Cucumber target leaf spot caused
by Corynespora
cassiicola has devastated greenhouse cucumber production.
In our previous study, the resistance monitoring of C. cassiicola to carbendazim was carried out, and
a large number of resistant populations carrying various mutations
(M163I&E198A, F167Y&E198A, F200S&E198A, or E198A) in β-tubulin were detected. However, the single-point mutations
M163I, F167Y, and F200S have remained undetected. To investigate the
evolutionary mechanism of double mutations in β-tubulin of C. cassiicola resistance to benzimidazoles,
site-directed mutagenesis was used to construct alleles with corresponding
mutation genotypes in β-tubulin. Through PEG-mediated
protoplast transformation, all the mutants except for the M163I mutation
were obtained and conferred resistance to benzimidazoles. It was found
that the mutants conferring the E198A or double-point mutations showed
high resistance to carbendazim and benomyl, but the mutants conferring
the F167Y or F200S mutations showed moderate resistance. Except, the
F200S mutants showed low resistance, the resistance level of the other
mutants to thiabendazole seemed no difference. In addition, compared
to the other mutants, the F167Y and F200S mutants suffered a more
severe fitness penalty in mycelial growth, sporulation, and virulence.
Thus, combined with the resistance level, fitness, and molecular docking
results, we concluded that the field double mutations (F167Y&E198A
and F200S&E198A) evolved from the single mutations F167Y and F200S,
respectively.