A new multiplex polymerase chain reaction assay for simultaneous detection of five soil-borne fungal pathogens in winter wheat

Abstract

Wheat (Triticum spp.) is often coinfected by several soil-borne fungal pathogens, causing serious crop yield and economic losses. Rapid diagnosis of infected fungi is critical for the application of disease control mechanisms. Therefore, we established a multiplex PCR method using specific primers for the simultaneous detection of five common soil-borne fungal pathogens affecting wheat: Rhizoctonia cerealis, Bipolaris sorokiniana, Gaeumannomyces graminis var. tritici, Fusarium pseudograminearum and F. graminearum. The specific primers were designed based on the sequences of several unique genes: ITS (internal transcribed spacer) of R. cerealis, URP-1F (universal rice primer) of B. sorokiniana, β-tubulin of G. graminis var. tritici and TEF1-α (translation elongation factor) of F. pseudograminearum and F. graminearum. During optimization, sensitivity assays showed that the detection limit of multiplex PCR was as low as 0.39 ng of mycelium DNA from a known pool of fungal DNA. To detect pathogens in wheat field, the multiplex PCR method was applied to infected wheat samples collected from wheat fields in the Fengqiu and Taikang counties in Henan Province, China. The R. cerealis and F. graminearum pathogens were identified in samples from Fengqiu, while the R. cerealis, G. graminis var. tritici, B. sorokiniana and F. graminearum pathogens were identified in samples from Taikang. In conclusion, the results indicated that our multiplex PCR method was efficient and specific for the detection of soil-borne pathogens.