Deciphering β-tubulin gene of carbendazim resistant Fusarium solani isolate and its comparison with other Fusarium species.

Abstract

Exploration of molecular structure of β-tubulin is key to understand mechanism of action of carbendazim since its activity depends on strong binding to β-tubulin. Resistance against the fungicide is often associated with mutation in β-tubulin gene. A full-length (1619bp) β-tubulin gene has been cloned and sequenced from a carbendazim resistant and a sensitive isolates of F. solani isolated from agricultural fields of Murshidabad (24.23°N, 88.25°E), West Bengal, India. Phylogenetic position of the isolates was confirmed using internal transcribed spacer and β-tubulin gene sequences. In the β-tubulin based phylogenetic tree, Fusarium species with available data were clustered in nine species complexes and members of both F. solani species complex and F. fujikuroi species complex were distributed into three clades each. The β-tubulin gene of F. solani was found to be shortest due to least number of non-coding sequences indicating its primitiveness among the Fusarium species. The coding region (G + C 58.54%) was organized into five exons. The protein has 446 amino acid, 49.834 KD molecular weight and 4.64 isoelectric point. Amino acid sequence of the resistant and the sensitive isolates were identical, suggesting that the mechanism of carbendazim resistance in the F. solani isolate was not due to point mutation in β-tubulin gene. The secondary and tertiary structure of β-tubulin were similar in all the species except F. oxysporum f.sp. cubense. The identification of binding sites for GDP, carbendazim and α-tubulin would resolve how carbendazim prevents tubulin polymerization. All the data are useful to design tubulin-targeted fungicide with better performance.