InstitutfỹrNutzpflanzenwissenschaftenundRessourcenschutzPhytomedizin Comparativestudiesontheinfectionandcolonization ofmaizeleavesbyFusariumgraminearum, F.proliferatumandF.verticillioides InauguralDissertation zurErlangungdesGrades DoktorderAgrarwissenschaften (Dr.agr.) derLandwirtschaftlichenFakultọt derRheinischenFriedrichWilhelmsUniversitọtBonn von NguyenThiThanhXuan aus Angiang,Vietnam Referent: Prof.Dr.H.W.Dehne Korreferent: Prof.Dr.J.Lộon TagdermỹndlichenPrỹfung: 18.12.2013 Erscheinungsjahr: 2014 Abstract ComparativestudiesontheinfectionandcolonizationofmaizeleavesbyFusarium graminearum,F.proliferatumandF.verticillioides Infection of Fusarium species causes quantitative along with qualitative damage on small grains and maize plants. This is due to leaf damage together with contamination by formationofdifferentmycotoxins.Becausethevegetativeaswellasthereproductiveplant partsofmaizeareusedespeciallyforanimalfeedandcanbeaffected,informationabout theinfectionprocessanddamageoftheentireplantsneededfurtherelucidation. The infection and colonization of maize leaves by the most important three Fusarium species provided insights in a role of the spread of Fusarium species from the different leaves into the cobs. Using microbiological assessments maize plants inoculated by Fusariumatthegrowthstage(GS)15reachedhigherinfectionratesthanthoseinoculated at GS 35. Higher spore concentration and increased relative humidity resulted in more intensivecolonization.Lightregimeshadnoeffectontheinfectionofdifferentcultivarsby Fusarium.Thecolonizationoflowerleaveswashigherthantheinfectionofupperleaves. The lesion development of maize plants infected by Fusarium occurred especially on the immatureleaves.Diseaseseverityshowednodifferenceamongthreespecies.Colonization was higher on symptom leaves than on symptomless leaves, but nevertheless even symptomless infections resulted in further propagation. Disease symptoms appeared on leavesinoculatedbyF.graminearum45daysafterinoculation(dai)andbyF.proliferatum and F. verticillioides 78 dai. F. graminearum caused small watersoaked lesions and the lesionsturnedintoyellowspots.F.proliferatumandF.verticillioidescausednecroticlesions, smallholesandstreaks. ThegerminationofconidiaofallFusariumspecieswaspresentat12hoursafterinoculation. The penetration of all three Fusarium species was quite similar: All species were able to penetrateintothetissuethroughcuticles,epidermalcells,trichomes,butalsoviastomata. Formingappressoria,infectioncushionsordirectpenetrationdemonstratedthebroadhost tissue these species resembled a high potential leading to symptomatic as well as asymptomaticinfections. All pathogens showed intercellularand intracellular infection of epidermal and mesophyll cells.Additionally,F.graminearumhyphaewerefoundinsclerenchymacells,xylemandthe phloemvesselsofdetachedleaves.Thesuperficialhyphaeandreemerginghyphaeofthe three species produced conidia. Especially, macroconidia of F. graminearum produced secondary macroconidia and F. proliferatum formed microconidia inside tissues and sporulatedthroughstomataandtrichomes. According to quantitative fungal DNA the biomass of Fusarium species increased until the 5thdaibutafterwardsdecreasedfromthe5thdaitothe20thdaiandincreasedagainuntil the40thdai.Diseaseseverityandfungalbiomass,diseaseseverityandcolonizationofthe6th and7thleavesweresignificantlypositivecorrelationat10daiand40dai,respectively. TheinfectionofmaizeleavesbythethreeFusariumspeciesandtheirsporulationindicated aninoculumcontributiontocobandkernelinfectionwhichmayleadtoreduceyield,quality andincreaseinpotentialmycotoxincontaminationonmaize. Kurzfassung Vergleichende Untersuchungen zur Infektion und Besiedlung von Maisblọttern durch Fusariumgraminearum,F.proliferatumundF.verticillioides InfektionenvonFusariumArtenverursachenquantitativeundqualitativeSchọdenanGetreideundMais. Diese Beeintrọchtigungen erfolgen durch Blatt und Kolbenschọden, vor allem aber auch durch die Kontamination der Pflanzenteile mit sehr unterschiedlichen Mykotoxinen. Von Mais werden sowohl vegetativealsauchreproduktivePflanzenteiledesMaisbeslastetseinkửnnenunddiesewerdenvorallem in Gọnze in die Tiernahrung eingebracht werden. Daher galt es Informationen ỹber den Blattbefall an MaiszugewinnenunddaherdenInfektionsprozessunddieSchadwirkunganMaisdetailliertzuverfolgen. DieInfektionundBesiedelungvonMaisblọtternwurdebezỹglichder3bedeutendstenFusariumArtenan Mais verfolgt und ergaben wesentliche Rỹckschlỹsse ỹber die Ausbreitung von FusariumArten an MaispflanzenvonBlọtternbishinzumKolben.MitmikrobiologischenErhebungenanMaisplanzenkonnte nach Inokulationen geklọrt werden, dass junge Maispflanzen (inokuliert im Stadium GS 15) deutlich anfọlliger waren als im Stadium GS 35. Die Erhửhung der Inokulumdichte und eine erhửhte Luftfeuchte fửrderten die Blattinfektionen. Belichtungsbedingungen lieòen keinen Einfluss auf die Infektionen erkennen.InallenErhebungenwarendieBefọllederunterenBlọtterderMaispflanzendeutlichhửherals dieInfektionenderoberenBlọtter. DieEntwicklungvonLọsionenaufdurchFusariuminfiziertenMaispflanzentratvorallemaufdenunreifen Blọttern auf. Die Befallshọufigkeit und Befallsintensitọt zeigte keinen Unterschied zwischen den drei Arten. Auch wenn die Besiedelung auf Blọttern mit Symptomausprọgung hửher war, fỹhrten auch die symptomlosen Infektionen zu einer weiteren Ausbreitung. Bei Fusarium graminearum traten die Symptome 45 Tage nach der Inokulation, bei F. proliferatum und F. verticiolliodies 78 Tage nach der Inokulation. F. graminearum verursachte Lọsionen, die anfangs aussahen, wie Verbrennungen durch heiòes Wasser und sich anschlieòend in gelbe Flecke verwandelten. F. proliferatum und F. verticilloides verursachtenNekrosen,diealskleineLửcherundStreifenerschienen. Die Konidien aller FusariumArten keimten im Zeitraum von 12 Stunden nach der Inokulation. Alle zu vergleichenden Arten wiesen ein ọhnliches Infektionsverhalten auf: Alle Arten konnten direkt in das Wirtsgewebeeindringen,penetriertwurdenCuticulen,Epidermiszellen,Trichomegelegentlicherfolgte auch eine Eindringung ỹber Spaltửffnungen. Dabei werden von den Pathogenen Appressorien gebildet, zudem Infektionskissen aber dennoch kamen stets auch direkte Infektionen vor. Dies bestọtigt das besonders breite Infektionsvermửgen der Fusarien. Vor allem wurden aber symptomatische und asymptomatischeInfektionenbeobachtet. AllePathogenezeigteneininterundintrazellulọresWachstuminEpidermisundMesophyllderBlọtter. Fusarium graminearum besiedelte auch Gefọssgewebe sowohl Xylem als auch Phloemgewebe. Die oberflọchlichen Hyphen sporulierten stets auf dem Blattgewebe. F. graminearum bildete sekundọre Makrokonidien. F. proliferatum bildete Mikrokonidien im Gewebe und sporulierte als ubiquitọrer PathogendurchStomataundTrichome. MittelsquantitativerPCRwurdediepilzlicheBiomasseerfasst.Biszum5.TagnachderInokulationstieg der Gehalt an die symptomlose Infektion in der Nekrotisierungsphase sank der Pilzgehalt um anschlieòendindersaprophytischenPhasederInfektionwiederanzusteigen. Die Infektion von Maispflanzen und insbesondere Blọttern durch reprọsentative Fusarium Arten und deren Sporulation sogar auf symptomlosen Blọttern belegt die Bedeutung latenter Infektionen fỹr die Kolben und Kửrnerinfektion dies gilt es zu vermeiden, um Ertragsbeeintrọchtigungen und EinschrọnkungenderQualitọtdesErntegutzureduzieren. Túmtt NghiờncusxõmnhimvkýsinhcanmFusariumgraminearum,F.proliferatumv F.verticillioidestrờnlỏngụ NhimnmFusariumgõyrathithivnngsutvchtlngngccvngụ.Nhiuloi ctcanmhỡnhthnhtrongquỏtrỡnhxõmnhim.Dongụcsdngchochnnuụi nờnnhimnmcúthnhhngnsckhevtnuụi.Vỡthquỏtrỡnhxõmnhimca nmvsthithicncnghiờncu. XõmnhimvkýsinhlỏngụbibaloiFusariumdnnphỏttỏnngunbnhtlỏn cỏc lỏ bờn trờnv lờn qu. S dngphng phỏp phõn lp nm sau chng bnh cho thycõyngụcchngbnhbinmFusariumgiaionsinhtrng15cúmcnhim caohnchngbnhgiaion35.Skýsinhxyravitnsutcaohnkhichngnng botnmcaovtngmtngi.Chỏnhsỏngókhụngnhhngns nhimnmFusariumtrờnhaigingngụ.NhnglỏbờndibFusariumkýsinhmnhhn lỏtrờn. Nhngvtbnhxuthintrờnlỏngụnon,cbittrờnlỏangmc.Tlbnhkhụngkhỏc bitýnghagiabaloiFusarium.Tlkýsinhcaohnivilỏcútriuchngbnhsovi lỏ khụng cú triờu chng. Triu chng bnh xut hin sm trờn lỏ ngụ c chng bi F.graminearum45ngysaukhichngnmv78ngysaukhichngF.proliferatumv F.verticillioides.TriuchngbnhgõyrabiF.graminearumbanulnhngmnh sng nc sau ú chuyn sang mu vng nht vi tõm xỏm trng. F. proliferatum and F. verticillioides gõy nờn cỏc m nh liờn tc v ni vi thnh nhng sc chy dc theogõnlỏhocmụlỏbthithihỡnhthnhcỏclthngtrờnlỏ,thnglhỡnhmtộn. Botnmca3loiFusariumbtunymm12gisaukhichng.BaloiFusarium cú kh nng xõm nhim mụ lỏ ngụ qua lp cutin, t no biu bỡ, lụng v khớ khng. Nm hỡnhthnhaỏphocmụmhocxõmnhimtrctipvolỏngụ.Cỏchxõmnhima dngcabaloiFusariumchothytimnngxõmnhimcaogõyratriuchngbnhtrờn lỏcngnhxõmnhimmkhụnggõyratriuchng.Fusariumspecieskýsinhtrongtbo hocgiacỏctbocalỏ.Hnna,nmF.graminearumóctỡmthytrongtbo cngmụvtbobúmchkhichngnmtrờnlỏngụtrongapetrivimcao. Sinmtrờnmtlỏvsinmmcratmụlỏbnhimcacbaloinmsinhbot. cbit,botcaF.graminearumhỡnhthnhthhbotthhaivF.proliferatum hỡnhthnhbotbờntrongmụlỏvphúngthớchrangoithụngquakhớkhnghoclụng calỏ. SdngqPCRỏnhgiỏsphỏttrincabaloinmtrờnlỏngụchothysinhkhica nmtngtlỳcchngchon5ngysaukhichngnhnggimtsau5ngyn20ngy vtngtrlisauú,40ngysaukhichng.Cústngquangiatlbnhvsinhkhi nm,10ngysaukhichngbnh,tlbnhvmckýsinh,40ngysaukhichngbnh. Sxõmnhimvkýsinhca3loinmFusariumtrờnlỏngụvphúngthớchbotócho thyõylngungõybnhiviquvhtngụvcúthdnngimnngsut,cht lngvtngnguycnhimctcanmtrờnngụ. Tableofcontents 1.Introduction 2.FactorsaffectingtheinfectionofmaizeleavesbyFusariumspecies 2.1.Introduction 2.2.Materialsandmethods 11 2.2.1.Fungalpathogenandinoculumpreparation 11 2.2.2.Plantcultivation 13 2.2.3.Experimentaldesign 14 2.2.3.1.Impactofgrowthstageofmaizeplantsoninfection 14 2.2.3.2.Impactofsporeconcentrationontheinfectionofmaizeleaves 15 2.2.3.3.Impactoflightoninfectionofmaizeleaves 15 2.2.3.4.Effectofinoculationsiteoninfectionandsymptommanifestationon maizeplants 16 2.2.3.5.Effectofinoculationsiteoninfectionandsymptommanifestationof differentspecies 16 2.2.4.1.Reisolationfrequency 17 2.2.4.2.Diseaseincidenceanddiseaseseverity 17 2.2.5.Dataanalysis 17 2.3.Results 19 2.3.1.Impactofgrowthstageofmaizeplantsoninfection 19 2.3.2.Impactofsporeconcentrationontheinfectionofmaizeleaves 21 2.3.3.Effectoflightregimesoninfectionofmaizeleaves 24 2.3.4.EffectofinoculationsiteonFusariuminfectionandsymptommanifestation 25 2.3.5.Effectofsiteofinoculationoninfectionandsymptommanifestationof differentspecies 27 2.4.Discussions 32 3.HistopathologicalassessmentoftheinfectionofmaizeleavesbyFusariumspecies 38 3.1.Introduction 38 3.2.Materialsandmethods 40 3.2.1.Fungalpathogenandinoculumpreparation 40 3.2.2.Cultivationofplant 40 3.2.3.Inoculationandsamplingcollection 40 3.2.3.1.Attachedleaves 41 3.2.3.2.Detachedleaves 41 3.2.4.Measurementofconidia 42 3.2.5.Microscopy 42 3.2.5.1.Lightmicroscopy 42 3.2.5.1.1.Freshspecimen 42 3.2.5.1.2.Wholespecimen 43 3.2.5.2.Scanningelectronmicroscopy 43 3.2.5.3.Transmissionelectronmicroscopy 44 3.2.6.Dataanalysis 46 3.3.Results 46 3.3.1.Morphologyofmaizeleaves 46 3.3.2.Conidialcharacteristics 48 3.3.2.1.Sizeandnumberofconidia 48 3.3.2.2.Germinationandgermtubeformation 49 3.3.3.ConidialcharacteristicsofFusariumspeciesonmaizeleaves 49 3.3.4.Infectionprocessonmaizeleaves 51 3.3.4.1.InfectionofmaizeleavesbyFusariumgraminearumandfungal sporulation 51 3.3.4.1.1.Germinationofmacroconidiaandmyceliagrowth 51 3.3.4.1.2.Infectionofasymptomaticmatureleaves 51 3.3.4.1.3.Infectionofimmatureleaveswithsymptoms 55 3.3.4.1.4.Infectionofdetachedleaves 63 3.3.4.1.5.Sporulation 63 3.3.4.2.InfectionofmaizeleavesbyFusariumproliferatumandfungal sporulation 67 3.3.4.2.1.Germinationofmicroconidiaandmyceliagrowth 67 3.3.4.2.2.Infectionofasymptomaticmatureleaves 67 3.3.4.2.3.Infectionofimmatureleaveswithsymptoms 67 3.3.4.2.4.Sporulation 73 3.3.4.3.InfectionandsporulationofF.verticillioidesonmaize 78 3.3.4.3.1.Germinationofmicroconidiaandmyceliagrowth 78 3.3.4.3.2.Infectionofasymptomaticmatureleaves 78 3.3.4.3.3.Infectionofimmatureleaveswithsymptoms 78 3.3.4.3.4.Sporulation 81 3.3.5.Comparisonofhyphalgrowthandmodesofinfection 85 3.3.5.1.Hyphalgrowth 85 3.3.5.2.Infectionoftrichomes 85 3.3.5.3.Infectionviastomata 87 3.4.Discussions 88 4.AssessmentofinfectionbyFusariumgraminearum,F.proliferatumandF. verticillioidesonmaizeleavesusingquantitativePCRandmicrobiologicalassays 93 4.1.Introduction 93 4.2.Materialsandmethods 95 4.2.1.Fungalpathogenandinoculumpreparation 95 4.2.2.Cultivationofplant 95 4.2.3.Experimentaldesign 95 4.2.4.Plantgrowth 96 4.2.5.Diseaseincidenceanddiseaseseverity 96 4.2.6.Reisolation 96 4.2.7.Microscopy 97 4.2.7.1.Stereomicroscopy 97 4.2.7.2.Lightmicroscopy 97 4.2.8.Fungalbiomassanalysis 97 4.2.8.1.DNAextractionfromfungalculture 97 4.2.8.2.FungalDNAextractionfromleafsamples 97 4.2.8.3.Polymerasechainreaction(PCR) 98 4.2.8.4.QuantificationofgenomicDNA 99 4.2.9.Dataanalysis 99 4.3.Results 100 4.3.1.Relationshipbetweenfungalbiomassandsymptommanifestationof infectedmaizeplantsbyF.graminearum,F.proliferatumandF.verticillioidesunder controlledconditions 100 4.3.1.1.Diseaseseverity 100 4.3.1.2.Fungalbiomass 100 4.3.1.3.Correlationsbetweendiseaseseverityandfungalbiomass 101 4.3.2.Relationshipsbetweenfungalbiomass,symptommanifestationandinfection ofmaizeplantbyF.graminearum,F.proliferatumandF.verticillioidesunderlow andhighhumidityconditions 102 4.3.2.1.EffectofFusariuminfectiononmaizeplantgrowth 102 4.3.2.2.EffectofFusariumspeciesondiseaseincidence,diseaseseverityand symptomdevelopment 102 4.3.2.3.Reisolationfrequency 107 4.3.2.4.BiomassofFusariumspeciesinmaizeleaves 108 4.3.2.5.Correlations:Colonization,fungalbiomass,diseaseseverity 109 4.4.Discussions 112 5.Summary 118 References 122 Appendix 142 Acknowledgements 144 Abbreviations C Celsius àg Microgram àl Microliter 15AcDON 15Acetyldeoxynivalenol 3AcDON 3Acetyldeoxynivalenol CZIDAgar CzapekDoxIprodioneDichloranAgar Dai Dayafterinoculation DNA Deoxyribonucleic acid GS Growthstage Hai Hourafterinoculation L Liter mg Milligram ml Milliliter MON Moniliformin NIV Nivalenol PCR PolymeraseChainReaction PDA PotatoDextroseAgar pg picogram qPCR TaqManđrealtimePolymeraseChainReaction RH relativehumidity rpm rotationperminute Sec second SEM Scanningelectronmicroscopy spp. species TEM 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Yoshizawa,T.,Yamashita,A.,Luo,Y.,1994.Fumonisinoccurrenceincornfromhighand lowriskareasforhumanesophagealcancerinChina.Appliedandenvironmental microbiology60,16261629. 141 B Figure4.2.Humidityandtemperatureinhighrelativehumidity.A:from0to11dai,B: 12to40dai.Datawererecordedbydatalogger. 40 20 Temp 80 60 40 20 35 33 31 29 27 25 23 21 19 17 15 Temp 80 29 27 60 25 23 40 21 19 20 Temperature (oC) 60 Temperature (oC) 33 31 29 27 25 23 21 19 17 15 Temperature (oC) 15-01-13 15-0113 14-0113 13-0113 12-0113 80 12-02-13 11-02-13 14-01-13 13-01-13 13-01-13 12-01-13 11-0113 10-0113 09-0113 08-0113 %RH 10-02-13 09-02-13 08-02-13 07-02-13 06-02-13 05-02-13 04-02-13 %RH 03-02-13 %RH 02-02-13 11-01-13 10-01-13 09-01-13 08-01-13 07-0113 06-0113 05-0113 04-0113 Relative humidity (%) 100 01-02-13 31-01-13 30-01-13 29-01-13 100 28-01-13 27-01-13 26-01-13 25-01-13 07-01-13 06-01-13 05-01-13 04-01-13 Relative humidity (%) 100 24-01-13 23-01-13 22-01-13 21-01-13 20-01-13 19-01-13 18-01-13 17-01-13 A 16-01-13 Relative humidity (%) Appendix 4.2.1.Temperatureandhumidityrecordedbydatalogger Temp 35 Figure4.1.Humidityandtemperatureinnormalchamberconditionfrom0to11dai. Datawererecordedbydatalogger. 33 31 17 15 142 4.2.2.FungalDNAextractionfromleafsamples LyophilisedleavesweregroundtoafinepowderusinganunltracentrifugalmillMM200 (Retsch,Germany).1820mgofgroundmaizeleaftissuewasusedforDNAextraction.A Qiagen DNeasy plant mini kit was used to perform DNA extractions. The extraction processfollowedinstructionsoftheproducer(Qiagen,2012)asbelow. 1.Add400lBufferAP1and4lRNaseAstocksolution(100mg/ml)toamaximumof 20mg(dried)disruptedplantorfungaltissueandvortexvigorously. 2. Incubate the mixture for 10 at 65C. Mix or times during incubation by invertingtube. 3.Add130lBufferAP2tothelysate,mix,andincubatefor5minonice. 4.Centrifugethelysatefor5minat14,000rpm. 5.PipetthelysateintotheQIAshredderMinispincolumn,placedina2mlcollection tube,andcentrifugefor2minat14,000rpm. 6. Transfer the flowthrough fraction from step into a new tube (not supplied) withoutdisturbingthecelldebrispellet. 7.Add1.5volumesofBufferAP3/Etotheclearedlysate,andmixbypipetting. 8. Pipet 650 l of the mixture from step 7, including any precipitate that may have formed, into the DNeasy Mini spin column placed in a ml collection tube. Centrifugefor1minat8000rpmanddiscardtheflowthrough. 9.Repeatstep8withremainingsample.Discardflowthroughandcollectiontube. 10.PlacetheDNeasyMinispincolumnintoanew2mlcollectiontube(supplied),add 500lBufferAW,andcentrifugefor1minat8000rpm. Discardtheflowthrough andreusethecollectiontubeinstep11. 11.Add500lBufferAWtotheDNeasyMinispincolumn,andcentrifugefor2minat 20,000xg(14,000rpm)todrythemembrane. 12.TransfertheDNeasyMinispincolumntoa1.5mlor2mlmicrocentrifugetubeand pipet100lBufferAEdirectlyontotheDNeasymembrane.Incubatefor5minat roomtemperatureandthencentrifugefor1minat8000rpmtoelute. 13.Repeatstep12once. 143 Acknowledgements IwouldliketoexpressmysincereappreciationandgratitudetoProf.Dr.W.H.Dehne forguidanceandsupportallfacilitiestocarryoutthisstudy.Thanksforgivingmethe chancetobeoneofyourPh.Dstudentsandyourhelpinanyproblems.ThanktoProf. Dr.J.Lộonforbeingmycosupervisor. I would like to express my deepest gratitude to PD. Dr. U. Steiner for her careful scientific advice, guidance, and forher daily assistance during the whole period of my researchinGermany.Sheshapedandgaveadviceformyscientificstudyingandwriting. A special note of thanks goes to PD. Dr. J. Hamacher for help in TEM and scientific advice. IwouldliketothanktoProf.Dr.R.SikoraandDr.J.Akelloforassistanceinmywriting. MyspecialthanksgotoallmembersattheINRESPhytomedizin,myfriendsforhelp, sharingexperiences,joysandsorrowsduringmystayinBonn. With a deep sense of gratitude to Kirchner Bierschenk family thank for their help, kindness,andagreeablewarmthintheirhouse. Iamverythankfultomother,fatherfortheirloveforever,supportthroughoutmylife. Thankstomysistersandbrotherfortakingcareofmychildrenandencouragementme. ThankstomydaughterXuanHuongandmysonQuangDuymyhusbandQuangforyour love,understandingandencouragement. My gratitude goes also to the Vietnamese Ministry of Education and Training (MOET) theGermanAcademicExchangeService(DAAD)forfundingascholarshipformystudy. 144 [...]... 10  Factors affecting infection of Fusarium   determinants  affecting  Fusarium infection into  maize leaves.   The specific  objectives  were to:    i study  the effects  of plant  age,  leaf  position  and cultivar  on the infection of Fusarium species into maize leaves.   ii examine the effects of inoculum density on infection of maize leaves.    iii evaluate the effects of light on Fusarium infection of maize leaves.  ... Factors affecting infection of Fusarium     2.3.2. Impact of spore concentration on the infection of maize leaves The frequency  of re‐isolation  on non‐sterilized  leaves differed  significantly  across  treatments  and depended  on spore  concentration  (P=0.001).  Higher  spore  concentrations resulted into higher levels of infection.  Hence, re‐isolation of the fungus  of non‐sterilized  leaves ... Disease severity on the other hand was rather low and appeared mostly on very young  leaves.                 22  Factors affecting infection of Fusarium   Table  2.2.  Effect  of inoculum  concentration  of Fusarium on the infection of lower  and upper  leaves assessed  from  non‐sterilized  and sterilized  leaf  surfaces  (re‐ isolation frequency, %), 10 days after inoculation.  Spore conc./  mL  5 Fungi (1) ... Therefore,  the protection  of cereal  crops  from  mycotoxin producing species of Fusarium is needed for the production of healthier food  and animal feed.     Research objectives  Although many studies have described the infection of Fusarium into host plants, most  of these  reports  concentrated  on the infection and the symptoms  of Fusarium on kernels, seeds or crown. Conversely, only a few investigations have described Fusarium ... for  the re‐ isolation of Fusarium proliferatum.  A= symptoms on emerging leaves.  B= symptom  leaves in A separated into the 6th, 7th and 8th leaf (L6, L7 and L8).            18  Factors affecting infection of Fusarium   2.3. Results  2.3.1. Impact of growth stage of maize plants on infection Results of the re‐isolation frequency revealed that the growth stage of the plant had a  significant  effect ... and F. verticillioides infect maize leaves and disseminate inoculum to upper leaves and to ears.    The specific objectives of the study were to:    i study factors affecting the infection of Fusarium spp. into maize leaves.    ii investigate  the infection process  of the three  species  of Fusarium on maize leaves and iii assess  the development  of the three  Fusarium species  on maize leaves using  quantitative PCR and microbiological bioassays. ... dispersed  by wind  and/ or  water.  Upon      9  Factors affecting infection of Fusarium   landing on the host, they infect silks and then kernels (Gulya et al., 1980; Nelson, 1992;  Munkvold and Desjardins, 1997).    The infection of Fusarium into the host plant, however, is influenced by several factors  including environmental conditions, physiology of the host and spore condition among  others (Dodd, 1980; Magan and Lacey, 1984; Marin et al., 1995a; Doohan et al., 2003). ...   8  Factors affecting infection of Fusarium   2. Factors affecting the infection of maize leaves by Fusarium species  2.1. Introduction  Throughout the world, maize plays an important role in the livelihood of humans. Apart  from serving as a staple food and source of income for millions of people, maize also is  used  extensively  as  animal  feed  and as  a  substrate  for  biogas  production. ... evaluate the effects of light on Fusarium infection of maize leaves.     2.2. Materials and methods  2.2.1. Fungal pathogen and inoculum preparation  Fusarium proliferatum (Matsushima)  Nirenberg,  isolate  AG31g  and F.   verticillioides (Sacc.) Nirenberg, isolate AG11i were utilized for examining the effects of the different  factors on the infection of Fusarium into maize leaves. F.  graminearum was included in  the experiment on effect of inoculation sites of Fusarium infection and manifestation of ... total, eight treatments were used, with each treatment comprised of 6 plants and the experiment was repeated two times.      15  Factors affecting infection of Fusarium     2.2.3.4. Effect of inoculation site on infection and symptom manifestation on maize plants  Fusarium proliferatum, and the maize cv Tassilo were used to test the hypothesis that  Fusarium produced symptoms on very young leaves i.e. emerging or immature leaves.   The maize plants were grown inside growth chambers under similar growth conditions  ... FactorsaffectinginfectionofFusarium 2.2.3.2.Impactofsporeconcentrationontheinfectionofmaizeleaves To assess the impact of spore concentration on Fusarium infection of different maize cultivars, maize. .. 2.3.1.Impactofgrowthstageofmaizeplantsoninfection 19 2.3.2.Impactofsporeconcentrationontheinfectionofmaizeleaves 21 2.3.3.Effectoflightregimesoninfectionofmaizeleaves 24 2.3.4.EffectofinoculationsiteonFusariuminfectionandsymptommanifestation... 2.2.3.2.Impactofsporeconcentrationontheinfectionofmaizeleaves 15 2.2.3.3.Impactoflightoninfectionofmaizeleaves 15 2.2.3.4.Effectofinoculationsiteoninfectionandsymptommanifestationon maizeplants