Impact of fluazinam on morphological and physiological characteristics of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a necrotrophic and filamentous fungus with a broad host range. Fluazinam is a pyridinamine fungicide with a broad spectrum of antifungal activity and had a strong inhibition effect on mycelial growth of S. sclerotiorum populations. But the impact of fluazinam on morphological and physiological characteristics of S. sclerotiorum is little known. In this study, the EC50 values of fluazinam to three strains of S. sclerotiorum (CZ17S, YZ55S and SA42S) were 0.0084, 0.007, 0.0065 µg/ml respectively. After fluazinam treatment, hyphae of S. sclerotiorum became thinner, hyphal offshoot of top increased, the distance between one septum and another became shorter, cell membrane permeability increased markedly, exopolysaccharide (EPS) content and oxalic acid content decreased significantly, peroxidase (POD) activity increased significantly and mycelial respiration was inhibited. While the number and dry weight of sclerotia, glycerol content in the mycelia did not significantly change. In protective activity assay on detached rapeseed leaves, application of fluazinam at 40 µg/ml and 80 µg/ml, the control efficacy reached to 41.4% and 100%, respectively. In curative activity assay, application of fluazinam at 100 µg/ml, the control efficacy reached to 61.09%. In the same concentration, protective activity of fluazinam against S. sclerotiorum was higher than curative activity. These results will contribute to us on evaluating the potential of the fungicide fluazinam for management of Sclerotinia stem rot and understanding the mode of action of fluazinam against S. sclerotiorum.

Activity of the dinitroaniline fungicide fluazinam against Bipolaris maydis.

Fluazinam is a dinitroaniline fungicide with broad-spectrum activities. However, the activity of fluazinam against Bipolaris maydis which is the causal agent of southern corn leaf blight is unknown yet. In this study, baseline sensitivity of B. maydis to fluazinam was determined using 92 isolates collected during 2015 and 2016 from different geographical regions in Jiangsu Province of China, and the EC50 values ranged from 0.0396 to 0.9808 µg/ml with average value of 0.3853 ±â€¯0.2297 µg/ml, and 0.079 to 0.7832 µg/ml with average value of 0.3065 ±â€¯0.1384 µg/ml for mycelial growth and conidium germination respectively. Fluazinam did not affect the distribution of cell nucleus and the formation of septum of B. maydis. However, fluazinam could make mycelium of B. maydis contorted and the mycelial branches increased and inhibit the development of conidia. The result of transmission electron microscope showed that fluazinam damaged cell wall and cell membrane of mycelium, and make organelles in mycelial cell dissolved and vacuolated, and the cell almost broke up, which caused the intracellular plasma leakage increase. The protective activity test of fluazinam suggested that fluazinam had great control efficiency against B. maydis on detached corn leaves. Application of fluazinam at 10 µg/ml and 20 µg/ml, the control efficacy reached to 87.70% and 98.25% respectively. However, fluazinam had no curative activity against B. maydis on detached corn leaves. These results will contribute to us on evaluating the potential of the dinitroaniline fungicide fluazinam for management of diseases caused by B. maydis and understanding the mode of action of fluazinam against B. maydis.

Molecular and biological characterization of Sclerotinia sclerotiorum resistant to the anilinopyrimidine fungicide cyprodinil.

Cyprodinil belongs to the chemical class of anilinopyrimidines fungicides. In this study, baseline sensitivity of Sclerotinia sclerotiorum (Lib.) de Bary to cyprodinil was determined using 100 strains collected from the fields in Jiangsu Province of China. The EC50 (50% effective concentration) values ranged from 0.0636-0.8163 µg/ml with a mean value of 0.1869 (±0.1118) ug/ml for mycelial growth. Nine cyprodinil-resistant mutants (Range of resistance factor: 20.22-271.59) were obtained from sensitive strains exposed on PDA medium amended with cyprodinil and the resistance was stable after their ten transfers on PDA without the fungicide or stored at 4 °C for two months. There was positive cross-resistance between cyprodinil and pyrimethanil but not to fludioxonil, dimetachlone, procymidone, carbendazim and boscalid in S. sclerotiorum. Compared with the parental strains, all of the nine cyprodinil-resistant mutants decreased in sclerotial production. The dry weight of mycelia, pathogenicity and cell membrane permeability of most resistant mutants decreased. The mycelial growth, oxalic acid content, and the response to various stress for resistant mutants were almost the same as the sensitive parental strains. Sequencing alignment results showed that there was no alteration of amino acid in cystathionine γ-synthase (MetB) and cystathionine ß-lyase (MetC) between cyprodinil-resistant mutants and their sensitive parental strains, which indicated that MetB or MetC was not the molecular target of cyprodinil in S. sclerotiorum. The addition of amino acids L-methionine, L-cystine or L-cysteine decreased the inhibition of cyprodinil against mycelial growth of S. sclerotiorum, which indicated that cyprodinil could not only inhibited methionine biosynthesis but also suppressed cystine and cysteine biosynthesis. These results will contribute to evaluating the resistance risk of cyprodinil for management of the plant diseases of Sclerotinia stem rot caused by S. sclerotiorum and further increase our understanding about the mode of action of cyprodinil.

Resistance risk assessment for fluazinam in Sclerotinia sclerotiorum.

In the current study, sensitivity distribution of Sclerotinia sclerotiorum populations to fluazinam was determined using 103 strains collected from the fields of Jiangsu Province of China in 2016-2017 and the resistance risk of fluazinam was assessed. The average EC50 (50% effective concentration) values and MIC (minimum inhibitory concentration) values of 103 S. sclerotiorum strains against fluazinam were 0.0073±0.0045µg/ml and <0.3µg/ml for mycelial growth, respectively. Nine mutants with low resistance level were obtained from wild type sensitive strains exposed on PDA medium amended with fluazinam and the resistance was stable after their ten transfers on PDA without the fungicide. Compared with the parental strains, the nine fluazinam-resistant mutants decreased in mycelial growth, sclerotial production, pathogenicity and were more sensitive to 0.7M NaCl. In addition, cell membrane permeability of resistant mutants was higher than that of their parental strains. Cross resistance assay showed that there was no cross-resistance between fluazinam and fludioxonil, dimetachlone, prochloraz, tebuconazole, azoxystrobin, or procymidone in S. sclerotiorum. The above results indicated that there was a low resistance risk for fluazinam in S. sclerotiorum. However, the sensitivity of all fluazinam-resistant mutants to fludioxonil decreased. Sequencing alignment results showed that there were no mutations in the two-component histidine kinase gene (Shk1) of the resistant mutants and the expression levels of Shk1 of three resistant mutants were significantly up-regulated while others were almost the same as their parental strains. These results will contribute to evaluating the resistance risk of fluazinam for management of diseases caused by S. sclerotiorum and further increase our understanding about the mode of action of fluazinam.

Activity of a novel succinate dehydrogenase inhibitor fungicide pyraziflumid against Sclerotinia sclerotiorum.

Pyraziflumid is a novel member of succinate dehydrogenase inhibitor fungicides (SDHI). In this study, baseline sensitivity of Sclerotinia sclerotiorum (Lib.) de Bary to pyraziflumid was determined using 105 strains collected during 2015 and 2017 from different geographical regions in Jiangsu Province of China, and the average EC50 value was 0.0561 (±0.0263)µg/ml for mycelial growth. There was no cross-resistance between pyraziflumid and the widely used fungicides carbendazim, dimethachlon and the phenylpyrrole fungicide fludioxonil. After pyraziflumid treated, hyphae were contorted with offshoot of top increasing, cell membrane permeability increased markedly, oxalic acid content significantly decreased and mycelial respiration was strongly inhibited. But the number and dry weight of sclerotia did not change significantly. The protective and curative activity test of pyraziflumid suggested that pyraziflumid had great control efficiency against S. sclerotiorum on detached rapeseed leaves, and protective activity was better than curative activity. These results will contribute to us on evaluating the potential of the new SDHI fungicide pyraziflumid for management of diseases caused by S. sclerotiorum and understanding the mode of action of pyraziflumid against S. sclerotiorum.

Crucial Role of the Ca2+/CN Signaling Pathway in the Antifungal Activity of Seselin against Botrytis cinerea.

Botrytis cinerea causes gray mold in many fruit and vegetable crops. We previously found that Seselin (SL) displayed antifungal activity against B. cinerea (EC50 = 6.1 µg·mL-1), and this study investigated the effects of Ca2+ and the Ca2+/CN signaling pathway on its antifungal activity against B. cinerea. The results indicated that exogenous Ca2+, Cyclosporine A, and Verapamil reduced the sensitivity of SL against B. cinerea; SL significantly reduced the intracellular Ca2+ concentration in the hyphae; the sensitivity of strains ΔbcCCH1 and ΔbcMID1 to SL were significantly increased; and the expressions of CCH1, MID1, CNA, PMC1, and PMR1 genes of the Ca2+/CN signaling pathway were significantly downregulated by SL treatment. Hence, SL is a potential compound for developing fungicides against B. cinerea. SL dramatically reduces intracellular Ca2+ concentration and disturbs Ca2+ homeostasis, leading to cell death. The Ca2+/CN signaling pathway plays an important role in the antifungal activity of SL against B. cinerea.

Crucial role of Ca2+ /CN signalling pathway in the antifungal activity of disenecioyl-cis-khellactone against Botrytis cinerea.

BACKGROUND: Botrytis cinerea causes grey mould and is one of the most destructive fungal pathogens affecting important fruit and vegetable crops. In preliminary studies, we found that disenecioyl-cis-khellactone (DK) had strong antifungal activity against several fungi species including B. cinerea [half maximal effective concentration (EC50 ) = 11.0 µg mL-1 ]. In this study, we aimed to further evaluate the antifungal activity of DK against B. cinerea and determine the role of calcium ion/calcineurin (Ca2+ /CN) signalling pathway on its antifungal effect. RESULTS: DK was effective against B. cinerea in both in vitro and in vivo assays. Exogenous Ca2+ reduced the antifungal activity of DK. The combination of DK and cyclosporine A (CsA) did not exhibit an additive effect against B. cinerea. In contrast to CsA, DK reduced the intracellular Ca2+ concentration in B. cinerea. DK bound to calcineurin A (cnA) and up-regulated the expression of PMC1 and PMR1 genes. Moreover, DK sensitivity of △bccnA significantly decreased compared with that of Bc05.10 strain. CONCLUSION: DK is a promising lead compound for developing fungicides against B. cinerea. The Ca2+ /CN signalling pathway plays a crucial role in the DK antifungal activity, and cnA is one of the targets of DK against B. cinerea. DK directly reacts with cnA, which up-regulates the transcription of Ca2+ /CN-dependent target genes PMC1 and PMR1, decreasing the intracellular Ca2+ concentration and disturbing the intracellular Ca2+ balance, leading to cell death. © 2022 Society of Chemical Industry.

Resistance mechanism of Fusarium fujikuroi to phenamacril in the field.

BACKGROUND: Rice bakanae disease, mainly caused by Fusarium fujikuroi, is an important disease of rice. Phenamacril has been used to control the disease for a few years in China. In 2016, nine phenamacril-resistant strains were found in the field in Zhejiang Province. The aim of the study was to clarify the mechanism of resistance of F. fujikuroi to phenamacril and the fitness of resistant strains. RESULTS: The nine F. fujikuroi strains examined were highly resistant to phenamacril. Eight of them had the point mutation TCA (Ser) → CCA (Pro) at codon 219 in the Myosin-5 protein, while the other had the point mutation TCA (Ser) → TTA (Leu) at codon 219. Myosin-5 replacement between resistant and sensitive strains confirmed that the point mutation in Myosin-5 caused the resistance of F. fujikuroi to phenamacril. Docking of phenamacril into the modeled binding pocket of Myosin-5 showed that the affinity between phenamacril and Myosin-5 decreased and a hydrogen bond could not be formed between phenamacril and the amino acid at codon 219 after it changed to Pro or Leu. There was no cross-resistance between phenamacril and other fungicides. The eight resistant strains containing the point mutation S219P had almost the same fitness as the sensitive strains, while the one resistant strain containing the point mutation S219 L showed decreased mycelial growth, sporulation and pathogenicity. CONCLUSION: In the field, the point mutation S219P or S219 L in Myosin-5 conferred high resistance to phenamacril in F. fujikuroi. The point mutation S219P did not affect the fitness of F. fujikuroi, while the point mutation S219 L decreased its fitness. © 2017 Society of Chemical Industry.