Detection and fitness comparison of target-based highly fludioxonil-resistant isolates of Botrytis cinerea from strawberry and cucumber in China.

Botrytis cinerea has a high risk of developing resistance to fungicides. Fludioxonil, belonging to phenylpyrroles, has been used for more than three decades, however, only few cases of field resistance against phenylpyrroles have been reported. In this study, the highly fludioxonil-resistant (HR) isolates of Botrytis cinerea were firstly detected in the commercial greenhouses of strawberry in China in 2015, and biochemical characterization differences in high fludioxonil-resistance from strawberry and cucumber were compared. All of the five HR isolates from greenhouses of strawberry and cucumber could grow on PDA amended with 100 µg/mL fludioxonil, and exhibited a positive correlation between the resistance of dicarboximide fungicides and fludioxonil. Sporulation and sclerotium production of the strawberry-originated HR isolates were increased in comparison with the cucumber-originated HR isolates. No matter how the HR isolates were from strawberry and cucumber, all the HR isolates showed enhanced sensitivity to the osmotic agents, but with significant difference. Based on sequence alignment of the BcOS1 which codes protein bound by fludioxonil, two genotypes of the strawberry-originated HR isolates were observed, i.e., (F127S + I365N + S426P) and (G538R + A1259T), which were totally different from those of the cucumber-originated HR isolates. Molecular docking of fludioxonil to the binding site of BcOS1 protein from the five HR isolates illustrated that all the HR isolates had less affinity than the sensitive isolates. Our data indicated that genotypes of the HR isolates match the corresponding fludioxonil-selection pressure on the field populations of B. cinerea in the commercial greenhouses of the two host plants.

Resistance risk assessment of Fusarium oxysporum f. sp. melonis against phenamacril, a myosin inhibitor.

Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (FOM) is one of the most notorious seed-borne diseases worldwide. Phenamacril is a cyanoacrylate fungicide with novel chemical structure and strong inhibitive activity against FOM. To evaluate the risk of FOM developing phenamacril resistance, five phenamacril-resistant mutants with >800µgml-1 minimum inhibitory concentration were obtained by repeated exposure to the fungicide in the laboratory. Compared with the parental isolate, four of the five phenamacril-resistant mutants showed enhanced biological fitness in sporulation and virulence, but not in sensitivity to various stresses (oxidative and osmotic pressure, cell membrane and wall inhibitor). No positive cross-resistance was observed among phenamacril and the other five fungicides, including azoxystrobin, carbendazim, boscalid, fluazinam and tebuconazole. Sequencing alignment results of the myosin 5 from the five resistant mutants and the parental strain indicated that the three resistant mutants fo-2, fo-3 and fo-4 had a single point mutation (S175L), which may confer the resistance of FOM against phenamacril. Interestingly, the resistant mutant fo-4 harbored not only one mutation (S175L) at myosin 5, but also the other mutation (A52G) at ß2-tublin. Our data supported that resistance risk of Fusarium oxysporum f. sp. melonis against phenamacril was between the moderate to high level.

The Autophagy Gene BcATG8 Regulates the Vegetative Differentiation and Pathogenicity of Botrytis cinerea.

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 (ΔBcAtg8 mutant) blocked autophagy and significantly impaired mycelial growth, conidiation, sclerotial formation, and virulence. In addition, the conidia of the ΔBcAtg8 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. cinereaIMPORTANCE 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.

Ubiquitin-like activating enzymes BcAtg3 and BcAtg7 participate in development and pathogenesis of Botrytis cinerea.

In eukaryotes, the ubiquitin-like (UBL) protein-activating enzymes play a crucial role in autophagy process, however, it is poorly characterized in filamentous fungi. Here, we investigated the functions of two UBL activating enzymes, BcAtg3 (E2) and BcAtg7 (E1) in the plant pathogenic fungus Botrytis cinerea. The physical interaction of BcAtg3 with BcAtg7 was demonstrated by yeast two-hybrid system. Subcellular localization assays showed that BcAtg3 diffused in cytoplasm, and BcAtg7 localized in cytoplasm as pre-autophagosomal structures (PAS). Target gene deletion experiments revealed that both BcATG3 and BcATG7 are essential for autophagy pathway. Notably, the single deletion mutant of BcATG3 and BcATG7 displayed similar biological phenotypes, including the defects in mycelial growth, conidiation and sclerotial formation. Infection tests showed that both BcATG3 and BcATG7 were required for full virulence of B. cinerea. All of these defective phenotypes were rescued by gene complementation. These results indicate that BcATG3 and BcATG7 are necessary for autophagy to regulate fungal development and pathogenesis in B. cinerea.