Rapid detection of benzimidazole resistance in Botrytis cinerea by loop-mediated isothermal amplification

Fei Fan,Guo-Qing Li,Chao-Xi Luo,Matthias Hahn,Yang Lin

doi:10.1186/s42483-019-0016-8

Abstract

Benzimidazole fungicides (MBCs) have been widely used in agriculture since 1970s, and resistance to this class of fungicides in Botrytis cinerea is reported worldwide. Resistance to MBCs in B. cinerea is related to mutations in the target β-tubulin gene (TUB2). Compared with the mutation at codon 200, the substitutions from glutamic acid to alanine (E198A), valine (E198V), or lysine (E198K) at codon 198 are currently the predominant mutations in MBC resistant populations of B. cinerea. In this study, a loop-mediated isothermal amplification (LAMP) method was established for rapid detection of benzimidazole resistance in B. cinerea. On the basis of the three mutations at TUB2 codon 198, three sets of LAMP primers were designed, and each of these primer sets was able to specifically amplify the DNA containing its corresponding mutation, while no amplification was detected with other mutated or the wild type DNA. After optimization, the sensitivity and specificity tests illustrated that this LAMP assay had good sensitivity and specificity to detect specific resistant genotypes in B. cinerea. Result also showed that the LAMP assay had good reproducibility. Above all, boiled mycelia could be used as templates, which simplified the process and increased the efficiency of the assay. Considering its rapidity, simplicity, and high efficiency, the LAMP assay developed in this study is a promising tool for the diagnosis of benzimidazole resistance in B. cinerea, and will contribute to the monitoring of resistance development to MBCs in the future.

Highlights

Grey mold is a common disease on numerous crops worldwide, especially on vegetables and small fruits
After addition of SYBR Green I, the samples of E198A, E198K, sensitive isolate, and DNA-free control remained brown, but the sample of E198V changed to a fluorescent yellow color (Fig. 2a), which indicated the positive loop-mediated isothermal amplification (LAMP) reaction
After the successful design of Tub-E198V, the F2 primer was modified to fit the other two mutations (E198A and E198K) following the same strategy (Fig. 1 and Table 1). Both dye treatment and electrophoresis demonstrated that primer sets Tub-E198A and Tub-E198K were capable of recognizing their corresponding mutations respectively (Fig. 2)

Summary

Introduction

Grey mold is a common disease on numerous crops worldwide, especially on vegetables and small fruits. The causal agent of this disease is the ascomycete fungus Botrytis cinerea (Williamson et al 2007) To control this fungus, fungicides are widely used. Resistance to this class of fungicides in B. cinerea was reported (Bolton 1976; Tripathi and Schlösser 1982). This resistance was related to several mutations in the β-tubulin gene (TUB2) at codon 198, which resulted in amino acid replacement of glutamic acid by alanine (E198A), valine (E198V), or lysine (E198K) (Yarden and Katan 1993; Banno et al 2008). Our earlier research showed that mutations at codon 198 were predominant in the isolates with resistance to carbendazim in Hubei, China (Fan et al 2016; Fan et al 2017)

Methods

Results

Discussion

Conclusion