A Study On Molecular Detection For Brown Stem Rot Pathogen

Brown stem rot of soybean (BSR), is a relatively new soybean disease. It was first reported in United States in 1940. Brown stem rot is a vascular disease of soybeans caused by the soil borne fungal pathogen Phialophora gregata f.sp.sojae (Allington & Chamberlain) Gams. BSR can survive in soil for many years; it resulted in significant crop loss. P. gregata is a challenging fungus to study.It grows slowly, can be difficult to isolate from infected tissues, and due to long latent periods. The symptom of BSR is similar with sudden death syndrome of soybean. It is distributing in main soybean produce countries, United State, Brazil, Japan, Canada and Argentina. As the soybean trade, BSR can be introduced to China. In order to prevent BSR, this study is purposed to establish BSR detection method for quarantine and identification.Base on ITS sequence of the BSR pathogen, under the rulers of primer design, two PCR primers were designed for Phialophora gregata f.sp.soja. The code name and sequences for the primer, reverse primer were Pg1 (5'-agagcaaaggatagggagca-3'), Pg4 (5'-tggcatgtagacctgcca-3').DNAs of sets of samples from 13 cultures which were from 9 different species often infected soybean were extracted. Delveloping the PCR, the best anneal temperature is 59℃.And dilution was up to 4.2×10¹ pg/μL.At the same time, realtime-PCR primers and probe were designed. The code name and sequences for the primer, reverse primer, and Taqman probe were BSR-1F (5'-ccaaccagggccgatcag-3') , BSR-1R (5'-cggattcagcgtaaaaaatgg-3') , and Taqman BSR-1M (5'-6FAM-ctcccctatggtttct-MGBNFQ-3') . This assay was found that the specificity was high without any cross-reactions with other 12 fungus.Delveloping the realtime-PCR, reactions were conducted in a volume of 25μL, 0.4μmol/L each of the two primers, and 0.2μmol/L probe. And the dilution was up to 4.2×10^-4pg/μL,was 100000 times more sensitive than the standard PCR.At last, the pgs1 (ATCC204452) culture was inoculated in three different breeds' of soybean. And the primers pg1/4 was used to detect the pathogen in inoculated plant successfully.