrepeatedlybeenproventobeessentialforpathogenicity,andthereforetranscriptionalregulationplaysacriticalroleinestablishinginfection.However,weonlyhaveaverylimitedunderstandingofthemolecularbasisofA.fumigatuspathogenicity.Inthisstudy,wecarriedoutasystematicphenotypingofatranscriptionfactorknockout(TFKO)collectionofA.fumigatusmutantstodefinethegenomiccohortoftranscriptionfactorsrequiredforpathogenicity.Ahigh-throughputanalyticalpipelinewasdevelopedina96-wellplatebasedliquidcultureformattoanalyzerelativefitnessof401A.fumigatusTFKOstrains.GrowthtraitsoftheTFKOstrainswerethenanalyzedinparallelundereachofthefollowinghost-imposedstressconditions:ironstarvation,temperaturestress,andpHtolerance.Dataanalysiswasautomatedtoconvertopticaldensityreadingscollecteddirectlyfromthespectrophotometerintofitnessindices.Ourstudyidentifies,forthefirsttime,thefullcohortofA.fumigatustranscriptionfactorsthatgovernresponsestosignificanthost-imposedstressesandprovidesafirstgenome-scalefunctionalannotationoftranscriptionfactorsinA.fumigatus.
28
BSPPTalksUsingpopulationgenomicstodeterminethegeneticbasisofpathogenadaptationinagro-ecosystemsBruceA.McDonaldPlant Pathology Group, Institute of Integrative Biology, ETH Zurich, 8092 Zurich,SwitzerlandEmail:bruce.mcdonald@usys.ethz.chSeptoriatriticiblotch(STB)isthemostdamagingwheatdiseaseinEurope.ThepathogencausingSTB,Zymoseptoriatritici,isgloballydistributedandhasevolvedtobecomeresistanttofungicidesandvirulentonresistantwheatcultivars.WecoupledQTLmappingbasedon700progenyfromtwocrosseswithanalysesofgenome-wideassociations(GWAS)basedon150strainsdrawnfromfourglobalfieldpopulationstoelucidatethegeneticarchitectureofseveraltraits,includinghostspecialization,virulenceandfungicideresistance.FinishedgenomesequencesandextensiveRNA-seqprofilesobtainedforallfourparentsenabledidentificationofcandidategenesaffectingeachtrait.Severalofthesegeneswerefunctionallyvalidated.Whilegenotypinghasbecomerelativelyeasy,phenotypingremainsdifficultandlimitsprogressinmostmarker-traitassociationstudies.Wedevelopedhigh-throughputphenotypingmethodstoacceleratebreedingforSTBresistance.Automatedanalysisofdigitalimagesgenerated2.7millionphenotypicmeasurementsassociatedwithmelanization,virulence,hostspecialization,fungicidesensitivityandthermaladaptationintheQTLmappingpopulations.Thisapproachwasextendedtoanaturallyinfectedfieldplantedto335eliteEuropeanwheatcultivars,yielding>15millionphenotypicmeasurementsassociatedwithhostdamage(percentleafareacoveredbySTBlesions)andpathogenreproduction(numberofSTBpycnidiapercm2ofleaflesion)thatrankedtherelativeSTBresistanceamongthesewheatcultivars.AGWASusingthesedatasetsallowedustogeneticallyseparateresistanceaffectinghostdamagefromresistanceaffectingpathogenreproduction.OurepidemiologicalanalysesindicatedthatresistanceaffectingpathogenreproductionoffersthegreatestpotentialforlimitingdamageduetoSTB.WhydoesIPMnotleadtomorereductionsinfungicides?LiseNistrupJørgensenDepartmentofAgroecology,AarhusUniversity-Flakkebjerg,DK-4200Slagelse,DenmarkEmail:lisen.jorgensen@agro.au.dkCerealcropsaccountforthemajoruseoffungicidesinEurope.Despitethepoliticallydrivendesiretoreducedependenceonpesticidesingeneral,ithasproveddifficulttoreducetheconsumptionoffungicidessignificantly.Severalnon-chemicalmethodsare
29
knowntohelpreducediseasesincerealcrops.However,farmers’willingnesstogeneratemajorchangesintheircroppingsystemssuchasincludingnewcropsoradoptingnewtillagemethodsissteeredbyeconomicrationaleandfarmers’abilitytoprovideflexiblemanagement.Croppingvarietieswithgoodresistanceareknownasthemostwidelyadoptedandadvocatedmethodofreducingpathogenattack,andvarietyresistancehasinseveralcasesprovidedgoodpossibilitiesofreducingtherelianceonfungicides.Evenso,incerealcrops,nocultivarswithcompleteresistancetoallrelevantdiseasesexistandfungicidesarecommonlyusedtoensureagainstmajorcroplossesfromdiseases.Changesinpathogenvirulencehaveinmanycasesledtolossofeffectivecultivarresistance,whichagainrequiredadjustedinputoffungicides.AsanelementofIPM,farmersinmanycountriesoftenapplyreduceddoses,althoughtheymostlikelystillapplyhigherdosesthantheoptimumtoensureagainstlossesinseasonswithahighdiseaselevel.Unfortunately,reliabletools,whichcanimprovepredictionoffuturediseasedamageandaccountforthecombinedeffectofintegratedcontrolmeasuresarestilllacking.Ideally,reliablemodelsshouldpredicttheoptimumnumberoftreatmentsandtheoptimumdoseneededtominimizebotheconomiccostsandenvironmentalimpactinagivenseason.Identifyingrootresistancetotake-alldiseaseVanessaE.McMillan,GailCanning,RichardJ.GutteridgeandKimHammond-KosackDepartmentofBiointeractionsandCropProtection,RothamstedResearch,UKEmail:vanessa.mcmillan@rothamsted.ac.ukTake-all(Gaeumannomycestritici)isamajorrootdiseaseofwheatwhichisaseriousconstraintinshortwheatrotations,reducingyieldsof2ndand3rdwheatcropsby1to3tonnes/ha.Distinctivepatchesofdiseasedevelopinthefieldwithstuntedandprematurelyripeningplantswheretherootsystemisseverelyinfected.Moderateandseverediseaseoutbreaksalsoputfieldsatriskofnitrateleachingasexcessunusednitrogenfertiliserisleftbehindinthesoil.AspartoftheWheatGeneticImprovementNetwork(WGIN,www.wgin.org.uk)andWheatImprovementStrategicProgramme(WISP,www.wheatisp.org)protocolshavebeendevelopedtophenotypearangeofdiversegermplasmcollections,includingelitewheatvarieties,landracesandwheatrelativesforrootresistancetotake-alland/ortheireffectonfungalinoculumbuild-upinthesoil.Phenotypingwasprimarilycarriedoutunderfieldconditionswiththeaimofidentifyingresistancethatwasconsistentlyexpressedacrossmultiplefieldseasonsandsites.Since2008over700Watkinshexaploidwheatlandraceshavebeenphenotypedfortake-allresistance.Moderateresistancetotake-alldiseasewasidentifiedforasubsetofgenotypeswithinthecollection.AParagon(moderatelysusceptible)xWatkins777(moderatelyresistant)mappingpopulationiscurrentlybeingphenotypedtodissectthegeneticbasisofresistance.ResultsfromthefirstyearoffieldphenotypingrevealtwoQTLsconferringresistanceinalowdiseasepressuresituation.Workisnowunderwayto
30
confirmandvalidatethesegenomiclocationsacrossfurtherfieldseasonsandtoexploretherootresponseinmoredetail.Ignoreduntilrecently–thestoryofthefungalpathogenLeptosphaeriabiglobosaYongjuHuang,AsnaJavaid,LakshmiH.Gajula,ChinthaniS.Karandeni-Dewage,GeorgiaMitrousia,BruceD.L.FittSchoolofLifeandMedicalSciences,UniversityofHertfordshire,Hatfield,Hertfordshire,AL109AB,UKEmail:y.huang8@herts.ac.ukManyplantdiseasesarecausedbymorethanonepathogenandtherearemanyfactorsaffectingtheirco-existenceontheirhostthatleadtochangesintheirpredominance.Phomastemcankerisagloballyimportantdiseaseofoilseedrape,causedbytworelatedpathogens,LeptosphaeriamaculansandL.biglobosa.SinceL.maculansisoftenassociatedwithstembasecankerwhileL.biglobosaisassociatedwithsuperficialupperstemlesions,L.maculansisconsideredmoredamagingthanL.biglobosa.AlthoughL.maculansandL.biglobosaco-existontheirhost,previousworkhasmainlyfocusedonL.maculansandtheimportanceofL.biglobosainphomastemcankerepidemicshasbeenignored.ResultsofrecentworkshowthatL.biglobosahasincreasedinimportanceintheUK,causingbothdamagingupperstemlesionsandstembasecankers.Furthermore,L.biglobosaislesssensitivetosometriazolefungicidesthanL.maculans.PreviouscontrolofphomastemcankerintheUKbycultivarresistanceorfungicideshastargetedonlyL.maculans,disregardingL.biglobosa.RecentstudieshaveshownthatcultivarsresistantagainstL.maculansareoftenmoresusceptibletoL.biglobosa.Foreffectivecontrolofphomastemcanker,thereisaneedtotargetbothL.maculansandL.biglobosa.Inthistalk,thereasonswhyL.biglobosahasrecentlyincreasedinimportanceinphomastemcankerepidemicsintheUKwillbediscussed,basedonresultsfromfieldexperimentsoverthreeseasonsandcontrolledenvironmentexperiments.*PowderyMildew:ATaleofTwoFungicidesCorinneJ.Arnold,DianeG.O.SaundersandJamesK.M.BrownCropGenetics,JohnInnesCentre,Norwich,NR47UH,UKEmail:Corinne.Arnold@jic.ac.ukBlumeriagraminisisapowderymildewpathogenthatinfectsmanywildgrassesandcerealsacrosstheworld.Ifleftuncontrolled,itcancausesignificantyieldlossesincereals.OnemainmethodforcontrolistheapplicationoffungicidesbutresistancetostrobilurinandoldertriazolefungicidesalreadyoccursinB.graminis,andfewotherfungicidesareavailableformildewcontrol.Ihavecharacterisedisolatesfroman
31
outbreakofB.graminisf.sp.tritici(Bgt)onwheatwheretwoofthemajorfungicidesusedagainstBgtwereineffective:cyflufenamid(Cyflamid),aneradicantfungicidewithanunknownmodeofaction,andfenpropimorph(Corbel),amorpholinefungicidetargetingasterolreductase.AllBgtisolatesfromthestudysitewerecompletelyresistanttoCyflamidsprayedattherecommendedfieldrate.However,overthefollowingtwoyears,theBgtpopulationrecoveredsensitivitytoCyflamid,suggestingthatresistancemayhaveasignificantfitnesspenalty.Additionally,followingtwospraysofCorbel,theseBgtisolateshadsignificantlylowersensitivitytofenpropimorphthanfieldisolates.Overuseoffenpropimorphwouldalmostcertainlyleadtostrongresistanceinmildew,soitshouldbeusedaspartofarotationoffungicideswithdifferentmodesofaction.Likewise,Cyflamidcancontinuetobeusedaspartofanintegrateddiseasecontrolprogramme.Geneticandgenomicanalysisisinprogress,withtheaimofidentifyingcandidategenesforresistancetocyflufenamidandtofenpropimorph.*Blumeriametallo-protease-likeeffector;azincophoreactingasauniversalvirulencefactorinfungi?ShaoliDasGupta1,KateOrman1,MartinUrban2,GabrielScalliet3,KimHammond-Kosack2andLaurenceBindschedler11School of Biological Sciences, Royal Holloway, University of London, Egham, SurreyTW200EX,UK;2DepartmentofPlantBiologyandCropScience,RothamsteadResearch,Harpenden, Hertfordshire AL5 2JQ, UK;3Syngenta Crop Protection, Research Biology,Schaffhauserstrasse,Stein,SwitzerlandEmail:Shaoli.DasGupta.2015@live.rhul.ac.ukBlumeriaeffectorcandidate19(BEC1019)isavirulencefactorofthebiotrophicpathogenBlumeriagraminisf.sp.hordei(Bgh),acausalagentofbarleypowderymildew.BEC1019silencingledtoreducedBghgrowthanddiseasedispersion.BEC1019hashomologuesinonethirdofthesequencedfungalgenomes,includingthezincscavengingvirulencefactorsPRA1(Candidaalbicans)andAspf2(Aspergillusfumigatus).BEC1019homologueslackproteaseactivitybutshareaconservedzincbindingHRXXHdomain,relatedtotheHEXXH-containingM35zincmetallo-proteasesuperfamily.Therefore,BEC1019homologuesarepotentialfungal“zincophores”withauniversalvirulencerole.ThisprojectisinvestigatingthepotentialinvolvementofBEC1019homologuesinzincsequestrationforthevirulenceofeconomicallyimportantwheatfungalpathogens.WewillshowthatBEC1019silencinginB.graminisf.sp.tritici(Bgt)ledtoreducedinfectionofwheatpowderymildew,thusindicatingitsroleinvirulence.Fusariumgraminearum(Fg)andZymoseptoriatritici(Zt)BEC1019knockoutsarebeingproducedtoestimatetheroleofBEC1019duringFusariumheadblightandSeptoriatriticiblotchinfection.SinceBEC1019wasshowntobehighlyinducedduringZtgrowthunderzincdeficiency,theroleofBEC1019andotherfactorsinvolvedinzincsequestrationforpathogensuccessandcounteringplant
32
nutritionalimmunitywillbefurtherinvestigated,inthequestoffindingnoveltargetsforcropprotection.*Understanding the ecology and epidemiology of Pythium violae to enable diseasemanagementincarrotcropsKathrynR.Hales1,GaryDBending2,TimRPettitt3andJohnP.Clarkson11WarwickCropCentre,UniversityofWarwick,Wellesbourne,CV359EF,UK;2SchoolofLife Sciences, University of Warwick, Coventry, CV4 7AL, UK; 3National Pollen andAerobiologyResearchUnit,UniversityofWorcester,Worcester,WR26AJ,UKEmail:k.r.hales@warwick.ac.ukCavityspotisamajordiseaseofcarrotprimarilythoughttobecausedbythesoilborneoomycetepathogenPythiumviolae,althoughotherPythiumspeciesmayalsobeassociatedwiththelesions.Diseasemanagementischallenging,asfungicideefficacyisvariableandlongrotationsnotalwayspossible.NewapproachestocontrolhavebeenhamperedbyalackoffundamentalknowledgeconcerningtheepidemiologyofP.violaeandbasictoolsforresearch.ThisPhDprojectaimstoaddresssomeoftheseissuesbydevelopingmoleculardetectiontechniquesandartificialinoculationmethods.TodeterminetherangeofPythiumspp.associatedwithcavityspotsymptoms,acollectionof150isolateswasassembledfromdiseasedcarrots.FollowingDNAextractionandsequencing,61%wereidentifiedasP.violaewithP.sulcatumandP.intermediumbothaccountingfor14%.TheremaindercomprisedotherPythiumspecies.TodetectP.violaeinrelativelylargesamplesofsoil,asucroseflotation/filtrationmethodwasdevelopedtocaptureoosporesfrom10gsoil.WhencombinedwithaspecificPCRtest,initialresultssuggestedthatdetectionoflessthan10oosporesispossible.ThisapproachisbeingdevelopedfurtherforRT-PCRapplicationtoenableaccuratequantificationofP.violaeandmonitoringofpathogendynamicsinsoilandonroots.AmethodofinoculumproductionwasdevelopedforP.violaeandusedtoinoculatepot-growncarrotsintheglasshouseandsmallfield‘macrocosms’.Initialresultsrevealedeffectsoncarrotgrowthandthedevelopmentoftypicalcavityspotlesionsonroots.*Examining identity, phylogeny and pathogenicity factors in Fusarium speciesaffectingpeaSaschaJenkins1,RichardJ.Harrison2andJohnP.Clarkson11WarwickCropCentre,UniversityofWarwick,Wellesbourne,CV359EF,UK;2Genetics,Genomics&Breeding,NIAB-EMR,EastMalling,ME196BJ,UKEmail:sascha.jenkins@warwick.ac.uk
33
PeaisanimportantlegumecropfortheUK,worthanestimated£50millionperyear.TheUKisalsothelargestproduceroffrozenpeasinEurope,duetoidealconditionsalongtheEasternseaboard.Fieldsarelocatedclosetoprocessingfactories,whichreducesthepotentiallandavailabletogrowpeas.Soilborneplantpathogensinthe‘footrot’complexareoneofthemaincausesofcroplossinpeas,resultinginwilting,reductionsinpodnumbersandplantdeath.SeveralFusariumspeciesaremajorcomponentsofthiscomplexwithF.oxysporumf.sp.pisi(FOP)beingamongstthemostimportantworldwide.RacesofFOParedifficulttodistinguishwiththecurrentmethodofpathogenicitytestsusingpeadifferentiallines.PathogenicityfactorsincludingeffectorgeneshavebeenreportedinotherpathogenicF.oxysporumwithSecretedinXylem(SIX)geneswidelystudied.ThemainaimsofthisPhDprojectaretoidentifytherangeofFusariumspeciesaffectingpeaintheUKandidentifyeffectorgenesassociatedwithpathogenicity.AsurveyofUKpeafieldsidentifiedfootrotcausingspeciesincludingF.oxysporum,F.solaniandF.redolens,withthemostcommonbeingF.oxysporum.TheseisolatescollectedfromUKfieldsandFOPrace-typedisolateswereusedintwopathogenicityteststodistinguishwiltcausingandfootrotcausingisolates.InitialresultsfromwholegenomesequencingandPCRhaverevealedthepresenceof7SIXgenesinFOPraces1,2and5,andfurthertestswillstudytheirexpressionlevelspostinoculation.*Finemapping of a major gene locus for resistance against Pyrenopeziza brassicae(lightleafspot)inBrassicanapusandidentificationofcandidateresistancegenesChinthaniS.KarandeniDewage,HenrikU.Stotz,BruceD.L.FittSchoolofLife&MedicalSciences,UniversityofHertfordshire,Hatfield,Herts,AL109AB,UKEmail:c.s.karandeni-dewage@herts.ac.ukPlantmajorgene-mediatedresistanceisimportantformanagementofdiseasethreatstocrops.Finemappingandcloningofthesegenescanprovideinsightsintohost-pathogeninteractionstoimprovediseasecontrolstrategies.ThisworkontheBrassicanapus–Pyrenopezizabrassicaepathosystemaimstoimproveunderstandingoftheoperationofmajorgene-mediatedresistanceagainsthemibiotrophicplantpathogens.PyrenopezizabrassicaecauseslightleafspotdiseaseonwinteroilseedrapeintheUKaswellasdiseaseonvegetablebrassicas,includingBrusselssprouts.AmajorgenelocusmapstothebottomendofBrassicanapuschromosomeA01.UsingnovelgenomicinformationandmolecularmarkerdataonB.napus,improvedmarkerresolutionandunderstandingofthegeneticbasisofthisresistancecanbeobtained.WeidentifiedthephysicallocationoftheclosestflankingmarkertotheresistancelocusonB.napuschrA01usingmarkersequenceandthesyntenybetweenB.rapaandB.napusgenomes.Singlenucleotidepolymorphism(SNP)markerinformationonthecorrespondinggenomicregionwasobtainedandc.400candidateSNPsspecifictochrA01wereidentified.Initially,38SNPswereselectedfordevelopmentofKASPmarkers,which
34
weretestedonparentallinesoftheDHpopulationthatsegregateformajorgeneresistanceagainstP.brassicae.Polymorphicmarkersbetweenparentallineswereanalysedinthesegregatingpopulationtoimprovethegeneticmapresolution.WealsoanalysedthreedifferentBrassicanapusgenomesequencestoidentifygenecontentinthisgenomicregionandgenepredictionswillbeusedtoidentifycandidateresistancegenesagainstP.brassicae.*Fromthefieldtothelabandbackagain:exploringlegacyeffectsofrootgeneticsandfungicidefortake-allrootdiseasecontrolinwheatrotationsJosephMoughan1,VanessaMcMillan1,RodgerWhite2,JamesMelichar3,DavidRanner3andKimHammond-Kosack11Biointeractions and Crop Protection Department, Rothamsted Research, Harpenden,Hertfordshire, AL5 2JQ, UK; 2 Computational and Analytical Sciences Department,Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK; 3SyngentaUK Limited,Fulbourn,Cambridge,CB215XE,UKEmail:joseph.moughan@rothamsted.ac.ukGaeumannomycestriticiisasoil-borne,rootpathogenofwheat,causingtake-alldisease.Somemodern,elite,winterwheatcultivarspossessagenetictraitthatpromoteslowtake-allinoculumbuild-up(LowTAB)infirstwheat(McMillanetal.,(2011),PlantPathology,60:200-206).Thisleadstoreduceddiseaseifwheatisgrowninthesamefieldthenextyearandhasgiventwotonnes/hayieldadvantagesinseverediseaseyears(McMillan,submitted).Usingfieldtrials,thisresearchinvestigatedifenhancedorsynergisticreductionoftake-allinoculumbuild-up,couldbeachievedwithfoliarapplicationsoftake-allactivefungicide,azoxystrobin,incombinationwithLowTABgenetics.Subsequentdiseasewasmeasuredinsecondwheatoversowexperiments.Tocomplementthis,laboratoryscreenswereperformedtocheckforcommontargetsitemutationstotheazoxystrobinfungicide,innewandhistoricG.triticiisolates.Reducedsecondwheattake-alldiseasewasfoundresultingfromlegacyeffectsinthesoilfromthepreviousyear’sfirstwheattreatments.Individualeffectsoffirstwheatrootgenetics(LowTAB)(P<0.001)andazoxystrobinapplication(P=0.009)wereobserved,butnosynergywasidentified.Firstandsecondwheatchemistryeffectsdidnotcorrelate.Mid-seasonfirstwheatspraysloweredinoculumbuild-up(P=0.014),butonlyearlyfirstwheatsprayseffectivelyreducedsecondwheatdisease.Theseinconsistentfungicideeffectscouldnotbelinkedtotargetsiteresistance,butmaybeduetolowlevelsoffungicideinsensitivityresultingfromactivityofanalternativerespirationpathwayinG.tritici.Weconcludethatinthefield,azoxystrobinalteredthenormaltake-alldiseaseprogressioncycle.
35
ExploitingeffectorsinbreedinganddeploymentofRgenesinpotatoVivianne G.A.A. Vleeshouwers1 , Geert Kessel2, Theo van der Lee2, and Richard G.F.Visser11PlantBreeding,WageningenUniversity&Research,Wageningen,P.O.Box386,6700AJ,TheNetherlands;2Biointeractions,WageningenUniversity&Research,Wageningen,P.O.Box16,6700AA,TheNetherlandsEmail:vivianne.vleeshouwers@wur.nlTheIrishfaminepathogenPhytophthorainfestanscausesthedestructivelateblightdiseaseonpotato(Solanumtuberosum),amajorfoodcropworld-wide.Potatoplantsattainresistanceduetodiseaseresistance(R)genesthathaveco-evolvedwitheffectors,orso-calledavirulence(Avr)genesoftheoomycetepathogen.Sincethegenomicsera,sucheffectorshaveemergedastoolstoaccelerateandimprovetheidentification,functionalcharacterisationanddeploymentofRgenes.UpontheavailabilityofthereferencegenomeofP.infestans,wehavegeneratedagenome-wideinfection-readylibraryofcandidateAvrgenes.TheseareusedinfunctionalassaysfordetectingactivityofRgenes.ThiseffectoromicsstrategyhasacceleratedcloningandspecificityprofilingofRgenesfromwildpotatospeciesandproveneffectiveandcomplementarytoclassicalresistancebreeding.ForvariousR-AVRpairs,wearestudyingtheeffectordiversityandactivitytogetinsightinthemechanismsthatP.infestansemploysforevadingRproteinrecognition.WehavedesignedaPCR-basedmonitoringforcharacterizedAvrgenestodetectvirulentisolatesamonglargesetsofsamplesinfieldtrails.Theseeffectorprofilesareimplementedinanintegratedpestmanagement(IPM)strategy.Withtheseexperiments,weshowthatahigh-throughputspatio-temporalmonitoringofeffectorallelicdiversityinP.infestanspopulationscanenableamoreeducateddeploymentofRgenesinpotato.The RxLR Motif of the Host Targeting Effector AVR3a of Phytophthora infestans IsCleavedBeforeSecretionStephanWawra1,2,, Franziska Trusch1,, AnjaMatena3, Kostis Apostolakis1, Uwe Linne4,IgorZhukov5,6,JanStanek7,WiktorKoźmiński7,IanDavidson8,ChrisJ.Secombes9,PeterBayer3andPietervanWest11AberdeenOomyceteLaboratory,InstituteofMedicalSciences,UniversityofAberdeen,Aberdeen,AB252ZD,UK;2BotanicalInstitute,GeneticalInstitute,UniversityofCologne,50674,Cologne,Germany;3StructuralandMedicinalBiochemistry,CentreofMedicinalBiotechnology,University ofDuisburg-Essen, 45141Essen,Germany; 4Core Facility forMass Spectrometry, Chemistry, Philipps-Universität Marburg, D-35032, Marburg,Germany;5InstituteofBiochemistryandBiophysics,PolishAcademyofSciences,02-106Warsaw,Poland;6NanoBioMedicalCentre,AdamMickiewiczUniversity,61-614Poznań,Poland; 7Biological and Chemical Research Centre (CENT III), Faculty of Chemistry,UniversityofWarsaw,02-089Warsaw,Poland;8ProteomicsFacility,Institute
36
ofMedical Sciences,University ofAberdeen,Aberdeen,AB252ZD,UK; 9 Scottish FishImmunology Research Centre, Institute of Biological and Environmental Sciences,UniversityofAberdeen,Aberdeen,AB242TZ,UKEmail:franziska.trusch@abdn.ac.ukWhenplant-pathogenicoomycetesinfecttheirhosts,theyemployalargearsenalofeffectorproteinstoestablishasuccessfulinfection.Someeffectorproteinsaresecretedandaredestinedtobetranslocatedandfunctioninsidehostcells.ThelargestgroupoftranslocatedproteinsfromoomycetesaretheRxLReffectors,definedbytheirconservedN-terminalArg-Xaa-Leu-Arg(RxLR)motif.However,thepreciseroleofthismotifinthehostcelltranslocationprocessisunclear.HeredetailedbiochemicalstudiesoftheRxLReffectorAVR3afromthepotatopathogenPhytophthorainfestansarepresented.MassspectrometricanalysisrevealedthattheRxLRsequenceofnativeAVR3aiscleavedoffpriortosecretionbythepathogenandtheN-terminusofthematureeffectorwasfoundlikelytobeacetylated.High-resolutionNMRstructureanalysisofAVR3aindicatesthattheRxLRmotifiswellaccessibletopotentialprocessingenzymes.ProcessingandmodificationofAVR3aistosomeextentsimilartoeventsoccurringwiththeexportelement(PEXEL)foundinmalariaeffectorproteinsfromPlasmodiumfalciparum.ThesefindingsimplyarolefortheRxLRmotifinthesecretionofAVR3abythepathogen,ratherthanadirectroleinthehostcellentryprocessitself.Knowyourenemy–engineeringresistanceagainstFusariumwiltinbananaLola Penadez-Gonzales, Rafal Zdrzalek, Christopher Stevens, Peter van Esse, SarahM.SchmidtTheSainsburyLaboratory,Norwich,NR47UH,UKEmail:sarah.schmidt@tsl.ac.ukBananasarenotjustdeliciousfruits,butarethe4thmostimportantfoodcropintheworld.Inmanypartsoftheworldbananasarestaplecropsthatareimportantforfoodsecurity.Currently,thebiggestthreattoworldwidebananaproductionisFusariumwiltdisease,avasculardiseasecausedbythesoil-bornefungusFusariumoxysporumf.sp.cubense(Foc).Inthe1990sanewFocracecalledTropicalRace4(TR4)emergedinSouth-EastAsiathatisspreadingaroundtheglobe.Fusariumwiltcannotbecontrolledchemicallyandbreedingbananasisdifficultbecauseediblecultivarsaresterileanddonotproduceseeds.Inthe2Bladesgroup,Iamexploringnewroutesofengineeringresistanceinbananasthroughabetterunderstandingofthepathogenfocusingonthefollowingquestions;Whichvirulencegenesdoesitneedtoinfectbananas?ArethereresistancegenesknownagainstFusariumwiltdiseaseinotherplantspeciesthatwecanintroduceintobanana?Andfinally,canweuseantagonisticmicrobestocontrolFusariumwiltdiseaseofbananas?
37
TheagronomicpotentialofgenesilencingapplicationAlineKoch1,DagmarBiedenkopf1,ElkeStein1,LukasJelonek2,AlexanderGoesmann2andKarl-HeinzKogel11Department of Phytopathology, Justus Liebig University, D-35390 Giessen,Germany;2DepartmentforBioinformaticsandSystemsBiology,JustusLiebigUniversity,D-35392Giessen,GermanyEmail:karl-heinz.kogel@agrar.uni-giessen.deNon-codingRNAshaveemergedasapowerfulgenetictoolinresearchandpracticaluseinmedicineandagriculture.Thedemonstrationthatagriculturalpests/pathogens,suchasinsects,nematodes,andfungi,arekilledbyexogenouslysuppliedRNAtargetingtheiressentialgeneshasraisedthepossibilitythatnon-codingRNAcanbedevelopedintoanovelclassofpesticides.Recently,weprovidedproof-of-conceptthatdeliveryofdsRNAtargetingthethreecytochromeP450lanosterolC-14α-demethylases(CYP51)genesofthenecrotrophicfungalpathogenFusariumgraminearumeffectivelycontrolsFusariumHeadBlightandRootRotinbarley.1,2Here,wewilldiscussthemodeofactionofdsRNA-mediatedFusariumgrowthinhibitionindifferentexperimentalset-upsincludinginvitroliquidfungalculture,insituleafsprayapplication,andtransgenicdelivery.OtherlabsalsorecentlyshowedthatsprayapplicationsofRNAtoplants,includingArabidopsis,tomatoandtobacco,israthereffectiveincontrollingplantdiseasescausedbymicrobialpathogensandviruses.3,4Moreover,optimizationofRNAdeliverybycertainnanostructure-basedformulationswasdemonstrated.4Basedonthosefindingsandnewunpublisheddatafromourlab,wewillreportonadvancesandsetbacksincontrollingotherfungalpathogensbydsRNAapplication.Altogetherwewillprovideinformationonafundamentallynewplantprotectionstrategy,therebyopeningnovelavenuesforimprovingcropyieldsinanenvironmentallyfriendlyandsustainablemanner.1.Kochetal.(2013)PNAS110:19324-193292.Kochetal.(2016)PLOSPath.DOI:10.1371/journal.ppat.1005901:1be8a6b457c73d5c3.Wangetal.(2016)NaturePlants,doi:10.1038/nplants.2016.154.Mitteretal.(2016)NaturePlants,doi:10.1038/nplants.2016.207Combining proteomics of haustoria and gene silencing to discover key players ofvirulenceandsusceptibilityduringbarleypowderymildewinfection.Kate Orman, Sebastien Lambertucci-Bonnet, Shaoli Das Gupta, Laurence V.Bindschedler1SchoolofBiologicalSciences,RoyalHollowayUniversityofLondon,Egham,TW200EX,UKEmail:Laurence.bindschedler@RHUL.ac.uk
38
Cerealpowderymildews,Blumeriagraminis,arerecognisedasmajorcontributorsaffectingyieldandgrainqualityofwheatandbarley.Usingaproteomicsapproach,Blumeriaeffectorcandidates(BECs),someofwhicharealsodefinedasCandidatesecretedeffectorproteins(CSEPs),werespecificallyassociatedwithhaustoria(Bindschedleretal2009,2011,Spanu2010,Pliegoetal,2013).Haustoriaareatthecentreofthebiotrophicinteraction,developingintheplantepidermiswithoutdestroyingtheinvadedcells.Haustoriaaresurroundedbyanextra-haustorialmembrane(EHM),whichisincontinuumbutdistinctfromthehostplasmamembrane.BecauselittleisknownabouttheEHMcomposition,weinvestigatedtheEHMproteometounravelputativekeyplayersofsusceptibilityandresistance.SinceBlumeriaisanobligatepathogen,wehadtodeviseanewconvenient,cloning-andGM-freeRNAialternativemethodthatdeliverssmall,modifiedoligodeoxynucleotidestowholeleaves,tovalidateeffectorfunctionbytransienthostinducedgenesilencing.Themethodwasequallysuitableforthevalidationofknownsusceptibilitygenes.Usingthissilencingapproach,wechallengedtheroleofaPR5thaumatin-likeproteinisoformwhichinteractswiththeRNAse-likeeffector,BEC1054(Penningtonetal2016)andwhichislikelyassociatedwiththeEHM.Tooursurprise,weestablishedthatthisPR5actsasasusceptibilityfactor,sinceitssilencingledtodecreasedBlumeriainfection.ThisincreaseinhostresistancebytargetingPR5wasassociatedwithadecreasedamountofthecognatemRNAandanincreasedpercentageofinfectedepidermalcellsproducinghydrogenperoxide.Directandindirecteffectsoffungalendophytesonplant-parasiticnematodesAlexanderSchoutenLaboratory of Nematology, Experimental Plant Sciences, Wageningen University,Droevendaalsesteeg1,6708PBWageningen,TheNetherlandsEmail:sander.schouten@wur.nlAlthoughnotorious for theirpathogenicity, it isbecomingevident thatmostFusariumoxysporum isolates encountered in nature are harmless to plants, someofwhich canevenplayabeneficial roleas trueendophytes,meaning that theycancoloniseplantswithouteverprovokingadiseasephenotype,byantagonizingplant-parasiticnematodes.The involved mechanisms in this quantitative antagonism are only superficiallyunderstood. There is evidence that the presence of the endophyte increases plantvigour and induces plant defence responses toward the nematode. Direct negativeeffectsoftheendophyteonnematodeperformanceandbehaviourcannotberuledoutaswell.Metabolites,producedbythefungalnematodecontrolendophyte,F.oxysporum162(Fo162),wereisolatedandfullycharacterized.Oftheelevenresolvedcompounds,sixwereisolatedfromaFusariumspp.forthefirsttime.Toxicityassaysshowedthat,4-hydroxybenzoicacid(4-HBA),indole-3-aceticacid(IAA)andgibepyroneDwerethemostpotenttowardnematodes,beingalmostasgoodasthebestcommercialnematocide.
39
IAAisawell-knownphytohormoneandknowntoplayaroleinplantdevelopmentandtriggeringdefences.4-HBAisreportedtoincreaseabioticstresstolerance.Thisfindingthussuggeststhatindividualmetabolites,producedbyFo162,canhaveadualactivity,i.e.inducingdefencemechanismsand/ortolerancetowardnematodesinplantsanddirectlykillingorcompromisingnematodes.Suchcompoundsmayserveasimportantleadsinthedevelopmentofsustainableandenvironmentalfriendlynematocides.ImprovingPlantResistancetoFungalPathogensbyCalloseModificationTobiasHanak1andChristianA.Voigt21MolecularPhytopathology,BiocentreKleinFlottbek,UniversityofHamburg,Hamburg,22609,Germany;2DepartmentofAnimalandPlantSciences,P3CentreforPlantProductionandProtection,UniversityofSheffield,Sheffield,S102TN,UnitedKingdomEmail:c.a.voigt@sheffield.ac.ukTheplantcellwalliscomplexpolymernetworkthatformstheouterbarrierofplanttissuestothesurroundingenvironment.Itsarchitectureisofgreatimportancetoconferresistancetoinvadingpathogens,whichareamajorthreatforcropyieldandfoodsecurity.Plantdefenceresponsesincludecellwalladaptionatsitesofpathogeninteractionwithadecisiveroleofthecellwallpolymercallose,a(1,3)-β-glucan.Ourrecentresultshaveproventhatmodificationandoptimizationofcallosebiosynthesisprovidesresistancetoinvadingpathogensthroughnetworkformationwiththepreexistingcellulosiccellwall,notinthemodelplantArabidopsisthalianabutalsoinBrachypodiumdistachyonandwheat.Theapplicationoflocalizationmicroscopy,thefastestevolvingtechniqueinsuper-resolutionfluorescencemicroscopy,allowedvisualizationofpolymerrearrangementsinnanoscale3Dnetworksandfacilitatedinsitustructuralanalysisofcallosesynthasecomplexesinlivecellimaging.Withthis,insitulivecellsuper-resolutionmicroscopyrepresentsanewtoolinbridgingthegapbetweenthemolecularandsystemslevelinplantpathology.Understandingthesubcellularandmolecularmechanismsofplantcellwallbiosynthesisandarchitecturealsoonastructurallevelwillleadtotheidentificationofkeymodulatorsandfactorsforcellwallengineeringtosuccessfullydesigncropswithimprovedpathogenresistance.Understanding the genetic control of pathogenicity and resistance for FusariumoxysporuminonionA. Taylor1, A.C. Jackson1, G. Teakle1, A.D. Armitage2, R. de Heer3, N. Zhang3, W. vanLeeuwen3,S.Ott4,L.Baxter4,B.Greenfield2,R.J.Harrison2andJ.P.Clarkson11WarwickCropCentre,UniversityofWarwick,Wellesbourne,Warwick,CV359EF,UK;2 NIAB-EMR, New Road, East Malling, Kent, ME19 6BJ, UK; 3 Hazera Seeds B.V.,Schanseind 27, 4921 PMMADE, The Netherlands; 4University ofWarwick, Coventry,CV47AL,UK.
40
Email:andrew.taylor@warwick.ac.ukBulbonion(AlliumcepaL.)isagloballyimportantcropbutthelackofagenomesequencehashamperedgeneticresearch.Fusariumoxysporumisanimportantsoilbornepathogenofmanycropsandcomprisesdifferentformaespeciales(f.spp.)thatinfectparticularhosts.Inonion,F.oxysporumf.sp.cepae(FOC)isamajorconstrainttoworldwideproduction,principallycausingabasalrotofbulbswithestimatedlossesof£11MperannumintheUK.AsFOCproduceschlamydosporesthatsurviveinsoilformanyyears,diseasemanagementischallengingandthereforedevelopingresistantcultivarsishighlydesirable.Initially,onionlineswithhighlevelsofbasalrotresistancewereidentifiedwithinauniquediversityset.NewonionpopulationssegregatingforFOCresistancewerethendeveloped,genotypedusingKASPmarkersandQTLsidentifiedlinkedtoresistance.Associatedgeneticmarkersarenowbeingdevelopedtoacceleratebreedingofbasalrotresistantvarieties.Inparallelresearch,thegeneticbasisforpathogenicityinFOCwasinvestigatedandthepresenceofputativeeffectorsidentifiedbygenomesequencingandPCRusingarangeofpathogenicandnon-pathogenicF.oxysporumisolatesfromonion.Thepresenceof‘secretedinxylem’(SIX)effectorgenes,whichhavehomologsinotherF.oxysporumf.spp.,wasclearlyassociatedwithpathogenicityandthesegeneswerestronglyupregulatedduringinfection.EffectorgenecomplementsandsequencesmayalsoprovidethebasisforadiagnostictestforFOC.Knock-outmutantstrainshavenowbeendevelopedforseveraleffectorgenesasafirststeptowardfunctionalanalysis.Understanding appressoriumdevelopment inMagnaporthe oryzaeusing analysis ofnetwork-extractedontologiesandpathogentranscriptomics.RyanMAmesWellcomeTrustCentreforBiomedicalModellingandAnalysis,LivingSystemsInstitute,UniversityofExeter,StockerRoad,Exeter,EX44QD,UKEmail:r.ames@exeter.ac.ukMagnaportheoryzaeisthecausalagentofriceblastdisease,themostimportantinfectionofriceworldwide.Halftheworld’spopulationdependsonriceforitsprimarycaloricintakeand,assuch,riceblastposesaseriousthreattofoodsecurity.ThestagesofM.oryzaeinfectionarewelldefined,withtheformationofanappressorium,acelltypethatallowspenetrationoftheplantcuticle,particularlywellstudied.However,manyofthekeypathwaysandgenesinvolvedinthisdiseasestageareyettobeidentified.Inthiswork,Ihaveusednetwork-extractedontologies(NeXOs),hierarchicalstructuresinferredfromRNA-Seqdata,toidentifypathwaysinvolvedinappressoriumdevelopment,whichinturnhighlightsnovelgeneswithpotentialrolesinthisprocess.ThisstudyillustratestheuseofNeXOsforpathwayidentificationfromlarge-scalegenomicsdataandalsoidentifiesnovelgenesandpathwayswithpotentialrolesindisease.Themethodspresentedherewillbeusefultostudydiseaseprocessesinother
41
pathogenicspeciesandthesedatarepresentpredictionsofnoveltargetsforinterventioninM.oryzae.
Integrated deployment of host resistance and fungicides: managing evolution ofvirulenceandinsensitivityFrankvandenBoschDepartment of crop protection, Rothamsted Research, Harpenden, AL5 2JQ, UnitedKingdomEmail:Frank.vandenbosch@rothamsted.ac.ukSustainablecontrolofplantpathogensismaintainedbytwohighlydynamicinteractions:firstlybetweentheintroductionofnewhostresistancegenesandpathogenpopulationsovercomingthem;secondlybetweentheintroductionofnewfungicidemodesofactionandpathogenpopulationsbecominginsensitivetothem.Virtuallyallstudiesonsustainablediseasecontrolstudyoneofthecontroloptionsexclusively.Thisissurprising,giventhewidelyheldbeliefthatintegratedcontrolwillleadtomoredurablecontrolthanrelianceononecontroloption.Inpracticebothdiseasecontroloptionsareintegrated,buttheeffectsofintegrationonthesustainabilityofdiseasecontrolarenotsufficientlyunderstoodtopredictwhichintegrationstrategieswillbemosteffective.Weintroduceagoverningprinciplethatpredictsthedirectionofselectionforfungicideinsensitivityandthedirectionofselectionforpathogenvirulenceunderarangeofdiseasemanagementoptions.Thepredictionsarecomparedtoexitingevidence.Thisleadsustoformulatetwostrategiesimprovingdurablediseasecontrol:
1. Deploymentoffungicidesreducesselectionforvirulence.2. Deploymentofcropresistancereducestheselectionforfungicide
insensitivity.Usingthismechanisticunderstandingabouttheeffectofintegrationthenleadsustohypothesisethatitismoresustainabletointegrateandbalancechemicalandgeneticcropprotection,thantoattempttoreplacechemicalwithgeneticcontrolorrelyentirelyonchemicalcontrol.Resultsofmodellingstudiesontwocontrastingpatho-systemswillbeusedhere(andinthepaperbyPaveley)toexplorethepracticalimpactofexploitingthemutualprotectionmechanism.Integrated deployment of host resistance and fungicides: economics, efficacy andevolutionNeilD.PaveleyADAS,HighMowthorpe,Duggleby,Malton,NorthYorkshire,YO178BP,UKEmail:neil.paveley@adas.co.uk
42
Theaimofdiseasecontrolistoprotectmarketableyieldinordertominimisetheunitcostofcropproduction.Butcontrolbyhostresistanceandfungicidescreatesselectionpressureforpathogenevolutiontowardsvirulenceandfungicideinsensitivity.Thecommercialandsocietalneedistobalancebetweenachievingcost-effectivecontrolintheshorttermandmanagingpathogenevolutiontoensurecontrolisdurableinthelongterm.TheabilitytocontroldiseasesinsomemajorEuropeancropsistenuouslybalancedcurrently.FrankvandenBosch(theseproceedings)describeswhyintegratinggeneticandchemicalcontrolislikelytobemoredurablethaneitheralone.Farmuptakeofintegratedcontrolinmajorcropsdependslargelyontheshort-termeconomics(yieldvalueminusfungicidecosts)ofgrowingdiseaseresistantorsusceptible,butsometimeshigheryielding,cultivars.Thechallengeforpathologists,cropphysiologistsandplantbreedersistominimisethe‘yielddrag’,whichcanbeassociatedwithbreedingfordiseaseresistance,sothatresistantvarietiesaremoreeconomicallyattractive.Datafromfieldexperimentsandanalysisbymathematicalmodellingshowthat:(i)thecombinedefficacyofhostresistanceandfungicidesispredictableusingasimplemultiplicativesurvivalapproach,enablingthedesignofoptimalintegratedcontrolprogrammes,(ii)diseaseforecastingprovidesgreatereconomicbenefitsonpartiallydiseaseresistant,thansusceptible,varieties,and(iii)thecombinationofvarietyresistanceandforecastingtoguidefungicidetreatment,canreducetheselectionpressureforevolutionoffungicideinsensitivestrains,thusincreasinglongtermdurabilityofcontrol.Fenpicoxamid(InatreqTMactivefungicide),anewfungicidewithutility incereals forcontrolofZymoseptoriatritici(Septorialeafblotch)andotherdiseasesAndrewJ.Leader1andGregoryMKemmitt21Dow AgroSciences, CPC2 Capital Park, Cambridge, United Kingdom; 2 DowAgroSciences,3BParkSquare,MiltonPark,Abingdon,Oxfordshire,UnitedKingdomEmail:aleader@dow.comFenpicoxamid(Inatreq™Active)isthefirstmoleculefromanewclassoffungicides(picolinamides).FenpicoxamidisderivedfromanaturalcompoundUK-2AwhichisproducedbyfermentationofanActinomycete(Streptomycesspp.).UK-2AissubjecttominoralterationpostfermentationtoproduceFenpicoxamidandimpartadditionalstabilitytothemolecule.InatreqinhibitsfungalrespirationinthemitochondriaattheQiubiquinonereductasebindingsiteincomplexIII,whichisanewtargetsitewithinthecerealfungicidesegment.Mostimportantly,Inatreqshowsnocross-resistancetoanyexistingcerealfungicidechemistries,includingtriazoles,strobilurinsorSDHIsandwillbeanimportantnewtoolinthearmouryofcerealgrowers.IntheEuropeancerealsmarket,Zymoseptoriatritici(synonymMycosphaerellagraminicola,Septorialeafblotch),thepathogenofgreatestconcernintermsofyieldlossesinwinterwheatproduction,hasdevelopedwidespreadresistancetostrobilurinsclassandsignificantsensitivitylosstothetriazoles.Recentlyisolateswithreduced
43
sensitivitytoSDHI’shavebeendetectedintheUKandIreland.Anadditionalconcernisthepossibilitythatsomemoleculesthatarewidelyusedtodaywillbeunderregulatorypressureandmayexitthemarketthusreducingthenumberofsolutionsavailabletogrowers.FenpicoxamidwillbeaninnovativeandeffectivetoolinhelpingfarmerswhofaceincreasedresistancethreatsfromZ.triticiwithexistingtechnologies.However,asfenpicoxamidisasinglesiteinhibitor,theproductshouldonlybeusedincombinationwithotheractivestominimizetheriskofresistancedevelopment.ControllingStrawberryPowderyMildewrequiresallyearroundvigilanceHall,A.M.,Jin,X.,Liu,B.BiologicalandEnvironmentalSciences,UniversityofHertfordshire,Hatfield,AL109ABEmail:a.m.hall@herts.ac.ukStrawberryPowderyMildewcausedbyPodosphearaaphanisisthemostfeareddiseaseofprotectedstrawberriesintheUK,causinglossesof20%to70%ofthecropperannum,whichresultsinthelossof£56.8millionwithonly20%loss.WorkattheUniversityofHertfordshiresince2003/4hasresultedinthedevelopmentofanallyearroundcontrolstrategy,which,iffollowedbygrowers,canresultinsatisfactorydiseasecontrol.Commercially,therearemanydifferentgrowingsystemsforstrawberries;insomecasestheplantsare‘perennial’,plantedfor3wintersand4harvests,inothercasestheyareplantedinFebruary/MarchandgrownuntilSeptember/October.Inthecaseofoverwinteredcrops,thediseasecanoverwinteraschasmothecia,whereasplantsarrivingfromthepropagatorsarerarelyclean.Controllingthisinitialinoculumisvital.ThereisheavydiseasepressureinJuly,AugustandSeptember,buttheepidemicscanbereducedbya)theuseofasiliconnutrientandb)theuseofapredictionsystembasedondisease-conduciveconditionsoftemperatureandhumidity.ChasmotheciastartformingonleavesinAugustandSeptemberandgiverisetotheoverwinteringinoculumontheperennialcrops.Thesecanbecontrolledbyusingafungicideafterharvesthasfinished,andagaininthespring,asthecropiscoveredwithfleece.Theresearchreportedherehasresultedinanupdatedfactsheetforgrowers,whichincludesanintegratedprogrammeforallyearrounddiseasecontrol.
44
ClosingPlenaryTalksTheScienceofQuornRobJohnsonScienceManager-FermentationSpecialist,Quorn,StationRoad,Stokesley,NorthYorks,TS97BR,UKEmail:rob.johnson@quornfoods.comQuornhasamere50yearhistoryandisthefirstnewfoodtypetoenterthemarketsincethepotato.ThistalkwillgiveanoverviewofthehistoryofQuorn,itsrelevancetothemodernworldandmoveontothesciencebehindtheproduct,includinghowitismade,wherethetexturecomesfrom,andwhatchallengesarebeingaddressedforthefuture.Coveringgenetics,fermentation,environmentalimpactandhealthbenefitsanoverviewofoneofthefastestgrowingfoodproductsintheworldwillbeoutlined.FungalweaponstomanipulateplantsNataliaRequena,MeikeHartmann,RubenBetz,SvenHeidtandCarolinHeckMolecularPhytopathologydepartment,KarlsruheInstituteofTechnology,Karlsruhe,76228,GermanyEmail:Natalia.requena@kit.eduThearbuscularmycorrhiza(AM)symbiosisbetweenGlomeromycotinafungiandmostplantrootsisoneofthemostancientandwidespreadsymbiosesonearth.AMfungiprovidephosphatetotheirhostsinexchangeforcarbonandtheestablishmentandmaintenanceofthesymbiosisrequiresacomplexcommunicationexchangebetweensymbiontsandwiththeenvironment,becausehighconcentrationsofPiinhibitthesymbiosis.Interestingly,AMfungiareabletocolonizethemajorityoflandplantsincontrasttomostpathogenicfungithathaveaquitenarrowhostrange.WehypothesizethatAMfungiemployconservedeffectorproteinstorewiretheplantphysiology.WepreviouslyidentifiedtheeffectorproteinSP7andagroupofrelatedproteins(SP2,SP5andSP31)fromRhizophagusirregularis.Allmembersofthisfamilyshowthesamebasicproteinstructurewithtandemhydrophilicandimperfectrepeatsandanuclearlocalizationsignal.Hereweshowthatallthreeeffectorsredundantlymodifytheplantphosphatestarvationresponse(PSR).Expressioninrootsofanyofthemleadstoaccumulationofpre-miRNA399sinleavesthatultimatelydownregulatesubiquitinE2conjugasePHO2inrootscausingincreasedPSR.Weproposethatthismechanismallowsthefungustocolonizetherootsystemfurtherdespiteanet-increaseinphosphate.InteractionandlocalizationassayssuggestthattheseeffectorsmodulatethePSRbyintersectingwiththeplantmRNAtranslationmachinery.Theseresultshighlightthefinesseofsymbioticarbuscularmycorrhizalfungitomanipulatetheplantcellprogram,
47
Effects of Leaf Extracts ofOcimum gratissimum L. on Quality of Fresh Cut CucumissativusL.OluwagbengaO.Adeogun,AlfredMaroyiandAnthonyJ.AfolayanDepartmentofBotany,UniversityofFortHare,Alice,EasternCape,SouthAfricaEmail:adeogunoluwagbenga@gmail.com;oadeogun@ufh.ac.zaHighmoisturecontentandminimalprocessingoffruitsandvegetablesencouragemicrobialincursionandtheseraisedtheneedforpreservation.Ocimumgratissimumleaveswereextractedwithethanolandhexaneseparately.TheextractswereusedtoenhancethequalityoffreshcutCucumissativusstoredfor9daysusingdippingmethod.QualitativeandquantitativephytochemicalanalysesoftheextractsweredeterminedandessentialoilconstituentswereestablishedusingGC-MS.TheresultsshowedthatthemoisturecontentsofC.sativuswithextractswerelowercomparedtostoredC.sativuswithoutextracts.TheacidityofC.sativuswithextractswaslowerinrelationtothetestsamplewithoutextracts.TheincreaseintotalsolublesolidwashigherinC.sativuswithouttheextracts.Theascorbicacid,carotenoidsandphenoliccontentsofstoredC.sativuswithextractshadlowerrangeofreductioncomparedtoC.sativuswithouttheextracts.ThedifferencesinpotentialbrowningandturbidityofstoredC.sativuswithextractsduringstoragewerenotobvious.TherewerehigherloadsofmicrobesinC.sativuswithoutextractscomparedtoC.sativusstoredwithextracts.Theresultofthephytochemicalscreeningshowedthepresenceofflavonoid,alkaloid,cardiacglycoside,phenol,tannin,steroid,anthraquinoneandsaponinintheleaf.Someoftheessentialoilconstituentsareγ-Terpinene,Caryophyllene,Oleicacidand(E)-9-Octadecenoicacid.ThisstudywasabletoestablishthattheleafextractsofO.gratissimumhavebio-preservationpotentialthatcanbeusedtoenhancetheshelfofC.sativus.IntegratingfungicideswithvarietalresistanceagainstSeptoriatriticiblotchinwheatOlubukola.O.Ajigboye,DimitraJ.Angelopoulou,JohnFoulkes,Rumiana.V.RaySchool of Biosciences, University of Nottingham, Sutton Bonington Campus,Loughborough,Leicestershire,LE125RD,UnitedKingdomEmail:bukky.ajigboye@nottingham.ac.ukSeptoriatriticiblotch(STB),causedbytheascomycetefungusZymoseptoriatritici,isthemostimportantfoliardiseaseofwheat,reducinggrainyieldbyupto50%.Symptomsoftenoccurfollowingalatentphasebetween5-10daysbeyondwhichfungicideeffectivenessisimpaired.Varietalresistanceisnotyetfullyutilisedinfieldalthoughtherearenumberofcommerciallyavailablewheatcultivarswithmoderatetohighfieldresistance.IntegratingpreventativefungicideapplicationswithvarietalresistanceisessentialtooptimizecontrolofSTB.TheobjectivesofthepresentstudyweretodeterminethebesttimingandfungicideagainstSTBfortwodifferentwheatvarieties,onesusceptiblecv.Dickensandtheresistantcv.Cougar.Fieldexperimentswere
48
conductedduringtwoseasons(2016/17)usingnaturalinfectionbyZ.tritici.Combinationsoffungicidesincludingmulti-site,demethylationandsuccinatedehydrogenaseinhibitorswereappliedatdifferentcropgrowthstages(GS)30,31/32,33,37/39or59/61.TheeffectoftimingandfungicidetreatmentontheprogressandseverityofSTBinthecanopywereassessedatGS31,61and75whilefungalbiomassofZ.triticiwasquantifiedinasymptomaticflagleavesatGS61.Theseparametersalongwiththecropphotosyntheticefficiency,biomassandfinalgrainyieldwereusedtoidentifythebeststrategiesforintegratingfungicidetiming,modeofactionandvarietalresistance.AcknowledgmentsThisstudyispartofcollaborationworkbetweentheUniversityofNottingham,AgriiandRAGTSeeds,co-fundedbyInnovateUK.*PowderyMildew:ATaleofTwoFungicidesCorinneJ.Arnold,DianeG.O.SaundersandJamesK.M.Brown.CropGenetics,JohnInnesCentre,Norwich,NR47UH,UKEmail:Corinne.Arnold@jic.ac.ukBlumeriagraminisisapowderymildewpathogenthatinfectsmanywildgrassesandcerealsacrosstheworld.Ifleftuncontrolled,itcancausesignificantyieldlossesincereals.OnemainmethodforcontrolistheapplicationoffungicidesbutresistancetostrobilurinandoldertriazolefungicidesalreadyoccursinB.graminis,andfewotherfungicidesareavailableformildewcontrol.IhavecharacterisedisolatesfromanoutbreakofB.graminisf.sp.tritici(Bgt)onwheatwheretwoofthemajorfungicidesusedagainstBgtwereineffective:cyflufenamid(Cyflamid),aneradicantfungicidewithanunknownmodeofaction,andfenpropimorph(Corbel),amorpholinefungicidetargetingasterolreductase.AllBgtisolatesfromthestudysitewerecompletelyresistanttoCyflamidsprayedattherecommendedfieldrate.However,overthefollowingtwoyears,theBgtpopulationrecoveredsensitivitytoCyflamid,suggestingthatresistancemayhaveasignificantfitnesspenalty.Additionally,followingtwospraysofCorbel,theseBgtisolateshadsignificantlylowersensitivitytofenpropimorphthanfieldisolates.Overuseoffenpropimorphwouldalmostcertainlyleadtostrongresistanceinmildew,soitshouldbeusedaspartofarotationoffungicideswithdifferentmodesofaction.Likewise,Cyflamidcancontinuetobeusedaspartofanintegrateddiseasecontrolprogramme.Geneticandgenomicanalysisisinprogress,withtheaimofidentifyingcandidategenesforresistancetocyflufenamidandtofenpropimorph.
49
**Use of bulk segregant analysis to analyse azole drug resistance in AspergillusfumigatusGeorgeD.Ashton1,CélineM.O'Gorman1,DanielZadik2,MartinBlythe2,VictoriaWright2,SimonV.Avery1andPaulS.Dyer11School of Life Sciences, University of Nottingham, Nottingham, NG7 2UH, UnitedKingdom;2DEEPSeq facility, University of Nottingham, Nottingham, NG7 2UH, UnitedKingdomEmail:stxgdas@nottingham.ac.ukAspergillusfumigatusisanopportunistichumanfungalpathogencausingaspergilliosis.Infectionsarecontrolledthroughtheuseofazoleantifungaldrugs.Overthepast20yearstherehasbeenanincreaseinoccurrenceofazoleresistantstrains.Resistanceisduetoavarietyofdifferentgeneticmechanisms,someofwhichhavebeencharacterizedinclinicalandenvironmentalisolates.A.fumigatuswasoncethoughttoreproduceentirelyasexually.However,asexualcyclewasdiscoveredin2009.Thefungalsexualcycleisapowerfultoolandcanbeexploitedinanumberofdifferentareas.ItwashypothesizedthesexualcycleinA.fumigatusmightbeusedtoidentifyazoleresistancegenesusingthetechnique‘bulksegregantanalysis’(BSA)incaseswherethemechanismofresistanceisunknown.TheprocessofBSAinvolvesasexualcrossbeingperformedbetweenanazoleresistantandazolesensitivestrain.Followingthis,aseriesofbackcrossesbetweentheresistantprogenyandsensitiveparentareperformedtominimizegeneticdifferencesbetweenthem,withtheazoleresistancegene(s)andanylinkedgenesbeingtheonlydifferingregions.GenomesequencingisthenperformedontwopoolsofDNA:oneofprogenyexhibitingazoleresistance;andoneshowingazolesensitivity,aswellasgenomesequencingofthesensitiveparent.Fromthis,theaimistoidentifykeyareasandgenesthatmaybecausingazoleresistanceintheseisolates.Thismayleadtothedevelopmentofusefuldiagnosticstomonitorazoleresistanceandimprovethetreatmentofazole-resistantA.fumigatusinfections.*An investigation into silicon localization in strawberryplants linked to reduction indiseasesusceptibilitytoPodosphaeraaphanisC.I.Asiana,A.M.HallandK.DaviesDepartment of Biological and Environmental Sciences, School of Life and MedicalSciences,UniversityofHertfordshire,Hatfield,AL109AB,UKEmail:i.asiana@herts.ac.ukThemostimportantdiseaseofprotectedstrawberriesintheUKisstrawberrypowderymildewcausedbyPodosphaeraaphanis,whichhastobecontrolledbyfrequentuseoffungicides.Siliconisnotconsideredasessentialfortheplant;however,workcarriedoutattheUniversityofHertfordshirehasshownthatweeklyuseofasiliconnutrientinfertigationtubesatacommercialstrawberryfarmresultsinreducedsusceptibilitytothisdisease.Siliconcanonlybetakenupinabioavailableformandthenutrientusedin
50
thisexperimentisabioavailableformofsilicon.Asiliconlocalizationexperimentwasconducted.Siliconwasappliedviathesoilintheglasshouse,thefertigationtubesatthecommercialfarmandwassprayedoninafieldplot.Inthisstudy,crosssectionsofleaves,petiolesandrootswerestainedwithafluorescencedye,“lysotrackeryellowHCK-123”.Sampleswereexaminedusingaconfocalmicroscopeatx400andawavelength450nm.Sofar,resultsshowedintheleaf,siliconwasfoundinthecuticle,epidermis,palisadelayer,stomataandvasculartissue.Inthepetiole,siliconwasfoundintheepidermisandxylemandintheroots,thesiliconwasfoundintheepidermis,endodermisandvasculartissue.Thefluorescenceintensityofthecrosssectionswasquantifiedandthisshowsthatthesiliconwas5timeshigherinthetreatedplantsthantheuntreated.Inaddition,thesiliconfertigationfieldtrialhasshownthatplantswithhigherlevelsofsiliconarelesssusceptibletothedisease.Chrysanthemum ChloroticMottle Viroidmediated Trafficking of ForeignmRNA intoChloroplastsEseulBaek1,MinjuPark1,Ju-YeonYoon2andPeterPalukaitis11Dept. of Horticultural Sciences, SeoulWomen’sUniversity, Seoul, 01797, Republic ofKorea; 2National Institute of Horticultural & Herbal Science, RDA, Wanju, 55365,RepublicofKoreaEmail:eseul@swu.ac.krChrysanthemumchloroticmottleviroid(CChMVd),amemberofthefamilyAvsunviroidae,wasidentifiedinchrysanthemumplantscausingchloroticleafsymptoms.ToshowthatCChMVdcantrafficintochloroplasts,whichisthoughttobethesiteofitsreplication,CChMVdwasfusedtotheleadersequenceofareportergeneencodingmonomerredfluorescenceprotein(mRFP)andexpressedtransientlyinagroinfiltratedNicotianabenthamiana.FluorescencefrommRFPwasobservedinchloroplastsbyconfocalmicroscopy,butonlyiftheviroidtranscriptionfusionswerepresent.Theviroidcomponentsofthefusionswerefull-length400-ntCChMVd,oroneoftwopartialfragments(nucleotides125to2and231to372).ThemRFPanditsmRNAweredetectedbywesternblottingandRT-PCR,respectively,inwholetissueextractsofplantsinfiltratedbyeachfusionconstruct.IsolatedchloroplastswereshownbyRT-PCRtocontaintheRNAsequencesofbothCChMVdandmRFP,ifbothwerepresent,butnotthemRFPsequenceintheabsenceoftheviroidsequences.Eggplantlatentviroid(ELVd),inthesamefamilyAvsunviroidae,hasbeenshowntotrafficmRNAintochloroplastsforexpressionofafluorescentmarkerproteininpreviousstudies(1-3).SincetherearedifferencesinsequencebetweenELVdandCChMVd,itsuggeststhattheabilityofbothRNAstotrafficRNAsintochloroplastswasprobablyduetoanRNAstructure,andnotaparticularsequence.1.Gómez,G.,Pallás,V.(2010).PLoSOne5(8):e12269.2.Gómez,G.,Pallás,V.(2010).PlantSignl.Behav.5(11):1517-1519.3.Gómez,G.,Pallás,V.(2012).PlantPhysiol.159:558-564.
51
WorkwasfundedbytheRuralDevelopmentAgencyoftheRepublicofKorea.Generation ofwheat plasmamembrane reporter lines to further understand fungalinfectionmechanisms.Laura Baggaley1, Alison Huttly1, Caroline Sparks1, Smita Kurup1, Christine Faulkner2,MichaelDeeks3andKimHammond-Kosack11 Rothamsted Research, Harpenden, AL5 2JQ, UK; 2 John Innes Centre, Norwich, NR47UH,UK;3UniversityofExeter,Exeter,EX44QD,UKEmail:laura.baggaley@rothamsted.ac.ukThefungus,Fusariumgraminearum,istheprimarycauseoffloralcerealinfectionsworldwide,resultinginreductionsinwheatgrainquality,quantityandadverselyaffectinghuman,animalandecosystemhealth.HowF.graminearumestablishesinfectionwithinwheathasbeenwellstudied,howeverwhatcausesthetransitionbetweentheasymptomaticandsymptomaticphasesremainsunclear.Thistransitionininfectioncorrespondswithashiftfromprimarilyinter-tointracellularfungalgrowth,leadingtoextensivecelldeath.Ithasbeenhypothesisedthatthischangeinfungalgrowthisduetotheutilisationofplasmodesmata–thinstreamsofcytoplasmbetweenadjacentplantcellwallsvitalforcell-to-cellcommunication.IdentificationofthecellularandmolecularmechanismsinvolvedintheF.graminearum-wheatfloralinteractionhaspreviouslyrelieduponhistologicalstainingandfungalreporterstrains[1].Wehaveattemptedtocreatetwowheatplasmamembranereporterlines,usingthegenesROP7[2]andLTI6b,toallowforamoreefficient,detailed,spatio-temporalstudy,oftheinfectionprocess.Duetotheautofluorescentnatureofwheattissues,tissuespecificautofluorescentprofileshavealsobeenestablishedusingconfocalmicroscopy,toaidimagecaptureandanalysis.Thisnewinformationwillbecriticalfortheevaluationofthewheatplasmamembranereporterlinesandfungalreporterstrains.Togetherthesenewtoolsanddatasetswillallowfortheidentificationandcharacterisationofinfectionmechanismsinwheat,allowingforagreaterunderstandingofmultiplecrop-yieldlimitingpathogens.[1]Brownetal.(2010)FungalBiology114:555-571[2]Kirienkoetal.(2012)PlantPhysiology159:1309-1318
52
Howtocontrolleafdiseasesoffababeans?BirutaBankina,GunitaBimšteineandArtursKatamadzeInstitute of Soil and Plant Sciences, Latvia University of Agriculture, Jelgava, LV 3001,LatviaEmail:Biruta.Bankina@llu.lvFababean(ViciafabaL.varminor)hasbecomeanimportantfieldcropinLatviaduringthelastfiveyears.FourBotrytisspeciesasthecausalagentsofbeandiseaseshavebeendetermined:B.fabae,B.fabiopsis,B.cinerea,andB.pseudocinerea.Also,severalspeciesofAlternariaandStemphyliumhavebeenfound.Theaimofthepresentstudywastoclarifytheefficacyoffungicideapplication.TrialswereconductedattheResearchandStudyfarm“Peterlauki”oftheLatviaUniversityofAgriculturein2015–2016.Athree-factorfour-replicatetrialwasestablished:factorA–variety(‘Laura’,‘Boxer’,‘Isabell’);factorB–sowingrate(30,40,50germinatingseedsperm2);andfactorC–fungicide(boskalid267.0gkg-1andpyraclostrobin67.0gkg-1)0.75kgha-1atthebeginningofflowering.Diseaseassessmentwasstartedwhenthefirstsymptomsoccurred:20randomplantswerechosenineachplot,anddiseaseseveritywasnoted(0–9pointsscale).TotalimpactofleafdiseasesduringvegetationwasevaluatedbycalculatingtheAUDPC(areaunderdiseaseprogresscurve).Fungicideapplicationsignificantly(P<0.0001)decreasedthediseaselevel:efficacyofchocolatespotcontrolwas21.3%in2015and31.7%in2016,andefficacyofAlternarialeafblotchcontrolwas40.7%and35.3%respectively.Furtherinvestigationsarenecessarytoessentiallyimprovetheefficacyoffungicideapplication.SupportedbytheStateResearchProgramme“AgriculturalResourcesforSustainableProductionofQualitativeandHealthyFoodsinLatvia”:project“Sustainableuseofsoilresourcesandabatementoffertilisationrisks”.HostandnonhostsinteractionsofpowderymildewswithvariouscropplantsBalázsBarna,CsillaJuhász,AdélNémethandGáborGullnerPlant Protection Institute, Centre for Agricultural Research, Hungarian Academy ofSciences,Budapest,P.O.box102,HungaryEmail:barna.balazs@agrar.mta.huPowderymildewsareoneofthemostdevastatingpathogensofcropsallovertheworld.Triticale,anartificialhybridofwheatandrye,wasresistanttopowderymildewuntil2001,butsincethenthisfungusbecomeaserioustriticalediseaseinEurope.RecentlyitwasfoundthatBlumeriagraminisf.sp.triticaleisahybridbetweenwheatandryepowderymildews.Noneof11testedtriticalegenotypesshowedsusceptibilitytoourwheatpowderymildewisolate.Inordertogetinformationontheroleofproteinkinasesandphosphatasesonmildewdevelopmenttheeffectofpre-treatmentofhostplantswiththeinhibitorsstaurosporineorokadaicacidwasevaluated.Furthermore,the
53
electrophoreticribonuclease(RNase)enzymepatternsofbarleyandwheatwereverydifferentfromeachotherandfromBrachypodiumdistachyonplantextractsthatshowedhighRNaseactivity.Allinvestigatedhostandnon-hostplantspeciesreactedwithanincreaseofRNaseactivitytopowderymildeworrustinfections.Wefoundthatheatpre-pretreatmentincreasedboththesusceptibilityofbarleytopowderymildewanditsRNaseactivity,whichconfirmsthepossibleroleofRNasesinsusceptibility.InteractionsofGolovinomycesorontiiwithvarioustobaccolinesinducedastrongincreaseofRNaseenzymeactivity,andanewRNasebandappearedintheextractsfromallinfectedtobaccosfollowingPAGEandspecificstaining.Interestingly,powderymildewinfectionsup-regulatedtheexpressionofPR-1b,PR-1candWRKY12genesinallsusceptibletobaccolinestovariousextents.Thepossibleroleoftheabovechangesinplant-powderymildewinteractionisdiscussed.Phenotypic, comparative genomic and transcriptomic analysis of recently collectedEuropeanZymoseptoriatriticistrains.HongxinChen1,CarlosBayon1,RobertKing2, StefanoTorriani3, StephaneBieri3,KostyaKanyuka1,KimE.Hammond-Kosack1andJasonJ.Rudd11DepartmentofBiointeractionsandCropProtectionand2DepartmentofComputationaland Analytical Sciences, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ,United Kingdom; 3Syngenta Crop Protection AG, Schaffhauserstrasse, CH-4332 Stein,SwitzerlandEmail:hongxin.chen@rothamsted.ac.ukSeptorialeafblotch(STB),causedbytheascomycetefungusZymoseptoriatritici,isoneofthemostimportantfoliardiseasesofwheat,especiallyinEuropeandUSA.Duetothepoorgeneticbasedresistanceofwheattothispathogen,extensiveapplicationoffungicidesiscurrentlythemostcommonwaytocontrolSTB.ThishasledtorapidevolutionoffungicideresistanceinmanyZ.triticipopulations.ToascertainthevariabilityinvirulenceprofilesofacollectionofrecentisolatesofZ.triticicollectedfromEuropeanfieldsites,42strainswereinoculatedonto21wheatcultivars,alongwiththereferencestrainIPO323asacomparison.Diseaseprogresswasmonitoreddailyanddateswererecordedwhen1stdiseasesymptomappearedandfullnecrosisdeveloped.Fungalsporulationwasalsomeasuredforeachinteraction.Theresultsshowdiversevirulenceprofilesofdifferentisolatesandarangeofinteractionoutcomes.ManyofthetestedisolatesdisplayedenhancedvirulencerelativetoIPO323.Noobviouscorrelationwasfoundbetweenvirulenceandsporulation,indicatingthesetwotraitsmightbeunderthecontrolofdifferentgeneticloci.Tobetterunderstandthegeneticbasisofdifferentialvirulence,genomicDNAofthetop12mostvirulentisolateswillbesubjectedtofullgenomesequencing,andRNAseqbasedgeneexpressionanalysisofinvitrocultures,andinplantasamples,willbeperformed.
54
Definingtheimpactofautophagyonthehost-pathogeninteractionofwheatandthefungusZymoseptoriatriticiHarryT.Child1,StevenBates1,JasonJ.Rudd2andKenHaynes11Biosciences, University of Exeter, Exeter, EX4 4QD, United Kingdom; 2Department ofBiointeractionsandCropProtection,RothamstedResearch,Harpenden,AL52JQ,UnitedKingdomEmail:hc515@exeter.ac.ukThefungalwheatpathogenZymoseptoriatriticicausesmajorcroplossesandcurrentlydemands€1billionworthoffungicidetocontrolinEuropeannually.TheinfectioncycleofZ.triticidisplaystwodistinctphases,beginningwithanextendedsymptomlessphaseofatleastaweek,beforethefungusinduceshostcelldeathandtissuecollapseintheleaf.Recentevidencesuggeststhatthefungususeslittlehost-derivednutritionduringtheearlystagesofinfection,insteadrelyingonmacromoleculeswithinthefungalspore.WehypothesisethatZ.triticiutilisestheself-degradativeprocessofautophagytorecyclestoredcellularresourcestosupportestablishmentofleafcolonisation.Usingmoleculartechniques,thisresearchwillassesstheimportanceoftheautophagypathwayduringZ.triticicellulardifferentiationandinfectionofwheat.Manipulatingthegenesinvolvedinautophagywillrevealitsimportancetofungalvirulence,whilefluorescentlytaggingkeyautophagyproteinswillvisualisethespatialandtemporaloccurrenceofautophagicprocessesinvivo.ThisresearchwillprovidefundamentalknowledgeofthemolecularmechanismsofZ.triticiinfectionaswellasidentifyingpotentialnewtargetsforcontrolofthisdevastatingdisease.*Resistance to Rhynchosporium: Characterisation and identification of candidategenesMaxCoulter1,MarkLooseley1,RobbieWaugh2andAnnaAvrova11TheJamesHuttonInstitute,Invergowrie,Dundee,UK;2UniversityofDundee,Dundee,UKEmail:Max.Coulter@hutton.ac.ukRhynchosporiumcommune,causingleafscald,isoneofthemostdestructivepathogensofbarleyworldwide,leadingtoyieldlossesofupto30-40%.Despiteadvancesinmolecularmarkertechnologyandthesequencingofthebarleygenome,understandingofbarleyresistancetoRhynchosporiumatthemolecularlevelislimited.TheSpanishlandraceCIho3515ishighlyresistanttoscald,containingresistancegenesRrs1andRrs18.Rrs1wasthefirstRgeneagainstRhynchosporiumtobeidentified,whileRrs18hasonlyrecentlybeencharacterised.Rrs18hasalsobeenidentifiedinaSteptoexMorexDHpopulation,andalargeSteptoexMorexBC1populationhasbeenusedtofinemapthegenetoa650Kbintervalon6HS,identifying17candidategenes.ToidentifyacandidateforRrs1,AssociationmappingusingelitespringbarleycultivarsandSyrianandJordanianlandraceshasnarrowedtheRrs1mappingintervaltoasinglegene
55
on3H,aWallassociatedKinase(WAK).AnalysisofexomecapturedataformultiplecultivarscarryingRrs1showedevidenceofaWAKparaloguenotpresentinthesusceptibleMorexreferencesequence.PCRandSangersequencingwascarriedouttodecipherthesequenceoftheWAKparaloguewiththeaimofcloningRrs1.CloningofbothRrs18andRrs1willfurtherourunderstandingofRhynchosporiumresistanceandenablediagnosticmarkerstobedevelopedhelpingbreederstopyramidbothgenestocreatedurableresistancetoRhynchosporiuminbarley.*Isolation and characterisationof a chrysovirus fromDothistroma septosporum, thecausativeagentofpineneedleblightJohnDaudu1,UnnatiShah1,Kotta-LoizouIoly2andRobertH.A.Coutts11Department of Biological and Environmental Sciences, University of Hertfordshire,Hatfield,AL109AB,UK;2DepartmentofLifeSciences,ImperialCollegeLondon,London,SW72AZ,UKEmail:j.o.daudu@herts.ac.ukDothistromaneedleblightcausedbyDothistromaseptosporumhasemergedintheBritishIslesasamajorthreattoCorsicanpine,lodgepolepineandScotspine.Thereisincreasingevidencethatmycovirusescanreducethegrowthandpathogenicityoffungalplantpathogens.Forinstance,avirulence-attenuatingRNAmolecule,Cryphonectriahypovirus1(CHV1)hasbeendescribedandsuccessfullyusedasabiologicalcontrolagentofCryphonectriaparasitica.Theaimofthepresentstudyistoisolateandcharacterisedouble-stranded(ds)RNAvirusespresentinacollectionofD.septosporumisolates.Tothisend22D.septosporumisolateswerescreenedusingarapidsmall-scaledsRNAextractionprocessandonlyonewasfoundtoharbourdsRNAelements.Subsequentlytheseelementswereclonedandsequenced,usingtargetedsequencingandRNAligase-mediatedrapidamplificationofcDNAends(RLM-RACE).ComputationalanalysisofthesequencesobtainedrevealedtheirsimilaritytomembersoftheestablishedfamilyChrysoviridae,whichhave4genomicsegmentsencapsidatedinisometricparticlesandareknowntoelicithypovirulenceintheirhosts.Generationofisogeniclinesofvirus-freeandvirus-infectedisolatesisinprogresstodetermineanyeffectsofthemycovirusonfungalfitnessandpathogenicity;thepathogenicityofthevirus-infectedisolatewillbeassessedincomparisontothevirus-freeone,inordertodetectputativehypovirulence,acommonfeaturenotedformycoviruses,whichmightbeusedforbiologicalcontroltoinvasionbymoreaggressivestrainsofD.septosporum.Inconclusion,thisstudyreportsthefirstmycoviruseverfoundinD.septosporum.
56
Anovelmodeofactioninoomyceteplantpathogencontrol:ORONDISTM
JudithSheldon,JillFoundling,MathiasBlum,SianDeller,DavidBeattieandMikeCsukaiSyngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire. RG42 6EY.UKEmail:sian.deller@syngenta.comOxathiapiprolin,discoveredbyDuPont,isthelatestoomycete-activecompoundwithanovelmodeofactiontoenterthemarket,andismarketedbySyngentasince2016asORONDISTM.DuPontinitiallyidentifiedthemoleculartargetofoxathiapiprolin.Thisreportconfirmsthatfindingbycharacterisingforwardgeneticsmutantswithanoxathiapiprolin-resistantphenotype.Non-synonymouspointmutationswerefoundinallresistantlabstrainsintheoxysterolbindingprotein-encodinggene(OSBP).OSBPisthoughttobeinvolvedinthemovementofsterolsandlipidsbetweenmembranes,maintaininglipidhomeostasis.Downstreamperturbationscausedbydisruptionofthisfunctionultimatelykillthecell.Toinvestigatethishypothesisintheoomycetepathogen,Phytophthorainfestans,wehaveusedmicroscopy-basedtechniquestoexaminetheultrastructureofgermlingstreatedwithsublethaldosesofoxathiapiprolinandcomparedthesetocontrolgermlings.ObservationsofdifferentialcytologywillprovideinsightstobetterunderstandtheroleofOSBPandthewayinwhichitsinhibitionbyoxathiapiprolincontrolsoomyceteplantpathogens.Anti-protozoal agents identified in the Pathogen Box can inhibit Aspergillus fumigatusgrowthThailaFernandadosReisandGustavoH.GoldmanFaculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,RibeirãoPreto,Brazil.E-mail:thailaf@hotmail.com
Aspergillusfumigatusisanopportunistichumanfungalpathogenandallergenresponsibleforinvasiveaspergillosis,animportantinfectionwhichcausesahighlevelofmortalityinimmunocompromisedpatients.Althoughtheaspergillosisismainlytreatedbyadministrationoftriazoles,epidemiologicalstudieshaveindicatedthattheemergenceofazole-resistantA.fumigatusisolateshasincreasedsignificantlyoverthelastdecade.Despitethegrowingneeds,treatmentsforinvasivefungalinfectionsremainunsatisfactoryandthediscoveryofnovelantifungalsdrugsishighlydesirable.ThePathogenBox(https://www.pathogenbox.org/)isalibrarycomposedby400syntheticchemicalsmoleculeswithactivityagainstdifferentpathogensresponsibleforimportantdiseasessuchasmalaria,tuberculosis,toxoplasmosis,andothers.AimingtodiscovernewpotentialagentsagainstaspergillosiswescreenedthePathogenBoxchemicalcompoundsforantifungalactivityagainstA.fumigatus.Weidentifiedmiltefosine(MMV688990)asanantifungicidalagentagainstaspergillosis.Usinginvitroassays,wedemonstratedthatthiscompoundhasaMIC(MinimalInhibitoryConcentration)of10
57
µM.Interestingly,anullmutantinthecAMPdependentproteinkinaseSchaSCH9hasaMICof20uM.Thisproteinkinaseinfluencessphingolipidsbiosynthesis,suggestingthatmiltefosinecouldactinhibitingthefungalgrowthviatargetingthelipids/sphingolipidssynthesis.Additionally,iodoquinol(MMV002817)showedanantifungistaticactionwithaMICof2µM.Basedonourresults,weproposethatthesetwocompoundscouldrepresentpotentialcandidatesforfurtherantifungaldrugdevelopmentagainstinvasiveaspergillosis.Financialsupport:FAPESPandCNPq,BrazilFunctional characterization of the Aspergillus nidulans non-essentialproteinkinasesinvolvedintheregulationofsorbicacidantifungalpropertiesThailaFernandadosReis1,IveyGeoghegan2,SimonAvery2andGustavoH.Goldman11Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,RibeirãoPreto,Brazil;2SchoolofLifeSciences,UniversityofNottinghamUniversityPark,NottinghamNG72RD,UK.E-mail:thailaf@hotmail.comDuetoaconstantlyincreasingoftheworldpopulation,thedemandforfoodisalsoontherise.However,ahighpercentageofthisfoodislostdue,partially,tofoodstuffspoilagebymicroorganisms,includingfilamentousfungi.Weakorganicacidssuchassorbic,benzoic,propionicandaceticacidareusedasantimicrobialagentsinordertopreventfoodandbeveragespoilagecausedbymicroorganisms.Unfortunately,themicroorganismsshowagreatresistancetoweakacidpreservativesandtheemergenceofresistantpopulationisoftenreported.Theexactmechanismbehindtheantifungalpropertiesofthesecompoundsisstillunknown.Aimingtobetterunderstandthemechanismsthatcontrolthesorbicacidantifungalpropertiesandidentifypotentialtargetsofthisweakacid,wescreenedanA.nidulansnullmutantlibrarycomposedof103non-essentialproteinkinases.Atotalof16nullmutantshadalteredgrowthinpresenceofdifferentsorbicacidconcentrations.Interestingly,03ofthemthatcorrespondtogenesencodingproteinsinvolvedintheHighOsmolarityGlycerol(HOG)pathwayweresensitivetosorbicacid.WeobservedthatA.nidulansHogAphosphorylationwasmodulatedinthepresenceofsorbicacidsuggestingtheinvolvementofthispathwayinthisprocess.TheacquisitionofmoredetailsaboutthemechanismsinvolvedintheantifungalpropertiesofsorbicacidaswelltheinvolvementoftheHOGpathwayinthisprocesswillclearlyhelponthedevastatingimpactoffungalfoodspoilageonglobalfoodsecurity.Financialsupport:BBSRC(UnitedKingdom),FAPESPandCNPq(Brazil)
58
FrontiersintheunderstandingandmanagementofArmillariainUKgardensJassyDrakulic,MatthewCromey,LizBeal1,GerardClover
PlantHealth,RoyalHorticulturalSociety,RHSGardenWisley,Woking,GU236QB,UK;Email:jassydrakulic@rhs.org.ukHoneyfungusrootrot,causedbyArmillariaspp.,isthediseasemostfrequentlyenquiredaboutbyUKgardenerstotheRoyalHorticulturalSociety(RHS).Armillariaepidemiologyhasbeenstudiedwithinforestryandagriculture,butdataislackingwithingardens,whichhavegreaterhostplantdiversitythanorchardsandvineyardsandgreaterdisturbancethanwoodlands.SurveysofmaterialsenttotheRHSbymembershashelpedanswerquestionsaboutwhichArmillariaspeciesareresponsibleforthediseaseingardens,andhowplantsfromthebroadrangeofsusceptibleornamentalsaredifferentiallyaffectedbythedisease.FurtherresearchdirectionswithintheRHSfocusonevaluationoftheprinciplesofmanagementpracticessuchasstudiesdeterminingthelifespanofrhizomorphsofdifferentsizewhenremovedfromafoodsource,screeningofTrichodermaendophytesforuseasbiocontrol,andelucidatingthenatureoftheinteractionsbetweendifferentArmillariaspeciesofdifferingaggressivenesstohostplants.Fullresultsfromthesurveyarepresentedalongsidepreliminaryfindingsfromthecurrentresearchprojectslistedabove.AphidinfestationincreasessusceptibilityofB.napustoRhizoctoniasolaniAG2-1FryniDrizou1,TobyJABruce2andNeilSGraham11Divisionof Plant andCrop Sciences, School of Biosciences,University ofNottingham,SuttonBoningtonCampus,Loughborough,Leicestershire,LE125RD,UK;2SchoolofLifeSciences,KeeleUniversity,Keele,Staffordshire,ST55BG,UKEmail:Fryni.Drizou@nottingham.ac.ukOilseedrape(OSR),Brassicanapus,istheprimaryhostofRhizoctoniasolaniAG2-1,asoil-bornepathogenthatinfectsyoungseedlingscausingdamping-offdiseaseandimpairedcropestablishment.Currentlythereisnoavailableresistantgermplasmanditisunknownhowthepathogenovercomesplantdefences.Additionally,OSRisoneofthesecondaryhostsofMyzuspersicae,anaphidknownforitsabilitytoadapthostplantdefences.TheaimofthepresentworkwastoidentifyifherbivorybyM.persicaeaffectsOSRresponsestoAG2-1.Weexaminedhowaphidinfestationaffectsresponseofcultivars‘Canard’and‘Temple’toAG2-1byvisuallyassessingdiseasesymptoms13dayspostinoculationonplantstemsandbyquantifyingthefungalDNA.AdditionallyweexaminediftherewasanalterationintheexpressionofthedefencerelatedgenesLOX3,MYC2,NPR1,PR1andWRKY38.Ourresultsshowthataphidinfestationresultedinincreasedsusceptibilityof‘Canard’plantstoAG2-1:plantswithaphidshadmoreseverediseasesymptomsbuttheamount
59
offungalDNAwasthesamebetweentreatments.GeneexpressionanalysisrevealedthatM.persicaeinfestationdecreasedtheexpressionofLOX3andMYC2inplantsinfectedwithAG2-1,hencepossiblealternationsinthejasmonatesignallingpathwaycausedtheincreasedsusceptibilitytothepathogen.Also,theelevationofPR1inAG2-1inoculatedplantsandNPR1underdualattack,impliesthatsalicylicacidmayplayaroleindefenceagainstthenecrotrophicfungiR.solaniAG2-1.AntifungalandPhytochemicalEvaluationofAnnonamuricataplantMargaret1A.Ebabhi.,KanifeU.Claris2,JuliusS.Omogbai3,NnabugwuE.Chinalurum2andRaheemL.Omobolaji2.1Department of Education Science, Distance Learning Institute, University of Lagos,Akoka, Lagos Nigeria; 2Department of Biological Science, Yaba College of Technology,PMB2011,Lagos,Nigeria;3DepartmentofPharmacy,FacultyofPharmacy,UniversityofLagos,Lagos,NigeriaEmail:ebabhi.meg@gmail.comTheincidenceoffungalinfectionisontheincreaseaspathogenicfungiarebecomingresistanttotheconventionaldrugs.Thisresearchexaminedtheantifungalpropertiesoftheaqueous,N-hexaneandethanolextractsoftheleaves,fruits,stem,barkandrootsofAnnonamuricatausingthediscdiffusionagarassay.Thezoneofinhibitionvariedforthetesteddermatophyticorganisms-Candidaalbicans,Trichodermasp.andTrichophytonmentagrophytes,withrespecttotheplantpartsandtypeofextract.Theaqueousextractsofthetestplantpartsshowedmorepotencyonthetestfungalspecies.PreliminaryphytochemicalstudiesoftheextractsoftheplantpartsshowedthatAlkaloid,Cardiacglycosidesandphenolwerehighlypresentinallpartswhilesteroidandphobataninarelesspresent.ThequantitativeresultsofvariouspartsoftheplantreviewedthatPhenolfromleafextractsarehighlyquantifiablethanotherphytochemicalcomponents.Inall,Alkaloids,CardiacGlycosideandphenolwerehighlyquantifiable,whileTannin,SteroidandPhobataninwerelessquantifiable.ThequantitativeanalysiswasdoneusingANOVAmethodat0.05levelofsignificance.ThesefindingsgivecredencetothetraditionaluseofAnnonamuricataforthetreatmentoffungalinfections.CharacteristicsandcontrolofAlternariaalternataaffectingsweetpepperinEgyptAbdulmageedM. Kamara1, Ahmed. E. Elkorany2, Eman E. Ramadan2, andGhonemM.Amer21 Department of Plant Pathology, University of Alexandria, Alexandria 21561, Egypt;2DepartmentofPlantPathology,UniversityofDamanhour,Damanhour22511,Egypt.Email:drelkorany125@yahoo.com
60
Thirty-nineisolatesofAlternariaalternatawererecoveredfromsweetpepperfruitsshowedblackmouldsymptomscollectedfromdifferentregionsinEl-Beheragovernorate,Egypt.Theisolateswerecharacterizedfortheircolonyphenotypesandvirulencewherenocorrelationwasdetected.TheRAPDanalysisrevealed100%polymorphismusingtheOPA-03primeramongsixA.alternateisolatesanalyzedandthedevelopeddendogramshowedpartialclusteringforregionsandvirulence.Alltestedcultivars,i.e.Emberu,Trezaa,Mazurka,andTarantoweresusceptibletotheartificialinfectionwithA.alternatatodifferentdegrees.Treatmentofpepperfruitswithresistanceinducers,i.e.salicylicacid(SA),abscisicacid(ABA),methyljasmonate(MJ),andcalciumchloride(CaCl2)significantlyinducedresistanceanddecreasedthedevelopedblackmouldwherecalciumchloride(1.5g/L)andSA(1mM)werethemosteffective.InductionofresistanceinpepperfruitswithCaCl2andSAaswellasthetolerancerevealedincv.Mazurkawasfoundtobeduetohighinductioninenzymeactivityofpolyphenoloxidase(PPO),peroxidase(POD),andphenylalanine(PAL)aswellasanincreaseinthetotalphenolicsinthetreatedpepperfruits.*PowderyMildewIdentification: ImprovingaccuracyusingpreviouslyuntappedDNAregionsOliverEllingham1,JohnDavid2,andAlastairCulham1
1School of Biological Sciences, University of Reading, Reading, RG6 6AS,UK;2DepartmentofPlantPathology,TheRoyalHorticulturalSociety,Wisley,GU236QB,UKEmail:o.h.ellingham@pgr.reading.ac.ukThepowderymildews(Ascomycota,Erysiphales)areagroupofobligatebiotrophicFungifoundonnearly10,000angiospermplanthostsglobally,manyofwhichareimportanthorticulturalandagriculturalplants.Infectioncanreduceattractivenessandyieldssignificantly.Areliablemethodisrequiredforunambiguousidentificationoftheseoftencrypticspeciessuchthatspreadtonewareasand/ornewhostscanbedetectedrapidlyandcontrolledearly.Thisprojecttakesauniqueapproachtosampling;usingacitizenscienceschemelaunchedin2014toassessthepresenceofthe876globalpowderymildewspeciesintheUKandtheefficacyoftechniquescurrentlyusedfortheiridentification–hostidentification,fungalmorphologicalanalysis,sequencingofthefungalrDNAITSregion.Thesurveywaspromotedviaanarrayofonlineandprintedmediaandover500infectedplantsampleswerereceived–mostwerepowderymildews.Ofthese94%wereidentifiedtogenusleveland79%tospecieslevel.ThisshowsthescopeforimprovementstothesetechniquesthroughanalysisofsupplementaryDNAregions.90differentpowderymildewspecieswerefoundoverthe2-yearperiod;60%ofthe200speciespreviouslyrecordedintheUK.DuringthesurveyperiodtwonewhostswerefoundforspeciesknowntooccurintheUK.ThesedataprovidethemostcomprehensivesurveyofpowderymildewspecieswithintheUK.This
61
knowledgeiscriticalforearlydetectionofnewlyarrivedspeciesofpowderymildewandofhostshiftsornovelhosts.**PhenotypicheterogeneityasanadaptationoffungitoenvironmentalstressDavidS.Foster1,SteveP.McGrath2,PaulS.Dyer1andSimonV.Avery11School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK;2SustainableAgricultureSciences,RothamstedResearch,Harpenden,AL52JQ,UKEmail:mbxdf@nottingham.ac.ukPhenotypicheterogeneitydescribesvariationinagivencharacteristicthatisevidentbetweenindividualcellsofanisogenicpopulation.Theextentofheterogeneityinaparticularphenotypemaybedeterminedbyepigeneticorgeneticfactors,e.g.specificgene-promotersequencesproducingnoisiergeneexpression.Anumberofcharacteristicsdisplayingphenotypicheterogeneityareknowntoinfluencecompetitivefitnessunderstress,thereforeheterogeneitylikelyhasabearingonsurvivalunderfluctuating-stressconditions.Recentevidencefromourlaboratorysuggeststhatenvironmentalstressselectsforincreasedlevelsofheterogeneityinresistancetocertainstressors.Wildyeastisolatesfrompollutedsiteswerefoundtoshowelevatedheterogeneityinresistancetopollutants,incomparisontoisolatesfromnearbycontrolsites.However,theinfluenceofenvironmentalstabilityonselectionforheterogeneityisnotunderstood,noritslong-termstability.Yeastandfilamentousfungalisolatesfromlong-termstressedexperimentalsiteshavebeenstudiedtoprovideinsight.Dose-responsegradients,amongstothermeasuresofcell-cellvariation,indicatethatalong-term,stablelevelofstressmayselectforadecreaseinheterogeneityofresistance.Thedegreeofresistanceheterogeneitymayhavethepotentialtoevolveindifferentdirectionsdependingonthestabilityofthestressordoseintheenvironment.DevelopmentofCRISPR/Cas9geneeditingtoolsforthericeblastfungusAndrewJ.Foster1,MagdalenaMartinUrdiroz1,NicholasJ.Talbot11SchoolofBiosciences,UniversityofExeter,Exeter,Devon,UnitedKingdom.Email:A.J.Foster@exeter.ac.ukCRISPR/Cas9geneeditingisarapiddevelopingfieldwhichholdsgreatpromiseforacceleratingthepaceoftargetedmutagenesisinfungi.Currently,althoughverymanyfungalgenesarepredictedbasedongenomesequencing,theprocessofspecificallymutatingselectedgenes,eveninthebeststudiedfungi,requiresmonthsratherthanweeks.ThericeblastfungusMagnaportheoryzaeisthemostseriouspathogenofrice.ToacceleratetheprocessoftargetedmutationandspecificgeneeditsinthisimportantspeciesweareworkingtowardsthedevelopmentofarapidCrispr/Cas9basedgeneeditingtechnology.Technologiesallowingpreciseeditstocodingsequences,gene
62
taggingaswellasdeletionsofsingleormultiplegenesarebeingoptimised.Thisresearchwillopenthedoortoanalysisofgenefamiliesorsubsetsoftargetgenes.Latestresultswillbediscussed.Tacklingresistancetofoodpreservatives:heterogeneityinfungalsporepopulationsIveyA.Geoghegan,MalcolmStratford,DavidB.ArcherandSimonV.AverySchoolofLifeSciences,UniversityofNottingham,Nottingham,NG72RD,UKEmail:mbzig@exmail.nottingham.ac.uk
Foodspoilagebyfungiisaglobalissuewithfar-reachingeconomic,environmentalandhealth-relatedconsequences.Spoilageisparticularlyseverewithinthesoft-drinksindustry,whereevenahandfulofspoiledbottlescanresultintherecallofanentirebatch,costingmillionsofpounds.Suchspoilageoccursdespitethepresenceoffoodpreservativesinsoftdrinks,whicharegenerallyeffectiveatrestrictinggrowthofyeastsandmoulds.However,certainspeciesoffungiarehighlyresistanttothesepreservatives,andareassociatedwithspoilageincidentsworldwide.Thus,effectivecontrolofthesefungiisanimportantavenueofresearch.Wearecharacterizingmechanismsofresistancetosorbicacid,awidelyusedpreservativeinsoftdrinks.ThesporesofthemouldAspergillusnigerarehighlyresistanttosorbicacid.Inaddition,A.nigersporepopulationsdisplayahighdegreeofheterogeneityinresistancetothisparticularstress.Bycombininganalysisofsinglesporephenotypeswithcutting-edgemolecularindexingtechnologyaswellasclassicalmoleculargeneticstechniques,wehavebeguntouncovertheinextricablelinksbetweenpopulationheterogeneityandpreservativeresistanceinthisspoilagefungus.*Xanthomonas citri pv. citri pathotypes: Lipopolysaccharide (LPS) structure andfunctionasmicrobe-associatedmolecularpatterns(MAMPs)LotteBettinaAndersenGersby1,OlivierPruvost2,AntonioMolinaro3andMari-AnneNewman11PLEN,UniversityofCopenhagen,Thorvaldsensvej40,1871Denmark;2CIRAD,PVBMT,SaintPierre,LaRéunion,France;3DepartmentofChemicalSciences,UniversityofNaplesFedericoII,Naples,ItalyEmail:lba@plen.ku.dkCitrusbacterialcanker(CBC)isadiseaseofcitrusplantswithremarkablesocioeconomicimpactonthecitrusindustry.ThecausalagentofAsiaticCBCistheGram-negativebacteriumXanthomonascitripv.citri(Xac).Xacstrainsareclassifiedintothreepathotypes.PathotypeAhasthebroadesthostrange,infectingalmostallCitrus,pathotypesA*andAWshowalimitedhostspecificity,onlyaffectingMexicanlime.
63
LPSisknowntoactasaMAMPandelicitsimmuneresponsesineukaryoticorganisms.MAMP-inducedresponsesincludeproductionofreactiveoxygenandnitrogenspecies,alterationsintheplantcellwall,inductionofantimicrobialcompoundsandexpressionofpathogenesis-relatedgenes.LPSiscomposedofapolysaccharideregion(O-chain),acoreoligosaccharideandaglycolipidregiontermedlipidA.AgenomicregioninvolvedinLPSbiosynthesisthatishighlyvariableamongtheXacpathotypeswasrecentlyidentified.Furthermore,partialstructureelucidationofXacLPSrevealedthepresenceofarhamnosehomo-polymerastheO-chainmoiety.AfullelucidationofthestructuresofLPSfromseveralXacstrainsmightclarifythedifferencesindefenceresponsesindifferenthostplants,aswellasthehostspecializationprocessoftheXacpathotypes.Here,wepresentthestructuresofLPSfromtwoXacstrainsofpathotypesAandA*andtheabilityofthewholeLPSandlipidAofbothstrainstoactasMAMPsinthemodelplantArabidopsisthalianaandinthetwohosts,Mexicanlime(Citrusaurantifolia,pathotypeA*host)andlemon(Citruslemon,pathotypeAhost).**FungalandOomyceteBiodiversityintheFalklandIslandsandSouthGeorgiaJacobHargreaves1,JimI.Prosser2,PaulBrickle3andPietervanWest11Aberdeen Oomycete Laboratory, College of Life Sciences and Medicine, Institute ofMedical Sciences, University of Aberdeen, Aberdeen, UK; 2Institute of Biological andEnvironmental Sciences, University of Aberdeen, Cruickshank Building, Aberdeen, UK;3SouthAtlanticEnvironmentalResearchInstitute,Stanley,FalklandIslandsEmail:j.hargreaves@abdn.ac.ukSoilmicrobialcommunitiesareextremelycomplexandheterogeneousentities,whicharenotfullyunderstoodatmostlevels,fromsoilporestructuralmicroclimatestoglobaldiversityanddispersion.Thisisdespitetheircrucialinvolvementinbiogeochemicalcycles,whichaffectsagriculturalandnaturalecosystems.Amajorcomponentofthemicrobialcommunityarefungi,whichhavecriticalrolesinbreakingdownorganicmatter.Asubgroupcanalsoformsymbioticrelationshipswithplants,knownasmycorrhizae,whichcanincreaseplantproductivity.TheFalklandIslandsandSouthGeorgiarepresentanisolatednaturalecosystemwithintroducedherbivoressuchassheepandreindeer.Toinvestigatefungaldiversityinthesecircumstances,soilsamplesfromWestFalklandandSouthGeorgiaweretakeninthe2017southernsummertocarryouthighthroughputsequencingofbarcoderegions.Thesesampleswillgiveasnapshotofthenaturaleukaryoticmicrofloraoftheregions,withareasofrelativelylittleandareasofalotofgrazingpressure.Thedatawillallowfungalcommunitystudiestobecarriedout,lookingattheimpactofvegetationandreindeergrazing.Thesubsequentanalyseswillallowinferenceofthefungalcommunityfunctiontobedetermined,withapplicationstowardsagricultureandbioremediation.Herewepresentourlatestfindings.
64
MappingtheAdaptiveLandscapeofZymoseptoriatriticiCYP51underAzoleFungicideSelectionNicholaJ.HawkinsandBartA.FraaijeBiointeractionsandCropProtection,RothamstedResearch,Harpenden,AL52JQ,UKEmail:nichola.hawkins@rothamsted.ac.ukFungicidesareakeycomponentofcontrolprogrammesformanyplantpathogens,buttheiruseexertsastrongselectivepressureforresistance.Forsomesite-specificfungicides,theevolutionofresistanceissimpleandrapid,withasinglemajortarget-sitemutationresultingineffectivelycompleteresistance.However,fortheazolefungicides,thepictureismorecomplex,withmultipletarget-sitemutationsandother,over-expressionbasedmechanismsaccumulatingtoproducequantitative,step-wisereductionsinsensitivity.Theeffectofmultiplemutationsonresistanceisoftenreferredtoas“additive”,butinfactthesituationisfurthercomplicatedbyepistaticinteractions,bothintermsoffungicideresistance,andofimpactsonenzymefunctionandstabilityofthetargetprotein.Thisresultsinaruggedadaptivelandscape.Consequently,adetailedunderstandingoftheeffectofeachmutation,individuallyandindifferentgeneticbackgrounds,onresistanceandfitness,isnecessaryinordertoattempttopredictevolutionarytrajectoriesunderdifferentfungicideselectionscenarios.WewillpresentanupdatedfitnesslandscapeofZ.triticiCYP51haplotypesreportedinfieldisolatestodate,discussingtheimplicationsforthepredictabilityoffutureevolutionandexperimentalapproachesfortestingthis.GeneticdiversityofFusariumspp.oncerealsinRussiaAleksandrIgnatov1,TatianaSuprunova2andNataliaZhemchuzhina31Russian University of People’s Friendship, Moscow, 117198, Russia and R&D Center“PhytoEngineering” LLC, Rogachevo, 141880, Russia; 2“Doka-Gene Technology” LLC,Rogachevo, 141880, Russia; 3 Russian Research Institute of Phytopathology, BolshieVyazemy,143050,Russia.Email:an.ignatov@gmail.comTherootrot,snowmold,andfusariumearblightcausedbyFusariumspp.andMicrodochiumspp.-relatedfungidamageawiderangeofcerealcropsinRussia.Thediseaseagentsarecharacteristictocertainecologicalandgeographicalareas.96strainsofFusariumspp.andMicrodochiumspp.fromtheStateCollectionofPlantPathogenicMicroorganismswereusedforMultiLocusSequenceTyping(MLST)analysisforgenesoftranslationelongationfactor-1alpha(TEF1),β-tubulin,ITSrRNA(with3differentprimersets),andRNApolymeraseII(RPB2)withprimersreportedelsewhere(O’Donnelletal.,2000;Debourgognetal.2010;CBS-KNAWFungalBiodiversityCentre’sFusariumMLSTwebsite).Resultsofpreviousphenotypicidentification,PCRFingerprint(IRAPandISSR),andMLSTanalysiswerecompared.StrainsofF.culmorumformedthemostpolymorphicgroupaccordingtoPCRfingerprintandMLST,whereasthemost
65
homogeneousgroupincludedF.sporotrichioides.StrainsofM.nivale,F.avenaceum,F.gibbosumwerenotsignificantlydifferentiatedstatisticallyfromotherspecies.SomestrainsofM.nivale,F.sporotrichoides,andF.oxysporumcouldnotbecorrectlyidentifiedbyeitherphenotypeorPCRfingerprinting.ThespeciesofthepathogenswidespreadinRussiahadvariouspathogenicandtoxicproperties,indicatingtheirstrongintraspecificvariability.Rarefungalspeciespossessedtoxicitytotest-plantseedlings,butwerenotpathogenic.ThegeneticsimilaritybetweennewlysequencesMLSTlociandclosestGenebank(NCBI)accessionsrangedfrom81to99%indicatingpresenceofbothpreviouslycharacterizedanduniquegeneticlinagesamongstrainsofFusariumspp.andMicrodochiumspp.infectingcerealsinRussianFederation.BiologicalmanagementofsouthernblightofchilibyPenicilliumoxalicumandleavesofEucalyptuscitriodoraArshadJavaid,RabiaAfzalandAmnaShoaibInstitute of Agricultural Sciences, University of the Punjab, Quaid-e-Azam Campus,Lahore,Pakistan.Email:arshadjpk@yahoo.com,arshad.iags@pu.edu.pkInthepresentstudy,leafdrybiomassofEucalyptuscitriodoraandabiologicalcontrolagentPenicilliumoxalicumwereevaluatedformanagementofsouthernblightdiseaseofchilicausedbySclerotiumrolfsii.Inapottrial,soilwasmadesickwithinoculumofS.rolfsii.SoilwasamendedwithdryleafbiomassofE.citriodora(1,2,3%)andabiologicalcontrolagentPacicilliumoxalicumaloneorincombination.Soilamendmentwith3%leafbiomassofE.citriodorasignificantlyreducedplantmortalityby65%overpositivecontrol.Consequently,rootandshootgrowth,fruityieldaswellaschlorophyllcontentweresignificantlyenhanced.P.oxalicumincombinationwith3%leafbiomassalsosignificantlyreducedplantmortalityandenhancedcropgrowthandyield.ProteincontentinP.oxalicuminoculatedtreatmentswasalsosignificantlyhigherthancontrol.S.rolfsiiinoculationsignificantlyenhancedperoxidase(PO),phenylalanineammonia-lyase(PAL),catalaseandpolyphenoloxidase(PPO)activitiesinleavesofchiliwhiletheseparametersweredrasticallyreducedbyapplicationofP.oxalicumandleafbiomassofE.citriodora.Inlaboratorybioassay,differentconcentrationsofmethanolicleafextractofE.citriodoraviz.1,2,…,5%reducedfungalgrowthby3to51%.Inmethanolicleafextract,compoundsnamely2-Furancarboxaldehyde,5-(hydroxymethyl),Cyclohexanol,2-(2-hydroxy-2-propyl)-5-methyl,Pentadecanoicacid,14-methyl,methylester,and14,17-Octadecadienoicacid,methylesterwereidentifiedthroughGC-MSanalysis.Thepresentstudyconcludesthat3%leafbiomassofE.citriodoracansignificantlyreduceplantmortalityduetoS.rolfsiiandenhanceplantgrowthandyieldinchili.
66
Tracing the transduction of a phenylpyrrole fungicide reveals its effect on cAMPsignallinginBotrytiscinereaJaafarKilani1,2,MonicaMiazzi3,ColetteAudéon1andSabineFillinger11UMRBIOGER, INRA,AgroParisTech,UniversitéParis-Saclay,Thiverval-Grignon,France;2Univ. Paris-Sud, Université Paris-Saclay, France; 3Department of Soil, Plant and FoodSciences,UniversityofBariAldoMoro,Bari,ItalyEmail:jaafar.kilani@inra.frFungirapidlyadapttotheirenvironmentinvolvingtosignallingpathwayslikethoseofmitogenactivatedproteinkinases(MAPKs).InBotrytiscinerea,thephenylpyrrolefungicidefludioxonilactivatestheSak1andBmp3MAPKs,whichareinvolvedinosmoregulation,cellwallintegrity,developmentandpathogenicity.Inordertotracethetransductionoffludioxonilandtoidentifythesignallingpathwaysinvolved,wehaveperformedcomparativephosphoproteomicsofthewild-typestrainsubjectedornottofludioxonil.Thisexperimentledtotheidentificationof“fludioxonil”candidatesignallingproteinssuchasthephosducin-likeproteinPhnA.Ineukaryotes,phosducinisinvolvedintheG-proteinsignallingandhencethecAMPpathway.InthephytopathogenicfungiCryphonectriaparasiticaandFusariumgraminearum,phosducinisinvolvedinpigmentation,sporulation,andpathogenicity.
WeperformedthefunctionalanalysisofphnAinB.cinerea.ItsdeletionrevealedthatphnAisinvolvedindevelopment,pathogenicityandadaptationtocellwallstress.TheroleofphsoducininG-proteinsignallingpushedustostudytheimpactoffludioxonilonthecAMPsignallingpathway.Inthewild-typeweobservedasteadydecreaseofintracellularcAMPaccumulationduringexposuretofludioxonil.WealsostudiedtheconnectionbetweenPhnAandSak1andweobservednoinfluenceofPhnAonSak1phosphorylation.
OuranalysesrevealedthatthephenylpyrrolefludioxonilactsoncAMPdependentsignaltransductioninadditiontoitsactivatingeffectontheosmosensingMAPKpathway.TheroleofPhnAandtheosmoticMAPKpathwayincAMPsynthesisunderfludioxoniltreatmentiscurrentlyunderinvestigation.*The impact of microbial communities on the incidence of foliar fungal diseasesymptomsinwheatGeorgeLund1,2,MatthewDickinson3,TimMauchline2andJasonRudd11Bio-interactionsandCropProtectionDepartment,RothamstedResearch,Harpenden,AL5 2JQ, UK; 2 Sustainable Agricultural Science Department, Rothamsted Research,Harpenden, AL5 2JQ, UK; 3 School of Biosciences, University of Nottingham,Loughborough,LE125RD,UKEmail:george.lund@rothamsted.ac.ukSeptoriatriticiblotchdisease(STB)causedbythefungusZymoseptoriatritici,isthemosteconomicallyimportantwheatdiseaseinWesternEurope.Withresistanceto
67
conventionalfungicidesbecominganincreasingproblem,alternativesolutionsarerequired.Primarystagesofinfectionoccurintheextracellularspacesofthewheatleaf,wherebacteriasuchasPseudomonasfluorescenscanalsobefound.InourfirstexperimentaPseudomonadlibrarywasscreenedfordirectinhibitionofZ.triticionagarplates,todetermineifanyexhibitantifungalactivity.Usingahigh-throughputscreeningmethodologydevelopedinthisprojectwhichtested460bacterialendophytesandrhizosphereassociatedPseudomonads,40isolatesshowedanabilitytodirectlyinhibitthegrowthofZ.tritici.Ourresultsraisethepossibilitythatbacteriapresentonwheatleavesmayaffecttheincidenceand/orseverityofSTBviadirectinhibitionoffungalgrowth,throughsecretedfungalinhibitors.Inasecondexperiment,weplantogrowsurfacesterilisedwheatseedsinsterilegrowthmediumamendedwithsyntheticmicrobialcommunitiesbeforeinfectingtheplantswithZ.tritici.Thiswillallowustomonitortheimpactofthebelowgroundmicrobiomeontheplant’sresponsetofoliardisease,throughprimingorothermechanisms.Thesecombinedapproachesmayleadtonovelmechanisms,whichcouldbeexploitedforchemicaland/orbiologicalcontrolofZ.tritici.ExploringNGS,HIGSandsi-RNAtechnologiesforthecontrolofFusariumearblightinwheatAnaK.Machado1,RobertKing2,WingShamLee1,CarolineSparks1,NeilBrown1,KostyaKanyuka1,MartinUrban1,JonWest1,EleneYamazaki-Lau3,CasianeS.Tibola3,MariaI.P.M. Lima3, Roberto Togawa4, Natalia Martins4, Francisco Aragão4, Camila P. Nicolli5,EmersonM. Del Ponte5, Dauri J. Tessmann6, JoséMauricio C. Fernandes3 and Kim E.Hammond-Kosack11 Biointeractions and Crop Protection Department and 2 Computational and SystemsBiology Department, Rothamsted Research, Harpenden, UK, 3 Embrapa Trigo, PassoFundo, Rio Grande do Sul, Brazil 4 Embrapa Genetic Resources and Biotechnology,Brasilia,Brazil,5UniversidadeFederaldeViçosa,MG,Brazil,and6UniversidadeEstadualdeMaringá,PR,Brazil.Email:ana.machado@rothamsted.ac.ukFusariumearblight(FEB)isamajorprobleminmostsmall-graincerealgrowingregionsandnowthreatensglobalfoodsecurity.CurrentlystrategiestocontrolFEBarenotveryeffective,fungicidesgivepartialprotectionanddevelopmentofgenetic-basedresistantcultivarshasproventobedifficult.Therefore,newwaystocontrolFEBareurgentlyrequired.Here,wedescribetheframeworkandinitialresultsfromabilateralUK-Brazilprojectthatisusingabespokewholegenomesequencing,inplantatranscriptomeandreversegeneticsguidedapproachtounderstandandpinpointtheFusariumgenesandpathwaysrequiredtocausediseaseinwheatheads.OurintentionistoidentifyanewsuiteofFusariumgenesforinterventionthatcansimultaneouslybetargetedviahostinducedgenesilencing(HIGS).HIGSconstructsarebeingtestedforefficacyinArabidopsis,lettuceandwheat,andpromisingHIGSconstructshavebeenstably
68
transformedintoacommercialmoderately-FEBresistantBrazilianwheatinpreparationforfieldtestinginsouthernBrazilduring2018and2019.BytakingaFusariumgenome/reversegeneticsguidedapproach,thisisenablingthedevelopmentofflexiblenewwaystocontrolFEBdiseaseinwheatcropsgrowninBrazil.Themainprerequisitesneededtoapplythisapproachinotherwheatgrowingregionswillbeaddressed,aswellasadditionalscientific,societalandindustrybenefitsthatcouldpotentiallyemergewhenusingHIGStechnologiesforplantdiseasecontrol.ThisresearchissponsoredbytheBBSRC,TheNewtonFundandEMBRAPA.HeterologousexpressionofferuloylesteraseofAspergillusterreusMiia R. Mäkelä1, Adiphol Dilokpimol2, Salla Koskela1, Ronald P. de Vries2, KristiinaHildén11Division ofMicrobiology and Biotechnology, Department of Food and EnvironmentalSciences, University of Helsinki, Helsinki, Finland; 2Westerdijk Fungal BiodiversityInstitute, Utrecht, The Netherlands; 3FungalMolecular Physiology, Utrecht University,Utrecht,TheNetherlandsEmail:miia.r.makela@helsinki.fiInthecellwallsofgramineousplants,hemicellulosesarecrosslinkedtothearomaticligninpolymerviahydroxycinnamicacids(ferulicacidandp-coumaricacid).Feruloylesterases(ferulicacidesterases,EC3.1.1.73),presentinCAZyfamilyCE1(www.cazy.org),areenzymesthatcatalysethecleavageofcovalentesterbondsbetweencarbohydrateandligninmoietiesinplantcellwalls.Duetotheabilitytospecificallycleaveesterlinkages,feruloylesterasesarepromisingbiocatalystsforabroadrangeofbiotechnologicalapplications.Theseincludee.g.pharmaceutical,agriculturalandfoodindustries,aswellastheproductionofbiofuel.AnalysisoftheCAZomesofeightAspergillusspeciesrevealedahighvariabilityinthegenesetsrelatedtoplantbiomassdegradation(1).OneofthesevariationswasintheputativeFAEsofCAZyfamilyCE1,whereallspeciescontainoneconservedfaegene,butsomespeciespossessadditionalcandidategenesforthisactivity.AspergillusterreuscontainstwoadditionalputativeFAEencodinggenes,oneofwhichhasorthologsinAspergillusoryzaeandAspergillusflavus,butnotinanyoftheothertestedspecies(Aspergillusniger,Aspergillusnidulans,Aspergillusclavatus,AspergillusfumigatusandAspergillusfisheri).OneoftheseputativeA.terreusFAEencodinggeneswaschosentobeheterologouslyexpressedinamethylotrophicyeastPichiapastoris.Therecombinantenzymewassuccessfullyproducedandfurtherstudiedforitsbiochemicalcharacteristics.(1)Closelyrelatedfungiemploydiverseenzymaticstrategiestodegradeplantbiomass.Biotechnol.Biofuels8,107.
69
MeasuringresistancetoSeptoriatriticiblotchinelitewheatcultivarsusingautomatedimageanalysisAlexeyMikaberidzePlantPathology,InstituteofIntegrativeBiology,ETHZürich,Universitaetstr.2,ETHZentrum,LFWB28,CH-8092Zurich,SwitzerlandEmail:QuantitativeresistanceislikelytobemoredurablethanmajorgeneresistanceforcontrollingSeptoriatriticiblotch(STB)onwheat.Earlierstudieshypothesizedthatresistanceaffectingthedegreeofhostdamage,asmeasuredbythepercentageofleafareacoveredbySTBlesions,isdistinctfromresistancethataffectspathogenreproduction,asmeasuredbythedensityofpycnidiaproducedwithinlesions.Wetestedthishypothesisusingacollectionof335eliteEuropeanwinterwheatcultivarsthatwasnaturallyinfectedbyadiversepopulationofZymoseptoriatriticiinareplicatedfieldexperiment.Weusedautomatedanalysisof21214scannedwheatleavestoobtainquantitativemeasuresofSTBconditionalseveritythatwereprecise,objective,andreproducible.Thesemeasuresallowedustoexplicitlyseparateresistanceaffectinghostdamagefromresistanceaffectingpathogenreproduction,enablingustoconfirmthattheseresistancetraitsarelargelyindependent.Thecultivarrankingsbasedonhostdamageweredifferentfromtherankingsbasedonpathogenreproduction,indicatingthatthetwoformsofresistanceshouldbeconsideredseparatelyinbreedingprogramsaimingtoincreaseSTBresistance.Wehypothesizethatthesedifferentformsofresistanceareunderseparategeneticcontrol,enablingthemtoberecombinedtoformnewcultivarsthatarehighlyresistanttoSTB.ThesedatacanalreadybeusedbybreedingprogramsaimingtoincreaseSTBresistancetochoosewheatcultivarsthatarebroadlyresistanttonaturallydiverseZ.triticipopulationsaccordingtothedifferentclassesofresistance.*In-Vitro Antifungal Screening of Radish (Raphanus sativus L.) Extracts as SafeBotanicalFungicidesagainstAlternariaalternataandSclerotiumrolfsiiHassanB.MohamedandRashidM.MamunurDepartment of Plant Pathology, Hajee Mohammad Danesh Science and TechnologyUniversity,Dinajpur,5200,BangladeshEmail:medbarkad1991@gmail.com
Twolabexperimentswereconductedonradish(Raphanussativusvar.BARIMula-1)extractionstoquantitativelyinvestigatephytochemicalsinmethanolicradishrootextract(MRRE)andscreenantifungalactivityofmethanolicandaqueousradishextractsagainstAlternariaalternataandSclerotiumrolfsiiinAgriculturalChemistryandPlantPathologyLaboratories,HajeeMohammadDaneshScienceandTechnologyUniversityDinajpur,duringSeptember,2015–September,2016.Methanolicsolventwasused
70
usingmacerationmethod,where15.12g(30.24%)brownpungentgreaselessjelly-likecrudephytochemicalsubstancewasextractedfrom50gradishrootpowderdissolvedwith200mlmethanol.TLCanalysisofMRREshowedat1:10ethylacetatehexane6spotsrecordedasRfvalues,wherehighestRfvaluewas0.91cmandlowestRfvalue0.29cm.In-vitroinhibitoryactivityoftheradishextractsagainstmycelialgrowthofthetestfungiwasscreenedusingpoisonedfoodtechnique.Threepoisonedmediaviz.methanolicradishrootextractmedia(MRREM,T1),aqueousradishrootextractmedia(ARREM,T2)andaqueousradishleafextractmedia(ARLEM,T3),andnon-poisonedPDAmedia(Tc)wereusedastreatments.Therewerehighlysignificancedifferencesamongthepoisonedmediainreductionofthemycelialgrowthoverthenon-poisonedcontrolPDAmedia.MRREMinhibitedthegrowthofA.alternataandS.rolfsii94%and58.1%,followedbyARREM93%and40%andbyARLEM90%and20%,respectively,overthecontrol.ItmaybeconcludedthatradishextractscanbeusedassafebotanicalfungicidestocontrolA.alternataandS.rolfsii.**ResistanceofSaccharomycescerevisiaetolignocellulose-derivedinhibitorsElenaMoreno-MartínezandSimonV.AverySchoolofLifeSciences,UniversityofNottingham,UniversityPark,NottinghamNG72RD,UKEmail:msxem3@nottingham.ac.ukProductionofbioethanolfromlignocellulosichydrolysatesisapromisingbiofueltechnologybutissubjecttoinhibitionbychemicalsreleasedduringthepretreatmentofbiomass.Weakacids,furansandphenolsimpairyeastgrowthandfermentation,thusreducingefficiencyoftheprocessandincreasingthecost.Someofthemolecularmechanismsofactionandresponsetothesestressorsarecharacterized,butlittleisknownabouthowuniformsucheffectsareacrossindividualcellswithinanisogeniccellpopulation.Non-genotypicheterogeneityreferstotheexpressionofdifferentphenotypeswithinaclonalcellpopulation,despiteallindividualsbeinggeneticallyidentical.Understandingindividual-celleffectsofhydrolysateinhibitorscouldhelpengineeringofpopulation-levelresistance.ThedegreeofheterogeneityinresistancetraitswasspecifictoparticularinhibitorsinthefermentingyeastS.cerevisiae.Inclusionofasecondinhibitorcouldalterheterogeneityrelativetoeitherinhibitoralone.Shortpretreatmentswithsub-inhibitoryconcentrationsoftheinhibitorsindicatedadaptiveresistanceandadaptiveheterogeneityresponses.RNAiassistedgenomeevolutionwasalsousedtorevealcombinationsofgeneknockdownsthatcansynergisticallyincreaseresistancetowardstheseinhibitors.Findingsderivedfromthisresearchwillprovidebetterunderstandingofthebehaviourofyeastinlignocellulosicethanolfermentations,andmighthelpimproveproduction.
71
Carbohydrate arrays coupled with mass spectrometry to detect activity of plantpolysaccharidedegradativeenzymesfromAspergillusnigerJolandavanMunster1,2,BaptisteThomas1,MichelRiese1,AdrienneDavis3,ChristopherGray1,DavidArcher2,SabineFlitsch11Chemical Biology,Manchester Institute for Biotechnology,University ofManchester,Manchester, M1 7DN UK; 2 Fungal Biology and Genetics, School of Life Sciences,UniversityofNottingham,Nottingham,NG72RD,UK;3SchoolofChemistry,UniversityofNottingham,Nottingham,NG72RD,UKemail:Jolanda.vanMunster@Manchester.ac.ukRenewables-basedbiotechnologydependsonenzymestodegradeplantlignocellulosetosimplesugarsthatareconvertedtofuelsorhigh-valueproducts.Identificationandcharacterizationofsuchlignocellulosedegradativeenzymescouldbefast-trackedbyavailabilityofanenzymeactivitymeasurementmethodthatisfast,label-free,usesminimalresourcesandallowsdirectidentificationofgeneratedproducts.TheobjectiveofthisworkwastodevelopsuchamethodbyapplyingcarbohydratearrayscoupledwithMALDI-ToFmassspectrometrytoidentifyreactionproductsofcarbohydrateactiveenzymes(CAZymes)ofthefilamentousfungusAspergillusniger.Wedescribetheproductionandcharacterizationofplantpolysaccharide-derivedoligosaccharidesandtheirattachmenttohydrophobicself-assemblingmonolayersonagoldtarget.Weverifiedeffectivenessofthisarrayfordetectingexo-andendo-actingglycosidehydrolaseactivityusingcommercialenzymes,anddemonstratehowthisplatformissuitablefordetectionofenzymeactivityinrelevantbiologicalsamples,theculturefiltrateofA.nigergrownonwheatstraw.WewillnowapplythismethodtocharacterisethedegradativepotentialofthebroadsetofA.nigerCAZymesthataresecretedinresponsetoplantlignocellulose.Inconclusion,wedescribeaversatilemethodthatisbroadlyapplicableinscreeningandcharacterisationofactivityofCAZymes,suchasfungalenzymesforplantlignocellulosedegradationwithrelevancetobiotechnologicalapplicationsasbiofuelproduction,thefoodandanimalfeedindustry.*Detection and Distribution of Aphanomyces Root Rot in Peas within the UnitedKingdomBrianÓLoinsigh1,LeaHerold2,DebbieSparkes1andMatthewDickinson11PlantandCropSciencesDivision,UniversityofNottingham,LE125RD,UK;2ProcessorsandGrowersResearchOrganisation,Peterborough,PE86HJ,UKEmail:stxbb13@nottingham.ac.ukRootrot,causedbyAphanomyceseuteiches,astrictsoil-borneoomycete,isoneofthemostdamagingdiseasesofpea(Pisumsativum)andsignificantlyreducespeaproductionglobally.Sincetheinoculumpersistswithinthesoil,andnoresistantcultivarsormeansofchemicalcontrolexists,thediseaseisdifficulttomanage.
72
Moreover,theUKviningpeaareasarefixedwithnoroomtomovefurtherafield.Forthesereasonsgrowersfinditusefultoobtaininformationonthedegreeofinfestationwithintheirfieldstoavoidthoseathighrisk.However,thespreadandseverityofA.euteichesacrosstheUKgrowingregionsiscurrentlyunknown.Inthisproject,soilsfrom38siteswerecollectedspanningtheviningpeaareasandtestedforthepresenceofAphanomycesrootrotthroughbaitingexperiments.ALoopMediatedIsothermalAmplification(LAMP)assayforthespecificdetectionofA.euteichesfrompeaplatebaitingexperimentswasdeveloped.TheresultssuggestthatthisLAMPassaycanbeusedtodetectA.euteichesDNAontherootsofinfectedseedlingsthathasbeenbaitedwithsoilwithindaysofreceivingthesamples.CombiningbothvisualdiseasescoresandLAMPresults,adistributionmapofthediseasewasdeveloped.WeshowthatA.euteichesisfoundacrossallareasoftheUKgrowingregionandismorewidespreadthanpreviouslyknown,withsomeareashavingahigherburdenthanothers.TheLAMPassayallowsforfastandspecificdetectionofthepathogen.ThisinformationcannowbeusedtofurtherunderstandthediseaseintheUKandhelpslowthespreadofthedisease.**Optimisation of sexual development in Pyrenopeziza brassicae to allow study ofhormonaltriggeredmorphogenesisTomPearson1,AlisonA.Ashby2,PaulS.Dyer11SchoolofLifeSciences,UniversityofNottingham,Nottingham,NG72RD,UK;2CrogganLtd,TheCoachHouse,LongLaneCambridgeshire,CB245LD,UKEmail:Stxtp4@nottingham.ac.ukFungicideresistanceisamajorproblemwithinagriculture,causingmillionsofpounds’worthofcroplossandthreateningfoodsecurity.Severaleconomicallyimportantfungalcroppathogenshavedevelopedsomelevelofresistancetocommonfungicides,oneexamplebeingPyrenopezizabrassicae,thecauseoflightleafspotonoil-seedrapeandotherBrassicaspecies.Itisclearthatnewmethodstocontroldiseaseneedtobedeveloped.ThereispreliminaryevidencethatP.brassicaeproducessexhormones(knownasSexFactor)duringmatingwhichalterdevelopment.Thesecouldbeutilisedasnovelfungicidestoinhibitasexualreproduction,thereforecontrollingthespreadofdisease.Toidentifymodelsystemsforhormonestudy,sexualcrossesarebeingsetupbetweenP.brassicaeisolatesofopposingmating(MAT)types.Currentworkisfocusedonoptimizingproductionoffertileapotheciainlaboratorycrosses,usingavarietyofcrossingmethodsandgrowthmedia.Ongoingresultswillbepresented.FutureworkwillfocusonfurtherpurificationandcharacterisationofSexFactor,whichpreviousstudiesindicateislipoidalinnatureandcanbeextractedusingethylacetateormethylchloroform.Bioassayswilllookforsuppressionofasexualsporulationandinductionofsex.
73
*Virus-andsalicylicacid-inducedchangesinplant-insectandplant-virusinteractionsAnna Platoni1,2, Sanjie Jiang1, Alex M. Murphy1, Adrienne E. Pate1, Ju-Yeon Yoon1,3,BeverleyJ.Glover1,JohnP.Carr1andGerardR.G.Clover21PlantSciencesDepartment,UniversityofCambridge,Cambridge,CB23EA,UK;2PlantHealth,RoyalHorticulturalSociety,RHSGardenWisley,Woking,SurreyGU236QB;3VirologyUnit,DepartmentofHorticulturalEnvironment,NationalInstituteofHorticulturalandHerbalScience,RDA,Wanju,55365,SouthKoreaEmail:amp93@cam.ac.ukInteractionsbetweenplantsandinsectsarecomplexandmediatedbyplant-derivedsignalmolecules,includingvolatileorganiccompounds.Infectionbyvirusesaltersplantvolatileemissionandconsequentlyaffectsplantinteractionswithinsectsthatvectorviruses,aswellaswithbeneficialinsects.Forexample,infectionwithcucumbermosaicvirus(CMV)affectsinteractionswithaphids(1),whileinotherworkwefoundthatbumblebees(Bombusterrestris)areattractedtoCMV-infectedtomato(Solanumlycopersicum)plants(2).Salicylicacid(SA)andjasmonicacid(JA)aredefensivephytohormonesthatinfluenceinteractionsofplantswithviruses,microbesandinsects.Intomatoandpepper(Capsicumannuum)weareinvestigatingtherolesofSAandJAinregulatingresistancetoCMVinfection,andincontrollingemissionofvolatilesthatinfluenceinteractionsofplantswithaphids(Myzuspersicae)orwithbumblebees.Inparticular,weareinvestigatingtheeffectsofexogenousapplicationofSAandJAonemissionofinsect-perceivablevolatiles,andtheeffectsofSAonCMVreplicationandmovement.ThewideraimofthisworkistofurtherunderstandtheeffectsoftheSA-andJA-dependentdefensivesignallingpathwaysoninteractionsbetweenplants,virusesandharmfulaswellasbeneficialinsectstoprovideamateurandprofessionalhorticulturalistswithbetteroptionsforenvironmentallyfriendlyintegratedpestanddiseasemanagement.1.Westwoodetal.2013PLoSONE8(12):e83066.2.Groenetal.2016PLOSPathogens12(9):e1005906.WorkfundedbytheRoyalHorticulturalSociety,BBSRC,LeverhulmeTrust,andtheRuralDevelopmentAgencyoftheRepublicofKorea.MechanisticbasisofepithelialdamageinresponsetoAspergillusfumigatusinfection**NormanvanRhijn1,TakanoriFurukawa1,PanagiotisPapastamoulis2,SayemaKhan1,MikeBromley1,MagnusRattray2andElaineBignell11Manchester Fungal Infection Group, University of Manchester, Manchester, UnitedKingdom;2DivisionofInformatics,ImagingandDataSciences,UniversityofManchester,Manchester,UnitedKingdom.Email:norman.vanrhijn@manchester.ac.ukThe human lung is continually exposed to spores of the airborne mouldAspergillusfumigatus. Inhaled spores are small enough to bypass mucociliary clearance
74
mechanisms and reach the alveoli of the lung where interaction between host andpathogen cells can lead to fungal clearance, or to development of inflammatory orinvasive fungal diseases. Fuelled by the results of extensive fungal transcriptomeresearchwehavedeveloped, inrecentyears,aprogrammeofresearchthatseeksthemechanistic basis of lung damage duringA. fumigatusinfection. In this study weintroduceanewhighthroughoutstudyaddressingtheA.fumigatusregulatorynetworkdriving epithelial damage in cultured human lung tissue and examine how thisknowledgemightleadtonoveltherapeuticinterventions.*InvestigatingeffectordeliverybythericeblastfungusMagnaportheoryzaeClaraRodriguez-Herrero,MagdalenaMartin-Urdiroz,GeorgeLittlejohn,MiriamOses-Ruiz,WasinSakulkoo,XiaYan,MichaelJ.Kershaw,DarrenM.SoanesandNicholasJ.TalbotSchoolofBiosciences,UniversityofExeter,EX44QD,UnitedKingdomEmail:cr425@exeter.ac.ukMagnaportheoryzaeisthecausalagentofriceblastdisease.ThelifecycleofM.oryzaestartswhenasporelandsonthesurfaceofaleaf.Thesporegerminatestoformagermtube,whichgeneratestheappressorium.Theappressoriumgeneratesenormousturgorpressurethatistranslatedintophysicalforcethatrupturesthericeleaf.Thefungusthendevelopsinvasivehyphae(IH),whichcolonizesplanttissue.M.oryzaesecreteseffectorstofacilitatehostcolonizationbysuppressingplantimmunityresponses.EffectorsinM.oryzaecanbeclassifiedascytoplasmic,orapoplastic,dependingonwheretheyaredelivered.Cytoplasmiceffectorscanbeseentoaccumulateinamembrane-richplantstructurecalledthebiotrophicinterfacialcomplex(BIC),whileapoplasticeffectorsareattheextra-invasivehyphalmembranesurroundingtheIH.TheBICisthoughttobeinvolvedintranslocationofeffectorsintothericecytoplasm.Cytoplasmicandapoplasticeffectorsfollowtwodifferentsecretionpathways.ApoplasticeffectorsaresecretedbytheconventionalER-Golgisecretorypathway,whilecytoplasmiceffectorsaresecretedviaanunconventionalroute.Littleisknownabouthowsecretedeffectorsaredeliveredtothecorrectdomains.Tounderstandthisprocess,wearetryingtoidentifyaspecifichosttargetingsequencebygeneratingalibraryofchimericeffector-encodinggenefusions.Weareanalysingthechimerasfortranslocationpotential.Inaddition,wearegeneratingstabletransgenicriceplantsinwhichwehaveGFP-taggedbothearlyandlateendosomalcompartmentstodeterminethepotentialroleofplantendocytosisineffectoruptakeintoplantcells.
75
Azole sensitivity in Leptosphaeria pathogens of oilseed rape: the role of lanosterol14α-demethylase.ThomasRSewell1,2,NicholaJHawkins3,YongJuHuang2,HenrikUStotz2,StevenLKelly4,DianeEKelly4,BartFraaije3,BruceDLFitt21Department of Infectious Disease Epidemiology, Imperial College London, UK; 2CropProtection Group, Centre for Agriculture, Food and Environmental Management,University of Hertfordshire, Hatfield, UK; 3Biointeractions and Crop Protection,Rothamsted Research, Harpenden, Hertfordshire, UK; 4Centre for Cytochrome P450Biodiversity,InstituteofLifeScience,SchoolofMedicine,SwanseaUniversity,Swansea,UKEmail:t.sewell@imperial.ac.ukLanosterol14-αdemethylase(erg11,CYP51)isakeyenzymeinthefungalergosterolbiosyntheticpathway,andisthetargetofazolefungicides.PriorstudieshaveshownthatLeptosphaeriamaculansandL.biglobosa,thecausalpathogensofphomastemcankeronoilseedrape(Brassicanapus),differintheirsensitivitytosomeazoles.Hereweusedheterologousexpressionandproteinmodellingtodeterminewhetherthereareinter-specificsensitivitydifferencesatthetarget-sitelevel.Moreover,wedeterminedthepracticalsignificanceoffungicidesensitivityusinganewcollectionofisolatesfromthesoutheastoftheUK.HeterologousyeastexpressionofLmCYP51BandLbCYP51Bwithfungicidesensitivitytestingofthetransformantssuggeststhatthetwoproteinsaresimilarlysensitivetoazolefungicidesflusilazole,prothioconazole-desthioandtebuconazole.Thesefindingsaresupportedbyhomologyproteinmodelling,whichpredictsthatLmCYP51BandLbCYP51Barestructurallyverysimilar,specificallyattheazole-bindingsite.Fungicidesensitivitytestingonthenewcollectionofisolatesshowsthattheysometimeshaveaminordifferenceinsensitivityinvitroandinplantabutatpresentbothspeciesremainverysensitivetosomeazolefungicides.TheseresultsshowthatazolefungicidesremainausefulcomponentoftheintegratedmanagementofphomastemcankerintheUKduetotheireffectivenessatcontrollingLeptosphaeriaspp.growth,andsuggestthatotherfactors,suchasvarietalresistanceorclimate,maybedrivingfrequencychangesinthetwospecies.BioactivecompoundswithantimicrobialactivityextractedfrommushroomsgrowninLaRiojaVanessa Grifoll1, Ana Gonzalo1, Maria Luisa Tello1,Margarita Pérez1, Carmen Lozano2,BeatrizRojo2,YolandaSáenz21CentroTecnológicodeInvestigacióndelChampiñónenLaRioja(CTICH).Ctra.Calahorra,KM 4, 26560 Autol, La Rioja, Spain; 2Centro de Investigación Biomédica de La Rioja(CIBIR).CallePiqueras,983º,26006Logroño,LaRioja,Spain.Email:biotecnologia@ctich.com
76
Theprolongeduseofantibiotics,inhumanmedicine,veterinaryandagriculture,hascausedanalarmingincreaseofantimicrobialresistantbacteriaworldwide.Concerningtothisseriousthreat,theWorldHealthOrganization(WHO)signedin2016anoverallagreementtoaddresstheproblemofantimicrobialresistance,andin2017publishedalistofprioritypathogensthatrequirethedevelopmentofnewantimicrobialagents.Inthatline,theaimofthisworkwastosearchnewantimicrobialalternativesobtainedfrommushrooms.Sixteenmushrooms(Pleurotuseryngii,Agaricusbisporus,Pleurotusostreatus,Pleurotussalmoneostramineus,Hypsizygusulmarius,Pleurotuscitrinopileatus,Agrocybeaegerita,Hericiumerinaceus,Lentinulaedodes,Agaricusbrunnescens,Agaricusbrazei,Hypsizygustessulatusgris,Hypsizygustessulatusblanca,Grifolafrondosa,GanodermalucidumandPholiotanameko)weregrownonsterilizedsubstratesinLaRioja(Spain)andsubsequentlyfreeze-driedtoobtainamushroompowder.Diversemethodologieswereusedtoobtainthelyophilisedpowdersandthedifferentaqueousandalcoholicextracts.AgardiffusionmethodswereusedtotesttheinvitroantimicrobialactivityagainstreferencestrainsofEscherichiacoli,Listeriamonocytogenes,Pseudomonasaeruginosa,Enterococcusfaecalis,MicrococcusluteusandStaphylococcusaureusspecies.PleurotuscitrinopileatusandLentinulaedodeslyophilisedpowderwereactiveagainstallstudiedbacteriaexcepttoP.aeruginosa.ThepowderofPleurotusostreatus,HypsizygustessulatusgrisandPholiotanamekosuppressedthegrowthofP.aeruginosaorS.aureus.Thefirstresultsareencouraging,andraiseapossibledevelopmentofnewextractswithantimicrobialpropertiesthatmaybeapplicableindifferentindustries.Methodologies development for the cultivation of new species of fungi in La Rioja(Spain)MariaLuisaTello,MargaritaPérezCentroTecnológicodeInvestigacióndelChampiñónenLaRioja(CTICH).Ctra.Calahorra,KM4,26560Autol,LaRioja,SpainEmail:biotecnologia@ctich.comThefungalkingdomischaracterizedbyitshighbiodiversity.Howeveronlyabout100speciesoffungihavebeencultivated.Inthelastyearsthenumberofcultivatedmushroomshasbeenincreasedgradually,optimizingtechniquesusedforgrowingtraditionalspeciestocultivatenewvarieties.Inaddition,themushroommarketinEuropehasgrownandconsumerssearchfornewhealthyproductswithhighqualityandthebestprice.Thus,themainpurposeofthisworkistodevelopthecultivationofnewmushroomspeciesendemictoLaRioja.Toachievethis,CTICHhaveselected12strainsfromthespeciesconservedintheMushroomsBiobankatCTICH.ThisbiobankisafungalgeneticresourcecollectionthatenablestheconservationofwildfungalbiodiversityintheregionofLaRioja(Spain).Thespeciesselectedwerethosewhosecultivationseemed
77
morefeasiblebearinginmindseveralfactors,suchas:costofrawmaterialsandproduction,simplicityofcultivationandcollection,marketingoracceptancebypotentialconsumers.AmongtheselectedspeciestherearespecieswithmedicinalpropertieslikeGanodermalucidum,protectedspeciessuchasRhodocybegeminaandediblemushroomslikeCalocybegambosa.Forthecultivationoftheselectedspeciesitwasnecessarytodeveloptechniquesforconservationofthemyceliumandspawnproliferationandnewsubstrateformulae,inadditiontodesigningandoptimizingtheconditionsforincubation,cultureandcropmanagement.Doesnitricoxideregulatecroppathogenresistance?AnnaM.M.TileyandAngelaFeechan
School of Agriculture and Food Science, University College Dublin, Dublin, Dublin 4,IrelandEmail:anna.tiley@ucd.ieZymoseptoriatriticiisapathogenicfungusthatcausesoneoftheworstdiseasesofwheatworldwide,SeptoriaTriticiBlotch.Theaimofourprojectistoinvestigatetheimportanceofnitricoxide(NO)duringZ.triticiinfection.NOisproducedasadefenceresponsebytheplant,andcanactdirectlyasanantimicrobialorasasignallingmolecule.WefirstlyaimtoassesswhetherNOmightregulatetheeffectorproteinsreleasedbyZ.triticiduringinfection.CandidateeffectorshavebeenidentifiedinZ.tritici,andarebeingstudiedtoassesswhethertheyareNO-regulatedduringplantinfection.ThisprojectalsoaimstoidentifywhetherZ.triticiemploysmechanismstocountertheplant’sNOburst.TheenzymeGSNOreductase(GSNOR)controlstheNOpoolinotherspecies,andsoweareinvestigatingtheeffectofdisruptingthegsnorgeneinZ.tritici.BioinformaticsstudieshaveidentifiedapotentialgsnorgenehomologinZ.tritici,ztgsnor.Knock-outplasmidshavebeenconstructedandtransformedintoZ.triticitocreateztgsnorknock-outmutants.Theresultsfromtheseexperimentsareongoing,andthemostrecentfindingsfromthesestudieswillbepresented.EvidenceforinvolvementofanRLXReffectorproteininPhytophthorainfestanssexualreproductionGeorgios Tzelepis, Anna Åsman, Ramesh Vetukuri, Johan Fogelqvist, and ChristinaDixeliusDepartment of Plant Biology, Uppsala Biocenter, Swedish University of AgriculturalSciences,LinneanCenterforPlantBiology,P.O.Box7080,S-75007,Uppsala,SwedenEmail:Georgios.tzelepis@slu.se
78
Phytophthorainfestansisanotoriousfungal-likeplantpathogen,responsibleformajoreconomiclossesinpotatoandtomatocultivationsworldwide.Inordertoestablishasuccessfulinfection,thispathogendeploysanarsenalofsmall-secretedproteins,termedeffectors.Amongthem,effectorsharbouringtheRXLRmotifarethebest-studiedones.Theyarehighlyinducedduringearlybiotrophicstageofinfectionandareabletomanipulateplantdefencemechanismsoperatinginsidethehostcell.InthisstudyweinvestigatedthetranscriptionprofilesofdifferentP.infestansisolates(originatedfromdifferentpartsinEurope)duringthematingprocess,sincematingfrequenciesareknowntodifferamongEuropeancountries.OurresultsrevealedthatanRXLReffector-encodinggeneishighlyinducedduringmatinginmostofourcrossings.Furtheranalysisshowedthatoosporesproductionissignificantlyreducedwhenthiseffectorhasbeensilenced.Moreover,weobservedthatthisproteinisaccumulatedinhyphaltipsduringmating,whileinfectionofN.bethamianaplantswiththeGFP-taggedstrainandtransientexpressioninpotatoleavesconfirmedthecytoplasmiclocalizationofthiseffectoruponinfection.Inconclusion,thisstudyrevealedapotentialmultifunctionalroleofanRXLReffector.Interestingly,thepresenceofthiseffectoronlyinheterothalliccloselyrelatedtoP.infestansspecies,supportsitspotentialroleinsexualreproduction.WecanspeculatethatcertaineffectorsmightbeinvolvedinotheraspectsofP.infestansbiologythaninoomycete-plantinteractions.Ifthisfunctionreflectsevolutionaryadaptationremainstobeclarified.PHI-base:HarnessingcommunityexpertisetofightplantdiseaseMartinUrban1,AlayneCuzick1,KimRutherford2,HelderPedro3,NishadiDeSilva3,PaulKersey3andKimE.Hammond-Kosack11DepartmentofBiointeractionsandCropProtection,RothamstedResearch,Harpenden,Herts, AL5 2JQ, UK; 2 Cambridge Systems Biology and Department of Biochemistry,University of Cambridge, Sanger Building, 80 Tennis Court Road, Cambridge,Cambridgeshire CB2 1GA, UK; 3 The European Molecular Biology Laboratory, TheEuropeanBioinformaticsInstitute,Hinxton,Cambridgeshire,CB101SD,UKEmail:Martin.Urban@rothamsted.ac.ukThepathogen-hostinteractionsdatabasePHI-base(www.phi-base.org1)isagold-standarddatabasestoringphenotypesongenesimplicatedinvirulence.Itisaprimaryinformationsourceforresearchersstudyingplant-pathogeninteractionsaswellashumanfungalpathogens.Morethan2300researcharticlesaremadeaccessibleforresearcherstoeasilyfamiliarisethemselveswithrelevantinformationontheirtargetgenesofinterest.PHI-baseprovidesbothmanuallycuratedmolecularandbiologicalphenotypeinformationonpathogengenes.Themulti-speciesdatabasesummarisesinformationfrom264pathogenstestedon173hoststoallowcomparativegenomicsapproachestofacilitatenovelfungicidetargetdiscoveryandidentifycriticallyimportantgenesforbiotech/breedingcontrolstrategies.Allmajorplantpathogensfromthebacterial,fungalandoomycetekingdomsareincluded.
79
HerewedescribeournewPHI-baseVersion4.3release(May2017).Ourcurrentdataplatformincludesimprovedsearch,filteringandextendeddatadisplayfunctions.Aprotein-to-phenotypeBLASTsearchfunctionisincluded.Developmentofacommonlanguage(ontology)todescribecomparablephenotypesinawiderangeofpathogen-hostinteractionsisdiscussed.ExemplarusecasesexploringthevastinformationinPHI-baseforfungaldiseasecontrolarepresented.1Urban,M.,Cuzick,A.,Rutherford,K.,Irvine,A.,Pedro,H.,Pant,R.,Sadanadan,V.,Khamari,L.,Billal,S.,Mohanty,S.,etal.(2017).PHI-base:anewinterfaceandfurtheradditionsforthemulti-speciespathogen-hostinteractionsdatabase.NucleicAcidsRes,doi:10.1093/nar/gkw1089.ThisworkissupportedbytheUKBiotechnologyandBiologicalSciencesResearchCouncil(BBSRC)(BB/I/001077/1,BB/K020056/1).PHI-basereceivesadditionalsupportfromtheBBSRCasaNationalCapability(BB/J/004383/1).*Effector discovery and characterisation in the Fusarium graminearum-wheatinteractionCatherineWalker1,2,Wing-ShamLee1,NeilBrown1,MartinUrban1andKim-HammondKosack11BiointeractionsandCropProtection,RothamstedResearch,Harpenden,AL52JQ,UK;2SchoolofBiosciences,UniversityofNottingham,SuttonBonington,LE125RD,UKEmail:catherine.walker@rothamsted.ac.ukFusariumgraminearum(Fg),thecausalpathogenofFusariumHeadBlight,exhibitsanunusualhemibiotrophiclifestylecharacterisedbyspatiallyandtemporallydistinctsymptomlessandsymptomaticphasesofinfection.Thesedistinctphasesarefacilitatedbythebiphasicsecretionofanarsenalofproteins,includingsmall,cysteine-richeffectors,whichmodifylivinghostplants,aidingcolonisationandinfection.Itishypothesisedthatthesecretionofeffectorsactstosubvertrecognitionbythehostand/orsuppresshostdefencesduringthesymptomlessphaseofinfection.Toidentifycandidateeffectors,aspecies-specificmicroarrayanalysiswasusedtointerrogatetheinvitroandinplantaexpressionpatternsofgenesbelongingtotheFgsecretome.TwentyputativeFusariumeffectorswereidentifiedandhavebeguntobecharacterisedusingtheBarley-StripeMosaicVirus-mediatedoverexpressionsystem(BSMV-VOX),stableArabidopsistransformationandviathegenerationofFggenedeletionmutants.InitialdatafromtheBSMV-VOXexperimentssuggeststhatseveralcandidateeffectorproteinsarebiologicallyactiveintheFg-wheatinteraction.DatafromtheseexperimentsindicatesthatviraltitreiscriticaltoproteinexpressionintheVOXexperimentsandthataninteractionbetweenthevirusandsomeproteinsofinterestmaybeoccurring.Preliminaryexperimentssuggestthatoverexpressionofthese
80
proteinsinthenon-hostNicotianabenthamianaresultsinthedifferentialexpressionofplantdefence-relatedgenes;ifso,thiscouldofferahigh-throughputmethodforeffectorscreening.Furtherworkisrequiredtodeterminewhetherthisisaviablerouteforeffectorcharacterisationandtomakeeffortstodecipherthefungal-viral-plantinteractionoccurringduringBSMV-VOXexperiments.AnalysisofpathogenicityshiftofCucumbermosaicvirusisolatesinCapsicumannuuminSouthKoreaJu-YeonYoon1,3,BongNamChung1,Seung-KookChoi2andJohnP.Carr31VirologyUnit,HorticulturalandHerbalCropEnvironmentDivision,NIHHS,RDA,Wanju55365 South Korea; 2 Vegetable Research Division, Department of Horticultural CropResearch, NIHHS, RDA, Wanju 55365, South Korea; 3 Department of Plant Science,UniversityofCambridge,Cambridge,CB23EA,UnitedKingdomEmail:juyeon74@gmail.comCucumbermosaicvirus(CMV),isanaphid-bornevirusthatinfectsawiderangeofplantspeciesincludingvegetablecropsandweeds.CMVisoneofthemostimportantviraldiseasesinpepper(Capsicumannuum)inSouthKorea.Since1990s,commercialchillipeppercultivarshavebeenbredforresistanceagainstCMVisolates(pathotype-0,CMV-P0)thatbelongtosubgroupIA(S-IA)becausetheS-IAisolatesweredominant.Recently,twoCMVisolates(CMV-CaP1andCMV-GTN),whichsystemicallyinfectthechillipeppercultivarsresistanttoCMV-P0,werereportedandbelongtoCMVsubgroupIB;theseweredesignatedaspathotype-P1(CMV-P1).Since2009,nationwideannualsurveysofpeppersfieldsshowedthemostdominantCMVpopulation(ca.90%)inpepperandweedswassubgroupIB.Inpathogenicityassays,CMV-GTNinducedseveremosaicandyellowingonchillipepper(cultivarCheong-Yang)21dayspost-inoculation(dpi),whileCMV-CaP1inducedmildoroccasionalmosaic.DetachedleafandDAS-ELISAassaysclearlyshowedthatCMV-GTNandCMV-Fny(S-IA)spreadfasterthanCMV-CaP1inthecultivarCheong-Yang,resultinginmoreseveresystemicsymptoms.PeppercultivarsManitta,BaerottaandLAM32containaresistancegene(s)againstCMV-P1.SystemicinfectionbyCMV-CaP1andCMV-GTNwasverifiedonupperleavesofboth‘Manitta’and‘Baerotta’butnoton‘LAM32’usingELISAanalysis.Interestingly,CMV-GTNinducedmoreseveresymptomsonupperleavesandhadfastersystemicmovementthanCMV-CaP1inBaerotta.TheseresultssuggestapathogenicityshiftinCMVisolatesinpepperplantsinSouthKorea.FundedbyRuralDevelopmentAgency(PJ012426)oftheRepublicofKorea.
81
Identification of QTLs underlying resistance to Cucumber mosaic virus in CapsicumannuumJu-YeonYoon1,3, SuKim2,Seung-KookChoi2,EunYoungYang2,BongNamChung1,andJohnP.Carr31VirologyUnit,HorticulturalandHerbalCropEnvironmentDivision,NIHHS,RDA,Wanju55365, South Korea; 2 Vegetable Research Division, Department of Horticultural CropResearch, NIHHS, RDA, Wanju 55365, South Korea; 3 Plant Sciences Department,UniversityofCambridge,Cambridge,CB23EA,UKEmail:juyeon74@gmail.comCucumbermosaicvirus(CMV)isoneofthemostimportantvirusesinfectingchilipepper(Capsicumannuum).RecentlyCMVisolatesthatbelongtosubgroupIB(namedCMV-P1pathotype)overcomeCMVresistanceofpepperinKorea.ResistanceevaluationofpeppergeneticresourcesidentifiedthatCapsicumannuum‘LAM32’showednoveltolerance(hereafterresistance)againstCMVisolatesCMV-P1pathotype.ToidentifyacandidateresistancegeneofLAM32forCMVresistanceQTL,C.annuum‘Subi’asasusceptibleparentwerecrossedandprogeniesofF1,F2andbackcross1populationswereproduced.ParentsandtheirprogenieswereinoculatedwithCMV-GTNisolatethatbelongstosubgroupIBandwereevaluatedforresistanceusinglateralflowimmunoassayandDAS-ELISA.TheresultsshowedthattheresistanceofC.annuum‘LAM32’wascontrolledbyasinglerecessivegene.Asubsetof119putativelymolecularmarkers,welldistributedoverthepeppergenome,wasselectedforQTLmappingusingthecompositeintervalmappingmethod.Sixgenomicregions,includingtwomajoreffectsandfourminoreffectQTLs,maybeassociatedwithresistanceagainstCMV.Subsequentmappinganalysis,basedonapepperlinkagemap,revealedthatgenescontrolledCMVresistancemightbelocatedatchromosome4&5.FurtherfinegeneticmappingforCMVresistanceisinprogress.FundedbyRuralDevelopmentAgency(PJ012426)oftheRepublicofKorea.
top related