Sharp eyespot is a crucial disease affecting cereal plants, such as bread wheat (Triticum aestivum) and barley (Hordeum vulgare), and is primarily caused by the pathogenic fungus Rhizoctonia cerealis. As disease severity has increased, it has become imperative to find an effective and reasonable control strategy. One such strategy is the use of the trehalose analog, validamycin, which has been shown to have a potent inhibitory effect on several trehalases found in both insects and fungi, and is widely used as a fungicide in agriculture. In this study, we demonstrated that 0.5 μg/mL validamycin on PDA plates had an inhibitory effect on R. cerealis strain R0301, but had no significant impact on Fusarium graminearum strain PH-1. Except for its inhibiting the trehalase activity of pathogenic fungi, little is known about its mechanism of action. Six trehalase genes were identified in the genome of R. cerealis, including one neutral trehalase and five acidic trehalase genes. Enzyme activity assays indicated that treatment with 5 μg/mL validamycin significantly reduces trehalase activity, providing evidence that validamycin treatment does indeed affect trehalase, even though the expression levels of most trehalase genes, except Rc17406, were not obviously affected. Transcriptome analysis revealed that treatment with validamycin downregulated genes involved in metabolic processes, ribosome biogenesis, and pathogenicity in the R. cerealis. KEGG pathway analysis further showed that validamycin affected genes related to the MAPK signaling pathway, with a significant decrease in ribosome synthesis and assembly. In conclusion, our results indicated that validamycin not only inhibits trehalose activity, but also affects the ribosome synthesis and MAPK pathways of R. cerealis, leading to the suppression of fungal growth and pesticidal effects. This study provides novel insights into the mechanism of action of validamycin.

GDP-mannose transporters (GMTs) have been implicated in the virulence of some important pathogenic fungi, and guanosine diphosphate (GDP) mannose transporters transport GDP-mannose from the cytosol to the Golgi lumen prior to mannosylation, where mannose attaches to the modified protein. GMTs could be potential targets for new antifungal drugs, as disruption of any step in GDP-mannose biosynthesis can affect fungal viability, growth, or virulence. To date, the GDP-mannose transporter has been extensively studied in yeast, but its biological function in fungi, particularly F. graminearum, is still unclear. In this experimental study, the role of the GDP-mannose transporter in F. graminearum was investigated by analysing the VRG4 gene. FgGmtA and FgGmtB were blastp-derived from their Scvrg4 protein sequences and proved to be their functional homologues. The mutant and complementary strains of FgGmtA, FgGmtB and FgGmtA&B genes were generated and used to evaluate the effect of the two GMTs genes on mycelial growth, asexual reproduction, sexual reproduction, cell wall sensitivity, glyphosate synthesis and drug susceptibility. Only in the FgGmtB and FgGmtA&B mutants was the rate of mycelial growth slowed, conidium production increased, sexual reproduction impaired, cell wall sensitivity increased, glycemic content decreased, and drug sensitivity reduced. The results of the pathogenicity assessment of GMTs showed that only FgGmtB affects the patogenicity of F. graminearum. At the same time, the effect of GMTs on the ability of rhinoceros to synthesise DON toxins was investigated and the results showed that the ability of ΔFgGmtB and ΔFgGmtA&B mutants to produce the DON toxin was significantly reduced, and the expression of toxin-related genes was also reduced.