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.
Highlights
Botrytis cinerea is a common airborne plant pathogen that causes serious pre- and post-harvest losses on at least 200 crops worldwide[1]
Based on a colony growth assay on potato dextrose agar (PDA) containing different concentrations of zoxamide or carbendazim, three phenotypes were detected among 161 B. cinerea field isolates obtained from 26 locations in China (Table 2)
In contrast with the high resistance risk of benzimidazoles, which is a severe problem for many plant pathogens worldwide[4,12,14,22,25], the resistance risk developing to zoxamide is defined as low[33], according to the uncommon appearance of resistant isolates in field and the low mutagenesis frequency in lab
Summary
Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a common airborne plant pathogen that causes serious pre- and post-harvest losses on at least 200 crops worldwide[1]. During the last 30 years, gray mold control in China has mainly depended on the application of systemic fungicides with single-site modes of action. These systemic fungicides (which include the benzimidazole fungicide carbendazim, the quinone outside inhibitor azoxystrobin, and the sterol biosynthesis inhibitors tebuconazole and prochloraz) are often mixed with the protective fungicides thiram, chlorothalonil, procymidone, and pyrimethanil[2,3,4,5]. Six different amino acid substitutions in conserved regions of β -tubulin were found in moderately zoxamide-resistant isolates with wild-type sensitivity to benzimidazoles, their contributions to the occurrence and development of zoxamide resistance have still been unclear[14]. The objectives of the study were to: i) determine the baseline sensitivity of B. cinerea to zoxamide; ii) generate zoxamide-resistant mutants with wild-type sensitivity to carbendazim, and characterize their fitness components; iii) investigate the molecular mechanism of zoxamide resistance in B. cinerea, and clarify how the point mutations might affect the binding of zoxamide and carbendazim by constructing docking models; iv) develop a rapid and reliable method for detection of zoxamide-resistant isolates in populations of B. cinerea
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