Abstract: Benzimidazole fungicides are important mixture components to control rape sclerotinia rot disease in China. Carbendazim-resistant isolates of Sclerotinia sclerotiorum have been detected in fields since 1996. In this paper, three pairs of degenerate primers were used to amplify theβ-tubulin gene from S.sclerotiorum. The gene from S.sclerotiorum has 1 685 bp, including 4 introns, encoding 447 amino acid. Except for the difference in the number of the introns, the deduced amino acid sequence of the β-tubulin gene from S.sclerotiorum are 95.78% to 97.66% identical to those of other six plant pathogenic filamentous fungi. Resistance was related to a point mutation in codon 198 where the glutamic acid has changed into alanine, which caused the occurrence of resistance field. A DNA fragment surrounding codon 198 was amplified directly from genomic DNA of sclerotinia using two pairs of Allele-Specific Oligonucleotide(ASO) primers to detect resistant frequency accurately. Using this method within 6 h, the detection rate for benzimidazole resistance was up to 100%; in comparison with the conventional assay procedure, which needs 1-2 weeks, the accuracy of the new method was 96%.

Key words: Sclerotinia sclerotiorum, β-tubulin, Carbendazim-resistance, Allele-Specific Oligonucleotide (ASO)-PCR