Illumina-based analysis of sorghum fungal pathogens ... · Mpumalanga Free State Limpopo North-...
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0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Blignaut Swart Du Preez Coetzer DuPlooy Westhuizen Potgieters Uitkyk Grobbler Groothout Phillip Rela%ve abundance % Neospora Tiarosporella Cladosporium Ramularia Epicoccum Psiloglonium Leptosphaerulina Phoma Leptosphaeria Alternaria Bipolaris Curvularia unclassified Pleosporales Exophiala Aspergillus Penicillium Pseudogymnoascus Acrostalagmus Simplicillium Eucasphaeria Sarocladium Fusarium Scopulariopsis Nigrospora Cornuvesica Cercophora unclassified Sordariomycetes Candida Wickerhamomyces Phanerochaete Coprinellus Dioszegia OcculUfur Malassezia 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Blignaut Swart Du Preez Coetzer DuPlooy Westhuizen Potgieters Uitkyk Grobbler Groothout Phillip Rela%ve abundance % Dothideomycetes EuroKomycetes LeoKomycetes Sordariomycetes Saccharomycetes Agaricomycetes Tremellomycetes Cystobasiomycetes Exobasidiomycetes B A Fig 2. Rela%ve abundance of different fungal classes (A) and genera (B) iden%fied from different farms. Background: Sorghum is an important crop in South Africa, but it is exposed to a variety of pathogenic fungi that cause many diseases leading to significant losses in yields and reduc%on in quality of the product, thereby threatening food security. Although several control methods that have been applied have been successful, pathogens adapt and evolve quickly. Precise knowledge of the disease causal agents is essen%al for the development of effec%ve counter measures to fungal infec%ons. The aim of the study was to survey local sorghum fungal pathogens and use morphological and molecular approaches for iden%fica%on. Results: Sorghum fungal pathogen diversity was inves%gated in eleven producing regions in South Africa using a culture-dependent technique. Morphological characteris%cs and ITS2 sequence data generated from Illumina MiSeq was used to iden%fy the fungal isolates. A total of 253 fungal isolates were obtained from leaf samples, from which opera%onal taxonomic units belonging to 36 genera were iden%fied as belonging to Ascomycota (98%) and Basidiomycota (2%) phyla. Fusarium was the dominant genera (38.9% rela%ve abundance of the detected sequences) followed by Curvalaria (14.7%) and Sarocladium (8.7%). The diversity and abundance of different taxonomic groups differed with sampling loca%ons. Significance: Studying the fungal pathogens, which cause diseases threatening the produc%on of one of the important crops, sorghum has a significant impact in crop produc%on. A beTer understanding of these pathogens will lead to effec%ve management and therefore food security for many leaving in poverty in South Africa. Pathogens cause diseases by producing enzymes that deconstruct the plant cell walls. Their ability to produce hydroly%c enzymes can be exploited for industrial bioconversion. The diversity of the fungi obtained in this study suggests that the enzymes produced are capable of providing the diversity and strength of ac%vity required for bioconversion. This study will have a significant contribu%on in food security and in energy produc%on. Abstract 213 ITS2 sequences clustered into 220 OTUs and 95% belonged to Fungi, while the other 5% were either plant or bacterial sequences. Members of the phylum Ascomycota were dominant in all samples, collec%vely accoun%ng for 98% of the total number of detected fungal sequences, and Basidiomycota accounted for 2%. Ascomycota sequences were largely iden%fied as members of the classes Sordariomycetes and Dothideomycetes (57.9% and 34% respec%vely), followed by Euro%omycetes (4.4%), Saccharomyctes (2.2%) and Leo%omycetes (0.4%). Within Basidiomycota phyla Agaricomycetes was the most abundant class with 0.6%, The rela%ve abundance of other classes Tremellomycetes, Cystobasiomycetes and Exobasidiomycetes was 0.2% (Fig. 2A). Further analysis of the iden%fied OTUs classified them into 36 genera which were characterized by the prevalence of Fusarium (38.9%), Curvularia (14.7%) Sarocladium (8.7%), Alternaria (6.9%), Nigrospora (5.4%), Bipolaris (5.3%) and Phoma (4.7%). The representa%on of other detected genera was limited with rela%ve abundance ranging from 0.1%-2.4 (Fig. 2B). Results Discussion Results indicate that a wide variety of fungal taxa inhabit sorghum plants. The high diversity of fungi iden%fied is comparable to the metagenomic studies done previously and this result was not expected considering the low diversity of isolated fungi reported in other studies. The abundance of Fusarium was expected as it has been reported as a natural contaminant in cereals, specifically maize and sorghum. The composi%on and diversity of fungi varied significantly in the sampling regions and this may be due to the differences in applied farm management methods. The disease severity and incidence levels corresponded with the diversity levels analyzed using morphological molecular methods. Leaf samples showing varying disease symptoms were selected (≈14) and isolated from each collec%on site. The disease symptoms included circular or elongated lesions, streaks, and veinal/midrib browning. Leaf lesions were the most observed disease symptom across all sampling loca%ons while leaf streaks were the least observed (Fig. 1). The incidence and prevalence of the disease symptoms showed correla%on. Fungal strains were isolated from the leaves and maintained on malt extract agar (MEA) and a total of 853 isolates were recovered from all collec%on sites. They were were grouped according to their morphological traits and 318 morphotypes were selected for DNA extrac%on. The universal primers ITS3-ITS4 targe%ng the ITS2 region of the ribosomal DNA were used and 253 amplified successfully and were sequenced using Illumina MiSeq. Results *Zanele Noqobo 1&2 , Idan Chiyanzu 1 , Jasper Rees 1 and SaneLe Marx 2 1 Agricultural Research Council, Biotechnology PlaDorm, Onderstepoort, 0110, South Africa 2 North-West University, School of Chemical and Minerals Engineering, Potchefstroom, 2520, South Africa *Correspondence: [email protected] , +27 781613694 Illumina-based analysis of sorghum fungal pathogens culRvated in vitro DNA extracRon and ITS2 amplificaRon Methodology Free State Gauteng North West Eastern Cape Western Cape Northern Cape Kwazulu Natal Mpumalanga Limpopo Parys Standerton Sasolburg Oranjeville SeClers Litchenburg Pederkop Sample collecRon sites Collected leaf samples Fungal isolaRon Illumina MiSeq sequencing amplicon sequencing FastQC- phred score =20; read length =200 bp SPAdes- conAg assembly Megablast- comparaAve sequence analysis OTUs – 97% similarity threshold Sequencing and bioinformaRcs Fig 1. Mean disease incidence % and severity % of observed disease symptoms on Sorghum leaves. The leaf samples were collected from farms in different provinces of South Africa. 0 10 20 30 40 50 60 70 80 90 100 BLIGNAUT SWART DU PREEZ COETZER DU PLOOY WESTHUIZEN POTGITERS UITKYK GROBBLER GROOTHOUT PHILIP Disease severity % 0 10 20 30 40 50 60 70 80 90 100 Disease incidence % Leaf lesions Brown midrib Leaf streaks Mpumalanga Free State Limpopo North- West Acknowledgments: Dr. K.O. Bezuidt for assis%ng with bioinforma%c analysis, BTP-core for sequencing and the farmers for samples. References: (1) Anjum, et al., (2013) An insight into the basis of resistance in Sorghum bicolor against Colletotrichum sublineolum. African journal of microbiology research,1397-1408. (2) AbdelfaTah, et al. ( 2016) Metagenomic analysis of fungal on Strawberry plants and the effect of of management prac%ces on the fungal community structure of aerial organs. Plos One,1-17.
Transcript of Illumina-based analysis of sorghum fungal pathogens ... · Mpumalanga Free State Limpopo North-...
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Fig2.Rela%veabundanceofdifferentfungalclasses(A)andgenera(B)iden%fiedfromdifferentfarms.
Background:SorghumisanimportantcropinSouthAfrica,butitisexposedtoavarietyofpathogenicfungithatcausemanydiseasesleadingtosignificantlossesinyieldsandreduc%oninqualityoftheproduct,therebythreateningfoodsecurity.Althoughseveralcontrolmethodsthathavebeenappliedhavebeensuccessful,pathogensadaptandevolvequickly.Preciseknowledgeofthediseasecausalagentsisessen%alforthedevelopmentofeffec%vecountermeasurestofungalinfec%ons.Theaimofthestudywastosurveylocalsorghumfungalpathogensandusemorphologicalandmolecularapproachesforiden%fica%on.Results:Sorghumfungalpathogendiversitywasinves%gatedinelevenproducingregionsinSouthAfricausingaculture-dependenttechnique.Morphologicalcharacteris%csandITS2 sequence data generated from IlluminaMiSeq was used to iden%fy the fungal isolates. A total of 253 fungal isolates were obtained from leaf samples, fromwhichopera%onaltaxonomicunitsbelongingto36generawereiden%fiedasbelongingtoAscomycota(98%)andBasidiomycota(2%)phyla.Fusariumwasthedominantgenera(38.9%rela%veabundanceofthedetectedsequences)followedbyCurvalaria(14.7%)andSarocladium(8.7%).Thediversityandabundanceofdifferenttaxonomicgroupsdifferedwithsamplingloca%ons.
Significance: Studying the fungal pathogens, which cause diseases threatening the produc%on of one of the important crops, sorghum has a significant impact in cropproduc%on.AbeTerunderstandingofthesepathogenswill leadtoeffec%vemanagementandthereforefoodsecurityformanyleavinginpovertyinSouthAfrica.Pathogenscause diseases by producing enzymes that deconstruct the plant cell walls. Their ability to produce hydroly%c enzymes can be exploited for industrial bioconversion. Thediversityofthefungiobtainedinthisstudysuggeststhattheenzymesproducedarecapableofprovidingthediversityandstrengthofac%vityrequiredforbioconversion.Thisstudywillhaveasignificantcontribu%oninfoodsecurityandinenergyproduc%on.
Abstract
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ITS2sequencesclusteredinto220OTUsand95%belongedtoFungi,while the other 5% were either plant or bacterial sequences.Membersof thephylumAscomycotaweredominant inall samples,collec%vely accoun%ng for 98% of the total number of detectedfungalsequences,andBasidiomycotaaccountedfor2%.Ascomycotasequences were largely iden%fied as members of the classesSordariomycetes and Dothideomycetes (57.9% and 34%respec%vely), followed by Euro%omycetes (4.4%), Saccharomyctes(2.2%) and Leo%omycetes (0.4%). Within Basidiomycota phylaAgaricomyceteswasthemostabundantclasswith0.6%,Therela%veabundance of other classes Tremellomycetes, CystobasiomycetesandExobasidiomyceteswas0.2%(Fig.2A).
Furtheranalysisoftheiden%fiedOTUsclassifiedtheminto36generawhich were characterized by the prevalence of Fusarium (38.9%),Curvularia(14.7%)Sarocladium(8.7%),Alternaria(6.9%),Nigrospora(5.4%), Bipolaris (5.3%) and Phoma (4.7%). The representa%on ofotherdetectedgenerawas limitedwith rela%veabundance rangingfrom0.1%-2.4(Fig.2B).
Results
DiscussionResults indicate that a wide variety of fungal taxa inhabit sorghum plants. The high diversity of fungi iden%fied is comparable to themetagenomicstudiesdonepreviouslyandthisresultwasnotexpectedconsideringthelowdiversityofisolatedfungireportedinotherstudies.The abundanceofFusariumwas expected as it has been reported as a natural contaminant in cereals, specificallymaize and sorghum. Thecomposi%on and diversity of fungi varied significantly in the sampling regions and this may be due to the differences in applied farmmanagementmethods.Thediseaseseverityandincidencelevelscorrespondedwiththediversitylevelsanalyzedusingmorphologicalmolecularmethods.
Leafsamplesshowingvaryingdiseasesymptomswereselected(≈14)and isolated from each collec%on site. The disease symptomsincluded circular or elongated lesions, streaks, and veinal/midribbrowning. Leaf lesions were the most observed disease symptomacross all sampling loca%ons while leaf streaks were the leastobserved (Fig. 1). The incidence and prevalence of the diseasesymptomsshowedcorrela%on.
Fungalstrainswereisolatedfromtheleavesandmaintainedonmaltextractagar (MEA)andatotalof853 isolateswererecoveredfromall collec%on sites. They were were grouped according to theirmorphological traits and 318 morphotypes were selected for DNAextrac%on.TheuniversalprimersITS3-ITS4targe%ngtheITS2regionoftheribosomalDNAwereusedand253amplifiedsuccessfullyandweresequencedusingIlluminaMiSeq.
Results
*ZaneleNoqobo1&2,IdanChiyanzu1,JasperRees1andSaneLeMarx21AgriculturalResearchCouncil,BiotechnologyPlaDorm,Onderstepoort,0110,SouthAfrica
2North-WestUniversity,SchoolofChemicalandMineralsEngineering,Potchefstroom,2520,SouthAfrica*Correspondence: [email protected], +27 781613694
Illumina-basedanalysisofsorghumfungalpathogensculRvatedinvitro
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Fig1.Meandiseaseincidence%andseverity%ofobserveddiseasesymptomsonSorghumleaves.TheleafsampleswerecollectedfromfarmsindifferentprovincesofSouthAfrica.
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Acknowledgments:Dr.K.O.Bezuidtforassis%ngwithbioinforma%canalysis,BTP-coreforsequencingandthefarmersforsamples.References: (1) Anjum, et al., (2013) An insight into the basis of resistance in Sorghum bicolor against Colletotrichum sublineolum. African journal of microbiology research,1397-1408. (2)AbdelfaTah,etal.(2016)MetagenomicanalysisoffungalonStrawberryplantsandtheeffectofofmanagementprac%cesonthefungalcommunitystructureofaerialorgans.PlosOne,1-17.
