A multiplex allele-specific PCR method for the detection of carbendazim-resistant Sclerotinia sclerotiorum

The benzimidazole fungicide carbendazim (CBD) has been used extensively in China for the control of sclerotinia stem rot (SSR) of rapeseed caused by Sclerotinia sclerotiorum. In this study, 245 isolates of S. sclerotiorum were examined for their sensitivities to CBD, and two resistance levels were identified. Among 245 isolates, 78 isolates were highly resistant to CBD. These CBD highly resistant (CBD-HR) isolates were more sensitive to the phenylcarbamate fungicide diethofencarb than CBD sensitive (CBD-S) isolates. One isolate was medium resistant to CBD, and insensitive to diethofencarb. Random amplified polymorphic DNA (RAPD) analysis showed the CBD-HR isolates had different genetic backgrounds. Analysis of the DNA sequence of the β-tubulin gene showed that all CBD-HR isolates had a point mutation at the codon 198, causing a substitution of glutamic acid to alanine. The CBD medium resistant (CBD-MR) isolate had a point mutation at the codon 200 causing a substitution of phenylalanine to tyrosine. Based on these point mutations, a multiplex allele-specific PCR method was developed to detect two different point mutations simultaneously in single PCR amplification.