Application of cycleave PCR to the detection of a point mutation (F167Y) in the β2‐tubulin gene of Fusarium graminearum

Abstract

Resistance of Fusarium graminearum to the benzimidazole fungicide carbendazim is caused by point mutations in the β2 -tubulin gene (FGSG_06611.3). The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field isolates in China. It is important to find a suitable method for rapid detection and quantification of this point mutation in the F. graminearum populations. A pair of primers, Codon167F/Codon167R, were designed to amplify a fragment containing the mutation site, and two cycling probes labelled with different fluorescent reporters were used to detect whether the mutation was present. A cycleave real-time PCR method was developed for rapid determination of the frequency of this point mutation in 282 F. graminearum perithecia collected from different fields in Jiangsu Province, China. The mutation frequency in ascospores from the perithecia to carbendazim by a spore germination assay was 6.0%, while the frequency of the point mutation at codon 167 by the cycleave real-time PCR assay was 3.9%. The cycleave real-time PCR method is suitable for accurate detection of the codon 167 point mutation. The frequency of this mutation in the β2 -tubulin gene represents the resistance frequency in F. graminearum populations to carbendazim.