M233I Mutation in the β-Tubulin of Botrytis cinerea Confers Resistance to Zoxamide

Abstract

Three phenotypes were detected in 161 Botrytis cinerea field isolates, including ZoxSCarS (sensitive to zoxamide and carbendazim), ZoxSCarR (sensitive to zoxamide and resistant to carbendazim), and ZoxRCarR (resistant to zoxamide and carbendazim), but not ZoxRCarS (resistant to zoxamide and sensitive to carbendazim). The baseline sensitivity to zoxamide was determined with a mean EC50 of 0.76 μg/ml. Two stable ZoxRCarS isolates were obtained with a resistance factor of 13.28 and 20.43; there was a fitness penalty in mycelial growth rate, sporulation, virulence and sclerotium production. The results suggest that the resistance risk of B. cinerea to zoxamide is low where benzimidazoles have not been used. E198V, E198K and M233I, were detected in the β-tubulin of ZoxSCarR, ZoxRCarR, ZoxRCarS, respectively. Molecular docking indicated that position 198 in β-tubulin were targets for both zoxamide and carbendazim. The mutations at 198 prevented formation of hydrogen bonds between β-tubulin and carbendazim (E198V/K), and changed the conformation of the binding pocket of zoxamide (E198K). M233I had no effect on the binding of carbendazim but resulted in loss of a hydrogen bond between zoxamide and F200. M233 is suggested to be a unique target site for zoxamide and be very important in the function of β tubulin.

Fungicide resistance of Botrytis cinerea from strawberry to procymidone and zoxamide in Hubei, China

“…The most predominant point mutation found in B. cinerea resistant isolates in Hubei Province to carbendazim was E198V (Fan et al 2017). E198V mutation is not related with zoxamide resistance (Dehne et al 2004;Malandrakis et al 2011;Cai et al 2015). In our findings, E198V point mutation was not detected among the zoxamide resistant isolates, indicating that zoxamide is a good alternative to control MBC resistant isolates with point mutation E198V.…”

“…Among eight B. cinerea sensitive isolates subjected to mycelial adaptation to zoxamide-amended medium, only one was mutated after ten transfers on zoxamide-containing media. The resulting two mutants showed significantly reduced growth and lack of sporulation (Cai et al 2015). Molecular docking analysis showed the formation of two strong hydrogen bonds between side-chain of E198 and carbendazim, but zoxamide failed to form such interactions, which could explain why mutations at codon E198 have less effect on the sensitivity of zoxamide as compared to carbendazim.…”

“…Molecular docking analysis showed the formation of two strong hydrogen bonds between side-chain of E198 and carbendazim, but zoxamide failed to form such interactions, which could explain why mutations at codon E198 have less effect on the sensitivity of zoxamide as compared to carbendazim. Authors proposed that difference in the binding-pocket of zoxamide and its target sites on BctubA protein make it different from MBCs (Cai et al 2015). In inner Mongolia and Liaoning, 84% of B. cinerea isolates were found to be resistant to carbendazim (Cai et al 2015).…”

“…Authors proposed that difference in the binding-pocket of zoxamide and its target sites on BctubA protein make it different from MBCs (Cai et al 2015). In inner Mongolia and Liaoning, 84% of B. cinerea isolates were found to be resistant to carbendazim (Cai et al 2015). High frequency of B. cinerea resistant isolates to carbendazim was also detected in Hubei Province (Fan et al 2017).…”

“…However, the situation was different for zoxamide Bi et al 2011;Malandrakis et al 2011;Bi et al 2014). Compared to the high frequency of MBCs resistant isolates, the frequency of Zox R isolates was low,which might be because the two fungicides are binding to different target sites of β-tubulin (Cai et al 2015).…”