title: Development and application of loop-mediated isothermal amplification for detecting the highly benzimidazole-resistant isolates in Sclerotinia sclerotiorum.
abstract: Resistance of benzimidazole fungicides is related to the point mutation of the β-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG → GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60-64 °C for 60 min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods.
authors.affiliation: College of Plant Protection, State &Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing, 210095, China.
authors.forename: Ya Bing
authors.initials: YB
authors.lastname: Duan
authors.affiliation: College of Plant Protection, State &Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing, 210095, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Yang
authors.affiliation: College of Plant Protection, State &Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing, 210095, China.
authors.forename: Jian Xin
authors.initials: JX
authors.lastname: Wang
authors.affiliation: College of Plant Protection, State &Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing, 210095, China.
authors.forename: Cong Chao
authors.initials: CC
authors.lastname: Liu
authors.affiliation: College of Plant Protection, State &Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing, 210095, China.
authors.forename: Ling Ling
authors.initials: LL
authors.lastname: He
authors.affiliation: College of Plant Protection, State &Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing, 210095, China.
authors.forename: Ming Guo
authors.initials: MG
authors.lastname: Zhou
mesh_terms: D001203:Ascomycota / Q000187:drug effects* / Q000302:isolation & purification*; D001562:Benzimidazoles / Q000494:pharmacology*; D017931:DNA Primers / Q000378:metabolism; D025141:Drug Resistance, Fungal / Q000187:drug effects*; D008826:Microbial Sensitivity Tests; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D013696:Temperature; D013997:Time Factors
chemical_list: D001562:Benzimidazoles; D017931:DNA Primers; C031000:benzimidazole