Analysis Of Fungal Diversity Of Imported Seeds Of Lolium Perenne And Trifolium Repens

There are a large quantity of grass seeds imported into China each year for demands of high quality grass seeds.Seed-borne fungal pathogens have negative effects on seed germination,seedling vigor and resulting in great economic losses.It is crucial to detect imported grass seeds rapidly and to understand seed-borne fungal diversity.In this study,three different DNA extraction methods were applied to extract the genomic DNA.The fungal diversity in four perennial ryegrass(Lolium perenne)and five white clover(Trifolium repens)seeds collected from different countries were analyzed by high-throughput sequencing,and compared with the results of traditional method.Meanwhile detecting and identifying the important plant pathogenic fungi Fusarium by DNA barcoding.The main results are as follows:1.The genomic DNA was extracted by CTAB,kit and urea method respectively,and the concentration and purity were determined by agarose gel electrophoresis and Nanodrop 1,000.Results showed that the urea extraction method had better efficacy,simpler and more higher extract rate(the concentration up to 886.5 ng·?L-1).The OD260/280 value was close to 1.80.Therefore,the urea extraction method could be satisfied requirement of the subsequent sequencing.2.The fungal diversity in four perennial ryegrass(L.perenne)and five white clover(T.repens)seeds were analyzed by high-throughput sequencing.Results showed that the fungal diversity of nine seed samples were different.The Ascomycota and Basidiomycota were predominant in all samples at phylum level(over 98.3%).A total of 69,40,75 and 41 genera were identified from JH(L.perenne imported from Canada),DH(L.perenne imported from Danmark),MH(L.perenne imported from America)and XH(L.perenne imported from New zealand),as well as 52,47,44,65 and 50 genera were identified from TB(T.repens imported from Argentina),DB(T.repens imported from Danmark),MB(T.repens imported from America),AB(T.repens imported from Australia)and XB(T.repens imported from New zealand),respectively.The major genera were Davidiella,Claviceps,Cryptococcus and Alternaria.3.Seed samples of four perennial ryegrassand five white clover were isolated and identified by morphology and ITS gene.Only 89 fungal strains belonged to 19 species of 9 genera were isolated.The major genus of AB was Stemphylium vesicarium(57.2%).The major genus of DB,XB and MH was A.infectoria(100%,100%,94.7%).The major genus of MB was Cladosporium tenuissimum(71.4%).The major genus of TB and JH was A.alternata(45.5%,66.7%).The major genus of DH was Aspergillus fumigatus(66.7%).The major genus of XH was Epicoccum nigrum(100%).The samples of AB,TB and JH have higher fungal diversity by traditional method,which show the similar results with the fungal diversity of high-throughput sequencing.4.To determine a suitable DNA barcode for the genus Fusarium,the ITS,EF-1?,mt SSU and ?-tubulin genes were selected as candidate markers.A total of 11 species of Fusarium isolated from different hosts as materials.The results showed that The PCR and sequencing success rate of EF-1? was 98.2%,higher than that of mt SSU and ?-tubulin genes.Among four genes mentioned above,the frequency distribution of the genetic distance of EF-1? gene was less in overlap than the other.According to the EF-1? tree,all of the species were separated.EF-1? gene could identify the genus Fusarium better than the other genes.Therefore,EF-1? gene was recommended as the DNA barcode to identify the genus Fusarium.