Validamycin A (VMA) is an aminoglycoside antibiotic used to control rice sheath blight. Although it has been reported that VMA can induce the plant defense responses, the mechanism remains poorly understood. Here we found that reactive oxygen species (ROS) bursts and callose deposition in Arabidopsis thaliana, rice (Oryza sativa L.) and wheat (Triticum aestivum L.) were induced by VMA and were most intense with 10 μg mL-1 of VMA at 24 h. Moreover, we showed that VMA induced resistance against Pseudomonas syringae, Botrytis cinerea, and Fusarium graminearum in Arabidopsis leaves, indicating that VMA induces broad-spectrum disease resistance in both dicots and monocots. Furtherly, VMA-mediated resistance against P. syringae was not induced in NahG transgenic plants, partially decreased in npr1 mutants, and VMA-mediated resistance to B. cinerea was not induced in npr1, jar1, and ein2 mutants. These results strongly indicated that VMA triggers plant defense responses to both biotrophic and necrotrophic pathogens involving in salicylic acid (SA), jasmonic acid/ ethylene (JA/ET) signaling pathway and dependent on NPR1. In addition, transcriptome analysis further revealed that VMA regulated the expressions of genes involved in SA, JA/ET, abscisic acid (ABA) and Auxin signal pathways. Taken together, VMA induces systemic resistance involving in SA, and JA/ET signaling pathways, and also exerts a positive influence on ABA and Auxin signaling pathways. Our study highlighted the creative application of VMA in triggering plant defense responses against plant pathogens, providing a valuable insight into applying VMA to enhance plant resistance and reduce the use of chemical pesticides.

Pydiflumetofen is a novel succinate dehydrogenase inhibitor fungicide. In the current research, we determined the sensitivity of 166 Sclerotinia sclerotiorum strains to pydiflumetofen using mycelial growth inhibition method. The results suggest that pydiflumetofen exhibited a strong inhibitory activity against S. sclerotiorum and the EC50 values ranged from 0.0058 to 0.0953 μg ml−1, with a mean EC50 value of 0.0250 μg ml−1. However, the baseline sensitivity was not normally distributed because of a high variation factor. After treatment with pydiflumetofen, cell membrane permeability increases, but exopolysaccharide and oxalic acid production decreases, which contributes to reduced virulence of S. sclerotiorum and leads to failure of disease infection. In addition, protective and curative activity was performed on detached oilseed rape leaves by artificial inoculation. Pydiflumetofen exhibited excellent protective and curative effects against S. sclerotiorum on oilseed rape, and the protective effect was better than the curative effect. Further, field trials were conducted to evaluate the potential of pydiflumetofen in controlling Sclerotinia stem rot (SSR) caused by S. sclerotiorum on oilseed rape. Compared with the currently used fungicides, pydiflumetofen not only exhibited excellent control efficacy against SSR, but also dramatically reduced the dosage of fungicides in the field. Thus, this study provides important references for revealing pharmacological mechanism of pydiflumetofen against S. sclerotiorum and managing SSR on oilseed rape caused by benzimidazole- and dicarboximide-resistant populations.

Deoxynivalenol (DON) is a vital virulence factor of Fusarium graminearum, which causes Fusarium head blight (FHB). We recently found that validamycin A (VMA), an aminoglycoside antibiotic, can be used to control FHB and inhibit DON contamination, but its molecular mechanism is still unclear. In this study, we found that both neutral and acid trehalase (FgNTH and FgATH) are the targets of VMA in F. graminearum, and the deficiency of FgNTH and FgATH reduces the sensitivity to VMA by 2.12‐ and 1.79‐fold, respectively, indicating that FgNTH is the main target of VMA. We found FgNTH is responsible for vegetative growth, FgATH is critical to sexual reproduction, and both of them play an important role in conidiation and virulence in F. graminearum. We found that FgNTH resided in the cytoplasm, affected the localization of FgATH, and positively regulated DON biosynthesis; however, FgATH resided in vacuole and negatively regulated DON biosynthesis. FgNTH interacted with FgPK (pyruvate kinase), a key enzyme in glycolysis, and the interaction was reduced by VMA; the deficiency of FgNTH affected the localization of FgPK under DON induction condition. Strains with a deficiency of FgNTH were more sensitive to demethylation inhibitor (DMI) fungicides. FgNTH regulated the expression level of FgCYP51A and FgCYP51B by interacting with FgCYP51B. Taken together, VMA inhibits DON biosynthesis by targeting FgNTH and reducing the interaction between FgNTH and FgPK, and synergizes with DMI fungicides against F. graminearum by decreasing FgCYP51A and FgCYP51B expression.

The high osmolarity glycerol (HOG) pathway, comprising a two-component system and the Hog1 MAPK cascade, plays a pivotal role in eukaryotic organisms. Previous studies suggested that the biological functions of some key genes in the HOG pathway varied in filamentous fungi. In this study, we characterized a putative mitogen activated kinase (MAPK) kinase kinase gene Ssos4 in S. sclerotiorum, which encoded a phosphotransferase in the MAPK cascade. Compared to the wild-type progenitor HA61, the deletion mutant ∆Ssos4-63 exhibited impaired mycelial growth, sclerotia formation, increased hyphal branches and decreased virulence. The deficiencies of the deletion mutant ∆Ssos4-63 were recovered when the full-length Ssos4 gene was complemented. Deletion of Ssos4 increased the sensitivity to osmotic stresses, cell wall agents and the resistance to fludioxonil and dimethachlon. Intracellular glycerol accumulation was not induced in the deletion mutant ∆Ssos4-63 when treated with fludioxonil and NaCl and the phosphorylation of SsHog1 was also cancelled by the deletion of Ssos4. Consistent with the glycerol accumulation, increased expression levels of SsglpA and Ssfps1, controlling glycerol synthesis and close of glycerol channel under hyperosmotic stress respectively, were detected in the wild type stain HA61 but not in the deletion mutant ∆Ssos4-63. Moreover, the relative expression level of Sshog1 significantly decreased, whereas the expression level of Ssos5 increased in the deletion mutant ∆Ssos4-63. These results indicated that SsOs4 played important roles in mycelial growth and differentiation, sclerotia formation, virulence, hyperosmotic adaptation, fungicide sensitivity and the phosphorylation of SsHog1 in S. sclerotiorum.

Cucumber target spot, caused by Corynespora cassiicola, is a devastating fungal disease in greenhouses in China. Lack of resistant cultivars and unscientific use of fungicides aggravated the difficulty to manage this disease. In recent years, resistance of C. cassiicola to benzimidazoles, quinone outside inhibitors, and succinate dehydrogenase inhibitors has occurred in China. Here, we tested the fluazinam sensitivity distribution of 79 C. cassiicola isolates from different provinces in China based on mycelial growth inhibition. The EC50 values of fluazinam ranged from 0.1002 to 0.3129 µg/ml with a mean of 0.2136 ± 0.0495 µg/ml, and the sensitivity frequency was normally distributed (P = 0.2083, Shapiro–Wilk test). Meanwhile, the EC50 values for spore germination inhibition ranged from 0.0992 to 0.2278 µg/ml with a mean of 0.1499 ± 0.0504 µg/ml. This indicated that fluazinam exhibited an excellent in vitro fungicidal activity on both mycelial growth and spore germination. In addition, fluazinam also exhibited a good in planta control efficacy on detached cucumber leaves in the protective and curative assays. Moreover, the biological and physiological characteristics of C. cassiicola as affected by fluazinam were determined. Fluazinam not only significantly inhibited respiration and adenosine triphosphate production but also caused the increase of cell membrane permeability and the dysfunctions of cellular homeostasis. Interestingly, we found that fluazinam especially damaged vacuole structures, causing the redistribution of vacuole substances. Taken together, our findings provide not only essential references for resistance management of C. cassiicola but also interesting insights for further revealing the action mode of fluazinam against plant pathogens.