Rapid identification of benomyl resistant strains of Botrytis cinerea using the polymerase chain reaction

A fragment of the beta-tubulin gene from Botrytis cinerea was amplified ina polymerase chain reaction (PCR) using generic primers, cloned and DNA sequenced. The sequence obtained was used to design beta-tubulin primers (BCF and BCR) more specific to the B. cinerea gene. These primers amplified a 381 base pair (bp) beta-tubulin gene product from B. cinerea strains isolated from a range of locations and hosts. Amplification products from 16 isolates were cloned and sequenced. All benzimidazole sensitive isolates (ben(S)) had the sequence GAG (Glu) at codon 198, while resistant isolates (ben(HR)) had a single base substitution to GCG (Ala) at this position. On the basis of this ben(HR) mutation, two rapid detection methods were designed. The first relied on the creation of a Tha I restriction site at codon 198 in ben(HR) strains, in which Tha I cleaved the 381 bp amplification product of BCF and BCR into 100 and 281 bp fragments, while products from ben(S) strains remained undigested. The second method relied on allele-specific PCR using an internal primer (BCM) with the codon 198 mutation at its terminal 3' base. A 381 bp fragment was amplified from all isolates using primers BCF, BCM and BCR, and an additional 281 bp fragment was amplified from ben(HR) strains. To improve the speed of both assays a simple microwave based procedure was developed, allowing the analysis of samples directly from fungal mycelium or from artifically infected plant tissue in approximately 5 h.