Botrytis cinerea is a filamentous phytopathogen with a high risk of developing resistance to fungicides. The phenylpyrrole fungicide fludioxonil has been reported to have excellent activity against B. cinerea and increasingly has been applied to control gray mold in China. In this study, molecular and biochemical characteristics of laboratory and field mutants of B. cinerea resistant to fludioxonil has been investigated. During 2012 to 2014, B. cinerea isolates collected from Jiangsu and Shandong Provinces in China were tested in vitro for sensitivity to fungicides commonly used to suppress gray mold of cucumber and tomato. Among the 75 isolates collected from cucumber in 2013, two were highly resistant (HR) to fludioxonil. Of the 308 isolates collected from tomato in 2014, four were fludioxonil-HR. This was the first time that B. cinerea isolates HR to fludioxonil had been detected in the field. Six fludioxonil-resistant mutants were obtained in the laboratory by selection on fungicide-amended media. These mutants exhibited stable resistance to fludioxonil, as indicated by resistance factor values that ranged from 34.38 to >10,000. In comparison with fludioxonil-sensitive isolates of B. cinerea, all field and laboratory mutants showed reduced fitness, as defined by mycelial growth, sporulation, virulence, and sensitivity to osmotic stress. When treated with fludioxonil at 1 μg/ml, sensitive isolates showed increased glycerol contents in mycelium and expression levels of Bchog1, while levels in field and laboratory HR mutants increased only slightly. Sequences of the Bos1 gene of field and laboratory fludioxonil-HR mutants showed that mutations in field mutants were located in the histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase (HAMP) domains of the N-terminal region, whereas mutations in the laboratory mutants were distributed in HAMP domains or in the HATPase_c domain of the C-terminal region. These results will enhance our understanding of the resistance mechanism of B. cinerea to fludioxonil.

Chili anthracnose caused by Colletotrichum spp. is an annual production concern for growers in China. Sterol C14-demethylation inhibitors (DMIs, such as tebuconazole) have been widely used to control this disease for more than three decades. In the current study, of 48 isolates collected from commercial chili farms in Jiangsu Province of China during 2018 and 2019, 8 single-spore isolates were identified as Colletotrichum gloeosporioides and the rest were identified as C. acutatum. To determine whether the DMI resistance of isolates develops in the field, mycelial growth of the 48 isolates was measured in culture medium with and without tebuconazole. In all, 6 of the 8 C. gloeosporioides isolates were resistant to tebuconazole, but all 40 of the C. acutatum isolates were sensitive to tebuconazole. The fitness cost of resistance was low based on a comparison of fitness parameters between the sensitive and resistant isolates of C. gloeosporioides. Positive cross-resistance was observed between tebuconazole and difenconazole or propiconazole, but not prochloraz. Alignment results of the CgCYP51 amino acid sequences from the sensitive and resistant isolates indicated that mutations can be divided into three genotypes. Genotype I possessed four substitutions (V18F, L58V, S175P, and P341A) at the CgCYP51A gene but no substitutions at CgCYP51B, while genotype II had five substitutions (L58V, S175P, A340S, T379A, and N476T) at CgCYP51A, concomitant with three substitutions (D121N, T132A, and F391Y) at CgCYP51B. In addition, genotype III contained two substitutions (L58V and S175P) at CgCYP51A, concomitant with one substitution (T262A) at CgCYP51B. Molecular docking models illustrated that the affinity of tebuconazole to the binding site of the CgCYP51 protein from the resistant isolates was decreased when compared with binding site affinity of the sensitive isolates. Our findings provide not only novel insights into understanding the resistance mechanism to DMIs, but also some important references for resistance management of C. gloeosporioides on chili.

Mutantions in ß2-tubulin relate to the MBC-resistance of Fusarium fujikuroi.

Autophagy is a conserved degradation process that maintains intracellular homeostasis to ensure normal cell differentiation and development in eukaryotes. ATG8 is one of the key molecular components of the autophagy pathway. In this study, we identified and characterized BcATG8 , a homologue of Saccharomyces cerevisiae (yeast) ATG8 in the necrotrophic plant pathogen Botrytis cinerea . Yeast complementation experiments demonstrated that BcATG8 can functionally complement the defects of the yeast ATG8 null mutant. Direct physical interaction between BcAtg8 and BcAtg4 was detected in the yeast two-hybrid system. Subcellular localization assays showed that green fluorescent protein-tagged BcAtg8 (GFP-BcAtg8) localized in the cytoplasm as preautophagosomal structures (PAS) under general conditions but mainly accumulated in the lumen of vacuoles in the case of autophagy induction. Deletion of BcATG8 (Δ BcAtg8 mutant) blocked autophagy and significantly impaired mycelial growth, conidiation, sclerotial formation, and virulence. In addition, the conidia of the Δ BcAtg8 mutant contained fewer lipid droplets (LDs), and quantitative real-time PCR (qRT-PCR) assays revealed that the basal expression levels of the LD metabolism-related genes in the mutant were significantly different from those in the wild-type (WT) strain. All of these phenotypic defects were restored by gene complementation. These results indicate that BcATG8 is essential for autophagy to regulate fungal development, pathogenesis, and lipid metabolism in B. cinerea . IMPORTANCE The gray mold fungus Botrytis cinerea is an economically important plant pathogen with a broad host range. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. Exploring the fundamental biology of B. cinerea can provide the theoretical basis for sustainable and long-term disease management. Autophagy is an intracellular process for degradation and recycling of cytosolic materials in eukaryotes and is now known to be vital for fungal life. Here, we report studies of the biological role of the autophagy gene BcATG8 in B. cinerea . The results suggest that autophagy plays a crucial role in vegetative differentiation and virulence of B. cinerea .