Laminarin‐hydrolysingactivitydevelopedintheendospermoftomato(Lycopersiconesculentum)seedsfollowinggermination.Theenzymewasbasic(pI>10)andtheapparentmolecularmasswasestimatedtobe35 kDabySDS‐PAGE.Itwasspecificforlinearβ-1,3‐glucansubstrates.Laminarinwashydrolysedbytheenzymetoyieldamixtureofoligoglucosides,indicatingthattheenzymehadanendo‐actionpattern.Thus,theenzymewasidentifiedasβ-1,3‐endoglucanase(EC3.2.1.39).TheactivityoftheenzymedevelopedintheendospermafterrADIcleprotrusion(germination)hadoccurredandtheenzymeactivitywaslocalizedexclusivelyinthemicropylarregionoftheendospermwheretheradiclehadpenetrated.Whenthelateralendospermregion,wherenoinductionoftheenzymeoccurred,waswounded(cutorpunctured),therewasamarkedenhancementofβ-1,3‐glucanaseactivity.Thusthepost‐germinativeβ-1,3‐glucanaseactivityinthemicropylarendospermportionmightbebroughtaboutbywoundingresultingfromendospermrupturebyradiclepenetration.

Anabundant,catalyticallyactiveβ-1,3-endoglucanase(EC3.2.1.39)hasbeenisolatedfromthepulpofripebananas.Biochemicalanalysisofthepurifiedprotein,molecularmodelling,andmolecularcloningofthecorrespondinggeneindicatethatthisbananaenzymecloselyresemblespreviouslycharacterizedplantβ-glucanaseswithrespecttoitsamino-acidsequence,structureandbiologicalactivity.Theresultsdescribedinthispaperdemonstrateboththeoccurrenceofanabundantactiveβ-1,3-endoglucanasesinfruitsandalsoreaddressthequestionofthepossIBLeinvolvementoftheseenzymesintheripeningand/orsofteningprocess.

LentinanisanantitumorproductthatispurifiedfromfreshLentinulaedodesfruitingbodies.Itisacellwallcomponent,comprisingβ-1,3-glucanwithβ-1,6-linkedbranches,whichbecomesdegradedduringpostharvestpreservationasaresultofincreasedglucanaseactivity.Inthisstudy,weusedN-terminalaminoacidsequencetoisolatetlg1,ageneencodingathaumatin-like(TL)proteininL.edodes.TheCDNAclonewasapproximately1.0kbwhereasthegenomicsequencewas2.1kb,andcomparisonofthetwoindicatedthattlg1contains12introns.Thetlg1geneproduct(TLG1)waspredictedtocomprise240aminoacids,withamolecularmassof25kDandisoelectricpointvalueof3.5.Theputativeaminoacidsequenceexhibitsapproximately40%identitywithplantTLproteins,andafungalgenomedatabasesearchrevealedthattheseTLproteinsareconservedinmanyfungiincludingthebasidiomycotaandascomycota.Transcriptionoftlg1wasnotdetectedinvegetativemyceliumoryoungandfreshmushrooms.However,transcriptionincreasedfollowingharvest.Western-blotanalysisdemonstratedariseinTLG1levelsfollowingharvestandsporediffusion.TLG1expressedinEscherichiacoliandAspergillusoryzaeexhibitedβ-1,3-glucanaseactivityand,whenpurifiedfromtheL.edodesfruitingbody,demonstratedlentinandegradingactivity.Thus,wesuggestthatTLG1isinvolvedinlentinanandcellwalldegradationduringsenescencefollowingharvestandsporediffusion.

Benzothiadiazole(BTH),asBionWG50,andacetylsalicylicacid(ASA)treatmentsofpotatofoliageoffield-andglasshouse-grownpotatoplantswerecomparedforcontroloftwofoliardiseases,earlyblight(Alternariasolani)andpowderymildew(Erysiphecichoracearum).Theeffectofthesetreatmentsonharvestedtuberswound-inoculatedwiththedryrotfungus(Fusariumsemitectum)wasalsoevaluated.BTH(50mga.i.L^-1)gavealmostcompletecontrolofbothfoliarpathogensoninoculatedglasshouse-grownplantsandreducedtheseverityofleafspottingdiseases(mainlyearlyblight)inthefield.BTH(100mga.i.L^-1)andASA(400mga.i.L^-1)reducedtheseverityofdryrotinfield-growntubersinsomepost-harvestwound-inoculatedtreatmentsbutnotothersandasimilarreductionoccurredwithtubersinoculatedpost-harvestfromBTH-treatedplantsgrownunderglasshouseconditions.BTHtreatmentincreasedβ-1,3-glucanaseactivityinleaves>stem>tubers>stolonsbutnotinroots.Increasedenzymeactivitywasrecordedforupto45dayspost-treatment.

ThecellwallofthefruitingbodyofthemushroomLentinulaedodesisdegradedafterharvestingbyenzymessuchasβ-1,3-glucanase.Inthisstudy,anovelendo-typeβ-1,3-glucanase,GLU1,waspurifiedfromL.edodesfruitingbodiesafterharvesting.Thegeneencodingit,glu1,wasisolatedbyrapidamplificationofcDNAends(RACE)-PCRusingprimersdesignedfromtheN-terminalaminoacidsequenceofGLU1.Theputativeaminoacidsequenceofthematureproteincontained247aminoacidresidueswithamolecularmassof26kDaandapIof3.87,andrecombinantGLU1expressedinPichiapastorisexhibitedβ-1,3-glucanaseactivity.GLU1catalyzeddepolymerizationofglucanscomposedofβ-1,3-linkedmainchains,andreactionproductanalysisbythin-layerchromatography(TLC)clearlyindicatedthattheenzymehadanendolyticmode.However,theaminoacidsequenceofGLU1showednosignificantsimilaritytoknownglycosidehydrolases.GLU1hassimilaritytoseveralhypotheticalproteinsinfungi,andGLU1andhighlysimilarproteinsshouldbeclassifiedasanovelglycosidehydrolasefamily(GH128).

Inmaturekernelsofoat(AvenasativaL.)andothercereals,mixed-linked(11→33;11→34)-β-glucansandarABInoxylansaremajorstructuralpolysaccharidesincellwallsoftheendosperm.However,(1→3)-β-glucansaredepositedtransientlyinwallsduringcellularizationofendospermearlyingraindevelopment[Planta202(1997)414–426].Theabsenceof(1→3)-β-glucansinmatureendospermcellwallssuggeststhat(1→3)-β-glucanasesareactiveduringendospermdevelopment.Toinvestigatetheroleofβ-glucanasesduringendospermdevelopment,a(1→3)-β-glucanasecDNA,Oglc13,wasisolatedfromanoat(A.sativaL.)kernelcDNAlibrary.Theenzymaticactivityoftheproteinproduct,OGLC13,expressedfromthecDNAinaninvitroexpressionsystem,exhibitedsubstratespecificityfor(1→3)-β-glucans.Oglc13transcriptsweredetectedintheendospermofportionsofdevelopingkernelswiththehigheststeadystatelevelofmRNAat15daysafteranthesis(DAA)andnotinvegetativetissues.AntibodiesraisedagainstOGLC13immunoprecipitated(1→3)-β-glucanaseactivityfromendospermextractsof10and15DAAkernelsandmilkyendospermextractedfrom15DAAkernels.TheOGLC13antibodiesdidnotprecipitate(1→3)-β-glucanaseactivityfromleafextracts.TheseresultsindicatedthatOglc13isaunique(1→3)-β-glucanaseexpressedearlyinendospermdevelopment.

Thedefencereactionsofsorghumseedlings7daysafterinoculationwithFusariumthapsinumandF.proliferatum,andinteractionswithwoundingandexposuretolightwerestudiedtodeterminewhetherresponsestothesefungidifferedfromthosetoabioticstresses.Innon-woundedplants,inoculationwithbothfungiincreasedconcentrationsofanthocyaninsandsolublephenolicsandactivitiesofperoxidase(POX),chitinaseandβ-1,3-glucanaseintheroots,andincreasedβ-1,3-glucanaseactivityinthemesocotyls.Therewasnoeffectofinoculationonphenylalanineammonia-lyase(PAL)activity.Woundingbyitselfincreasedanthocyanincontentofmesocotyls.Woundingalsohadavarietyofinteractionswithinoculation.Exposuretolighthadverylittleeffectonanydefenceresponsemeasured.Atimecourseexperimentshowedthatinductionofchitinaseandβ-1,3-glucanaseoccurredinlessthan24hafterinoculation.POXactivityincreased2daysafterinoculation,followedbyatransientincreaseinPALactivity.Thecontentofanthocyaninsandsolublephenolicsinrootsofinoculatedseedlingsincreasedgraduallycomparedwithcontrolsover6days.TheresponsesofsorghumseedlingstoinoculationwithF.thapsinumandF.proliferatumweresimilartothosefoundbyotherworkersfollowingchallengebynecrotrophicpathogensandweredifferentfromthoseinducedbywoundingandexposuretolight.

TheCentralAndeanHighlandsarethecenteroforiginofthepotatoplant(Solanumtuberosum).AgesofmutualismbetweenpotatoplantsandsoilbacteriainthisregionsupportthehypothesisthatAndeansoilsharborinterestingplantgrowth-promoting(PGP)bacteria.Therefore,theaimofthisstudywastoisolaterhizobacteriafromAndeanecosystems,andtoidentifythosewithPGPproperties.Atotalof585bacterialisolateswereobtainedfromeightpotatofieldsintheAndesandtheywerescreenedforsuppressionofPhytophthorainfestansandRhizoctoniasolani.AntagoNISTicmechanismsweredeterminedandantagonisticisolateswerefurthertestedforphosphatesolubilization,1-aminocyclopropane-1-carboxylate(ACC)deaminaseactivity,andproductionofNH₃-andindole-3-aceticacid(IAA).PGPwasstudiedinhealthyandR.solanidiseasedplantletsundergrowthroomconditions.PerformancewascomparedtothecommercialstrainB.subtilisFZB24®WG.Isolatesweredereplicatedwithmatrix-assistedlaserdesorption/ionizationtimeofflightmassspectrometry(MALDI-TOFMS),andidentifiedwith16SrRNAgenesequencingandmultilocussequenceanalysis(MLSA).Atotalof10%oftheisolateswereeffectiveantagonists,ofwhichmanywereabletosolubilizephosphate,andproduceIAA,ACCdeaminase,NH₃andhydrogencyanide(HCN).Duringgrowthroomexperiments,23antagonisticisolateswereassociatedwithplantgrowth-promotionand/ordiseasesuppression.Tenisolateshadastatisticallysignificantimpactontestparameterscomparedtotheuninoculatedcontrol.Threeisolatessignificantlypromotedplantgrowthinhealthyplantletscomparedtothecommercialstrain,andsevenisolatesoutperformedthecommercialstrainininvitroR.solanidiseasedplantlets.

Fourgreen-odourcompounds—trans-2-hexenal,cis-3-hexenol,n-hexanal,andcis-3-hexenal—wereapplied(0.85µgml^-1asvapour)toriceplantsinlaboratoryconditionstoobservetheirbiologicalactivityagainstthephytopathogenicfungusMaganportheoryzae,whichcausesriceblastdiseaseworldwide.Twocompounds,trans-2-hexenalandcis-3-hexenal,showedremarkablediseasesuppressionefficacy(99.7%and100%suppression,respectively),whilen-hexanalhadmoderate(86.5%)andcis-3-hexenolhadweak(20.8%)disease-suppressingeffects.Pre-applicationandpost-applicationoftrans-2-hexenalorcis-3-hexenalhadslighteffectsonblastincidence,suggestingthatthesecompoundshaddirecteffectstosuppressM.oryzaeinfection.Infact,trans-2-hexenalandcis-3-hexenalexhibitedagrowthsuppressioneffectonM.oryzae.Interestingly,thesetwocompoundsinhibitedappressoriumformationatlowerconcentrationsthanthegrowthsuppression.Studiesonthehypersensitiveresponse(HR)-likereactionandplantβ-1,3-glucanaseactivityinriceplantconfirmedthatinducedresistancewasnotthemajorfactorinvolvedinthediseasesuppressionmechanism.Resultsofthisstudyconclusivelyshowedthattrans-2-hexenalandcis-3-hexenalpossesspotentinhibitoryactivitiesagainstthegrowthandtheappressoriumformationofM.oryzaeandcouldbeusedasantifungalagentstosignificantlyreduceM.oryzaeinfectionsinrice.

ArhizobacteriumwithhighantifungalactivitywasisolatedfromapotatofieldatInneruulalik,SouthGreenland.PhylogeneticanalysisbasedonmultilocussequencetypingshowedthatthebacteriumwasaffiliatedwithstrainsofPseudomonasfluorescens.Thebacterium,denotedasPseudomonasfluorescensIn5,inhibitedinvitroabroadrangeofphytopathogenicfungi,andtheantifungalactivityincreasedwithdecreasingtemperature.MicrocosmexperimentsdemonstratedthatP.fluorescensIn5protectedtomatoseedlingsfromRhizoctoniasolani.TransposonmutagenesisshowedthatthemajorcausefortheantifungalactivityofP.fluorescensIn5wasanovelnon-ribosomalpeptidesynthase(NRPS)gene.Inaddition,transposonmutagenesisshowedthatP.fluorescensIn5alsocontainedaputativequinoproteinglucosedehydrogenasegene,whichwasinvolvedingrowthinhibitionofphytopathogenicfungi.AlthoughP.fluorescensIn5containedthecapacitytosynthesizehydrogencyanide,β-1,3-glucanase,protease,andchitinase,thesedidnotseemtoplayaroleintheinvitroandmicrocosmantifungalassays.

Fieldtrialswereconductedusing4-year-oldhorsechestnut(AesculushippocastanumL.)toassesstheefficacyofpotassiumandsiliconphosphiteasplantprotectionagentsagainstthebacterialpathogenPseudomonassyringaepv.aesculi(Pae)thecausalagentofPseudomonasbleedingcankerofhorsechestnut.Phosphiteswereappliedpreventatively,i.e.beforePaeinoculationoftrees,andcuratively,i.e.afterPaeinoculationoftrees,and,asbothafoliarspray(FS)androotdrench(RD).Applicationofbothphosphiteformsinducedpositiveeffectsonplantvitality(increasedleafchlorophyllcontent,leafchlorophyllfluorescence(F_v/F_m),enhanceddefensiveenzymeactivity(β-1,3-glucanase,peroxidase)andreducedPaelesionsize,themainproxyofPaesuccessoraggressiveness.PreventativeratherthancurativephosphiteapplicationresultedingreaterreductionsinPaeseverity.Littlesignificanceofmodeofapplication(FS,RD)andphosphiteanion(potassium,silicon)wasdemonstratedindicatingbothphosphitescanbefoliarappliedorrootdrenchedwithsimilardegreesofresultingPaecontrol.SignificantreductionsinPaeseverityrecordedinthisstudygavecredencetothepotentialofphosphitesasanalternativeorcomplimenttoconventionalbactericidesforPaecontrol.

Aspergillushancockiisp.nov.,classifiedinAspergillussubgenusCircumdatisectionFlavi,wasoriginallyisolatedfromsoilinpeanutfieldsnearKumbia,intheSouthBurnettregionofsoutheastQueensland,Australia,andhassincebeenfoundoccasionallyfromothersubstratesandlocationsinsoutheastAustralia.ItisphylogeneticallyandphenotypicallyrelatedmostcloselytoA. leporisStatesandM.Chr.,butdiffersinconidialcolour,otherminorfeaturesandparticularlyinmetaboliteprofile.Whencultivatedonriceasanoptimalsubstrate,A. hancockiiproducedanextensivearrayof69secondarymetabolites.Elevenofthe15mostabundantsecondarymetabolites,constituting90%ofthetotalareaunderthecurveoftheHPLCtraceofthecrudeextract,werenovel.ThegenomeofA. hancockii,approximately40Mbp,wassequencedandminedforgenesencodingcarbohydratedegradingenzymesidentifiedthepresenceofmorethan370genesin114geneclusters,demonstratingthatA. hancockiihasthecapacitytodegradecellulose,hemicellulose,lignin,pectin,starch,chitin,cutinandfructanasnutrientsources.LikemostAspergillusspecies,A. hancockiiexhibitedadiversesecondarymetabolitegeneprofile,encoding26polyketidesynthase,16nonribosomalpeptidesynthaseand15nonribosomalpeptidesynthase-likeenzymes.

Background:Improvedcarbohydrate-activeenzymes(CAZymes)areneededtofulfillthegoalofproducingfood,feed,fuel,chemicals,andmaterialsfrombiomass.Littleisknownabouthowthediversemicrobialcommunitiesinanaerobicdigesters(ADs)metabolizecarbohydratesorwhichCAZymesthatarepresent,makingtheADsauniquenichetolookforCAZymesthatcanpotentiatetheenzymeblendscurrentlyusedinindustry.Results:EnzymaticassaysshowedthatfunctionalCAZymesweresecretedintotheADenvironmentsinfourfull-scalemesophilicDanishADsfedwithprimaryandsurplussludgefrommunicipalwastewatertreatmentplants.MetagenomesfromtheADswereminedforCAZymeswithHomologytoPeptidePatterns(HotPep).19,335CAZymeswereidentifiedofwhich30%showed50%orloweridentitytoknownproteinsdemonstratingthatADsmakeupapromisingpoolfordiscoveryofnovelCAZymes.Afunctionwasassignedto54%ofallCAZymesidentifiedbyHotPep.Manydifferentα-glucan-actingCAZymeswereidentifiedinthefourmetagenomes,andthemostabundantfamilywasglycosidehydrolasefamily13,whichcontainsα-glucan-actingCAZymes.CellulyticandxylanolyticCAZymeswerealsoabundantinthefourmetagenomes.Thecellulyticenzymeswerelimitedalmosttoendoglucanasesandβ-glucosidases,whichreflectthelargeamountofpartlydegradedcelluloseinthesludge.NodockerindomainswereidentifiedsuggestingthatthecellulyticenzymesintheADsstudiedoperateindependently.OfxylanolyticCAZymes,especiallyxylanasesandβ-xylosidase,butalsoabatteryofaccessoryenzymes,werepresentinthefourADs.Conclusions:OurfindingssuggestthattheADsareagoodplacetolookfornovelplantbiomassdegradingandmodifyingenzymesthatcanpotentiatebiologicalprocessesandprovidebasisforproductionofarangeofadded-valueproductsfrombiorefineries.