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.

LAMP detection of the genetic element ‘Mona’ associated with DMI resistance in Monilinia fructicola

“…LAMP has previously been used in the detection of point mutation‐mediated fungicide resistance, for example MBC resistance in Fusarium species and S. sclerotiorum …”

“…Recently, there has been interest in using LAMP to detect fungicide resistance. For example, the method has been developed to detect the F167Y mutation in carbendazim‐resistant Fusarium graminearum and Sclerotinia sclerotiorum isolates . However, the LAMP method has rarely been adopted to detect non‐target site‐mediated resistance.…”

Genetic variation in Pythium myriotylum based on SNP typing and development of a PCR-RFLP detection of isolates recovered from Pythium soft rot ginger

“…; Duan et al . ). Thus, applications of novel techniques for fast and accurate detection of PSR, P. myriotylum strains will be warranted.…”

“…2) (Notomi et al 2000;Mitani et al 2007). Both techniques, LAMP and SMAPs have shown to be highly specific detection methods of pathogen targets with the capability of detecting SNPs (Mitani et al 2007;Duan et al 2015). Thus, applications of novel techniques for fast and accurate detection of PSR, P. myriotylum strains will be warranted.…”

Characterization of species associated with Pythium soft rot of ginger and evaluation of Pythium Oligandrum as a biocontrol

“…However, with rapid progress in developing novel molecular tools, PCR-RFLP is considered to be a time consuming approach compared to isothermal amplification of DNA techniques such as LAMP (loop-mediated isothermal amplification) and SMAP 2 (smart amplification process version 2) (Mitani et al, 2007;Notomi et al, 2000). Both techniques, LAMP and SMAPs have shown to be highly specific detection methods of pathogen targets with the capability of detecting SNPs (Duan et al, 2015;Mitani et al, 2007;Yabing et al, 2015). Thus, applications of novel techniques for fast, effective and accurate detection of PSR P. myriotylum strains will be warranted.…”