Molecular Identification And Rapid Detection Of Some Colletotrichum Species

Colletotrichum Cda.is one of worldwide plant pathogens causing anthracnose before or after maturity in lots of crops,which had led great economical lose.The classification and identification of plant pathogenic Colletotrichum species has been confusing,difficult to accurately identify pathogen species,brought disorder to study such diseases and affects the control effect.Therefore,it is very necessary to accurate identification and rapid detection studies of Colletotrichum.In this study on the basis of the traditional morphological classification,we combined ITS,GAPDH,ACT,TUB2,CAL,CHS-1 gene to establish the multi-gene phylogenetic trees to accuratly identify the taxonomic status of Colletotrichum species.Some incorrect colletotrichum pathogenic name will be revised on the basis of the molecular identification,simultaneously,it may be find new species and some new host species of Colletotrichum,and specific primers were designed for rapid detection of pathogens research on Dracaena sanderiana.Concrete results are as follows:In 2012-2014,we collected about 1000 samples from some areas of Henan,Guangdong,Guangxi,Beijing,Yunnan,Hunan and Hainan Provinces in China.The hosts plants included 36 families,59 genera,59 species.74 strains obtained by single spore isolation and tissue separation.We got 85 strains in total together with 11 strains from the laboratory of Plant Protection College.We had identified 21 species by morphological characteristics and multilocus molecular analyses,including four new species,Colletotrichum tamarindum Meng Zhang&C.D.Zhang,Colletotrichum sanderianum Meng Zhang&C.D.Zhang,Colletotrichum hostum Meng Zhang&C.D.Zhang,Colletotrichum racemosum Meng Zhang&C.D.Zhang.In addition,2 species,Colletotrichum fioriniae J.H.Simmonds,Colletotrichum scovillei Damm,P.F.Cannon&Crous are new records in China.We also found one kind of new Colletotrichum pathogen on Dracaena sanderiana:C.Cliviae.In this study,45 typical strains were divided into four species complex groups based on the morphological characteristics.Then,we had identified them by compared it with neighboring species on the basis of N-J phylogenetic tree constructed by multi-gene combining.By single-gene and multi-gene molecular sequence analysis,it is difficult to define a single strain taxonomic status by single-gene.For example,the strain ZM130987 of C.fructicola,the single-gene phylogenetic tree for ITS,GAPDH,ACT,TUB2 constructed not clustered together with the type strain,and the support of less than 50%(not shown),and CAL,CHS-1 can separate them(not shown),while assembled multi-gene can be well separated from it with similar species(Fig 1).These cases also appear in other taxa.The results showed that:Colletotrichum species can accurately be identified only by combination of multi-gene assembled analysis,morphoology and other evaluation methods.Pathogenicity tests resulted:C.cliviae and C.dracaehophilum on Dracaena sanderiana,C.scovillei on peppers and C.asianum on mango were proved to be pathogens by Koch's spostulate.Rapid detection technology studies of 2 pathogens of Dracaena sanderiana results:By gene sequence analysis,specific mutation sites of ITS region from C.cliviae and C.dracaehophilum had been found.Using Primer5.1 software,we designed two pairs specific primers of the two species,that were ZM3-7F/ZM3-7R and 710F/710R,the detection sensitivity with the two couples of primers were respectively 100pg/ul,1ng/ul.We used Nested-PCR technology,C.dracaehophilum sensitivity can be increased 1015times,reaching 10-6ag/ul.