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.
Highlights
Resistance of benzimidazole fungicides is related to the point mutation of the β-tubulin gene in Sclerotinia sclerotiorum
Since Loop-mediated isothermal amplification (LAMP) was reported in 2000 by Notomi, it has been widely applied in nucleic acid analysis because of its simplicity, rapidity, high efficiency, and outstanding specificity[12]
The application of LAMP in fungicide resistance of plant pathogens has not been reported until the detection of benzimidazole-resistant mutants of F. graminearum was disclosed by our group[26]
Summary
Resistance of benzimidazole fungicides is related to the point mutation of the β-tubulin gene in Sclerotinia sclerotiorum. 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). Loop-mediated isothermal amplification (LAMP) is an outstanding gene amplification procedure, which can amplify nucleic acids with high specificity, sensitivity, efficiency and rapidity under isothermal conditions[12]. This technique is characterized by the use of a DNA polymerase with strand displacement activity and a set of two specially designed inner primers (FIP and BIP) and two outer primers (F3 and B3)[13,14]. Nagamine et al had improved the method by putting forward loop primers (LF and LB) that accelerated the LAMP reaction[15]
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