Evolution of Benzimidazole Resistance Caused by Multiple Double Mutations of β-Tubulin in Corynespora cassiicola

Abstract

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.

Resistance Risk and Molecular Mechanism of Tomato Wilt Pathogen Fusarium oxysporum f. sp. lycopersici to Pyraclostrobin

“…The genomic DNA of the wild-type strains and resistant mutants was extracted by the CTAB method, as previously described. 14 The Cytb fragment (1985 bp, containing the complete coding sequence) was amplified using primers FoCytb-F (5′-ATGAGAATATTAAAAAATCACCCTTTATT-3′) and FoCytb-R (5′-TTATATATTTTTGGCATTATTTTGTAATTCT-3′). The amplification was performed with 2× Hieff PCR Master Mix (Yeasen, Shanghai) according to the protocol.…”

Expanding the Structural Diversity of Tubulin-Targeting Agents: Development of Highly Potent Benzimidazoles for Treating Fungal Diseases

“…As a crucial component of the cytoskeleton, microtubules are mainly formed by the polymerization of heterodimers of α-tubulin and β-tubulin. , Some literature indicates that microtubules are involved in the proliferation, transport and signaling of cells, as well as chromosome segregations during cell division. − One of the most representative examples of concern is tubulin-targeting fungicides (e.g., carbendazim, benomyl), which have been developed and extensively used. However, due to their simple molecular structure and single site of action, plant pathogenic fungi have developed severe resistance. − Therefore, the development of novel, potent antifungal agents specifically targeting microtubules is a crucial strategy for the prevention and controlling of plant fungal diseases.…”