Rapid detection of virulence-related genes by multiplex PCR in five pathogenic bacteria of mulberry bacterial wilt

Mulberry bacterial wilt is a devastating disease that is difficult to control and causes serious economic losses to the sericulture industry. This disease is mostly caused by a diverse group of pathogenic and opportunistic bacteria including, Ralstonia pseudosolanacearum, Pantoea ananatis, Enterobacter cloacae complex (ECC), Klebsiella pneumoniae species complex (KpSC), and K. oxytoca complex (KoC). Due to the lack of a rapid and reliable test to simultaneously detect these complex pathogens of mulberry wilt, we developed a multiplex PCR (mPCR) assay to detect five virulence-related genes carried by the pathogenic bacteria of mulberry bacterial wilt disease. The primers were designed for the virulence-related genes: pleD (GGDF structural domain-containing protein), yjfP (esterase), pelY (peripheral pectate lyase), ampD (N-acetyl-anhydromuranmyl-L-alanine amidase), and ripW (type III effector). Overall, the developed mPCR assay showed highly specific, sensitive and reproducible detection of target pathogens. Briefly, the results showed that the mPCR was highly specific in individual reactions, and the lowest detection concentration of the five pathogenic bacteria was 1.87 x 10(3) CFU/mL (DNA = 2.45 pg/mu L). From 46 natural mulberry wilt samples, the mPCR detection rates of P. ananatis, ECC, KpSC, KoC and R. pseudosolanacearum were 8.69, 91.3, 34.7, 23.9 and 65.21%, respectively. The traditional culture media isolation methods showed comparable results. The pathogenicity test of 84 suspected pathogenic bacteria revealed that the morbidity (average morbidity level) caused by the pathogenic bacteria detected by mPCR was >= 65.5%, while the morbidity of the undetected pathogenic bacteria was <= 35.5%. Based on these results, we believe that the mPCR developed in the present study will be useful in rapid, reproducible, and sensitive detection of the pathogenic bacteria causing mulberry bacterial wilt including, R. pseudosolanacearum, P. ananatis, ECC, KpSC, and KoC.