Abstract: This study uses the loop-mediated isothermal amplification to set up a fast,accurate and efficient way to detect Botrytis cinerea that confered high resistant to carbendazim. Carbendazim resistance analysis of B. cinerea was conducted in three produce bases of Dendrobium candidumin in Lin'an county. We found that local high-level carbendazim resistance of B. cinerea was due to 198 amino acids mutantion of β-tubulin encoding protein from E(GAG) to V(GTG). In this study,point mutant was designed in primer F2 to detect the B. cinerea E198V genotype that confered high resistant to carbendazim. The LAMP assay efficiently amplified the target DNA in 40 min under isothermal conditions at 65℃ and was evaluated for specificity and sensitivity. A positive colour(bright green) was only observed in the presence of B. cinerea E198V strain by addition of calcein dye as an indicator reaction prior to amplification,whereas none of other isolates showed a colour change(still orange). The detection limit of the LAMP assay for B. cinerea E198V was 10 pg/μL of genomic DNA per reaction. Agarose gel electrophoresis results were consistent with the LAMP test results. Our establishment of LAMP assay provides a new alternative method for rapid identification of carbendazim resistant strains of B. cinerea E198V strain of Dendrobium candidumin.

Key words: Loop-mediated isothermal amplification(LAMP), Botrytis cinerea, Carbendazim resistance detection

CLC Number: 

• Q78