Amplification and cloning of a β-tubulin gene fragment from strains of botrytis cinerea resistant and sensitive to benzimidazole fungicides

A series of oligonucleotide primers was used to amplify portions of the Botrytis cinerea β -tubulin gene, in particular, the region where mutations conferring resistance to benzimidazoles occur in other fungal species. The optimum primer pair produced three distinct fragments from B. cinerea DNA: A strong band of 440 base pairs (bp); a faint band of480 bp; and an additional band of 700 bp. There was no apparent difference between isolates that were resistant or sensitive to benzimidazoles. Amplifications from other fungal species produced a440 bp band (Moniliniafructicola and Phomopsis viticola), a 480 bp band (Aspergillus nidulans), or both (Penicillium digitatum), but did not produce a 700 bp band. Raising the primer annealing temperature from 55 to 58°C eliminated the 700 bp product from the B. cinerea amplifications. To test the identity of the amplified fragments, DNA transfers were probed with a biotin-labelled β-tubulin clone from Leishmania major. Both 440 and 480 bp products responded to the probe but the 700 bp product did not. The hybridisation and annealing data suggest B. cinerea has two distinct β-tubulin genes (represented by the 440 and 480 bp products), and that the 700 bp fragment may be a distantly related tubulin gene or the result of non-specific amplification. All products from the B. cinerea isolates were purified by electroelution and inserted into plasmid vectors. Insertion of the β -tubulin gene fragments was confirmed using restriction digestion and hybridisation with the L. major β-tubulin clone. The identity and relationship of the various amplified fragments will be tested using DNA sequencing, which will also determine if sequence changes to this portion of the β-tubulin gene are associated with benzimidazole resistance in B. cinerea.