Validamycin A Enhances the Interaction Between Neutral Trehalase and 14-3-3 Protein Bmh1 in Fusarium graminearum.

In agriculture, Trehalase is considered the main target of the biological fungicide validamycin A, and the toxicology mechanism of validamycin A is unknown. 14-3-3 proteins, highly conserved proteins, participate in diverse cellular processes, including enzyme activation, protein localization, and acting as a molecular chaperone. In Saccharomyces cerevisiae, the 14-3-3 protein Bmh1could interact with Nth1 to respond to specific external stimuli. Here, we characterized FgNth, FgBmh1, and FgBmh2 in Fusarium graminearum. ΔFgNth, ΔFgBmh1, and ΔFgBmh2 displayed great growth defects and their peripheral tips hyphae generated more branches when compared with wild-type (WT) PH-1. When exposed to validamycin A as well as high osmotic and high temperature stresses, ΔFgNth, ΔFgBmh1, and ΔFgBmh2 showed more tolerance than WT. Both ΔFgNth and ΔFgBmh1 displayed reduced deoxynivalenol production but opposite for ΔFgBmh2, and all three deletion mutants showed reduced virulence on wheat coleoptiles. In addition, coimmunoprecipitation (Co-IP) experiments suggested that FgBmh1 and FgBmh2 both interact with FgNth, but no interaction was detected between FgBmh1 and FgBmh2 in our experiments. Further, validamycin A enhances the interaction between FgBmh1 and FgNth in a positive correlation under concentrations of 1 to 100 µg/ml. In addition, both high osmotic and high temperature stresses promote the interaction between FgBmh1 and FgNth. Co-IP assay also showed that neither FgBmh1 nor FgBmh2 could interact with FgPbs2, a MAPKK kinase in the high-osmolarity glycerol pathway. However, FgBmh2 but not FgBmh1 binds to the heat shock protein FgHsp70 in F. graminearum. Taken together, our results demonstrate that FgNth and FgBmh proteins are involved in growth and responses to external stresses and virulence; and validamycin enhanced the interaction between FgNth and FgBmh1in F. graminearum.

PCR-RFLP for Detection of Fusarium graminearum Genotypes with Resistance to Phenamacril.

Phenamacril is a cyanoacrylate fungicide that provides excellent control of Fusarium head blight (FHB) or wheat scab, which is caused predominantly by Fusarium graminearum and F. asiaticum. Previous studies revealed that codon mutations of the myosin-5 gene of Fusarium spp. conferred resistance to phenamacril in in vitro lab experiments. In this study, PCR restriction fragment length polymorphism (RFLP) was developed to detect three common mutations (A135T, GCC to ACC at codon 135; S217L, TCA to TTA at codon 217; and E420K, GAA to AAA at codon 420) in F. graminearum induced by fungicide domestication in vitro. PCR products of 841 bp (for mutation of A135T), 802 bp (for mutation of S217L), or 1,649 bp (for mutation of E420K) in the myosin-5 gene were amplified by appropriate primer pairs. Restriction enzyme KpnI, TasI, or DraI was used to distinguish phenamacril-sensitive and -resistant strains with mutation genotypes of A135T, S217L, and E420K, respectively. KpnI digested the 841-bp PCR products of phenamacril-resistant strains with codon mutation A135T into two fragments of 256 and 585 bp. In contrast, KpnI did not digest the PCR products of sensitive strains. TasI digested the 802-bp PCR products of phenamacril-resistant strains with codon mutation S217L into three fragments of 461, 287, and 54 bp. In contrast, TasI digestion of the 802-bp PCR products of phenamacril-sensitive strains resulted in only two fragments of 515 and 287 bp. DraI digested the 1,649-bp PCR products of phenamacril-resistant strains with codon mutation E420K into two fragments of 932 and 717 bp, while the PCR products of phenamacril-sensitive strains was not digested. The three genotypes of resistance mutations were determined by analyzing electrophoresis patterns of the digestion fragments of PCR products. The PCR-RFLP method was evaluated on 48 phenamacril-resistant strains induced by fungicide domestication in vitro and compared with the conventional method (mycelial growth on fungicide-amended agar). The accuracy of the PCR-RFLP method for detecting the three mutation genotypes of F. graminearum resistant to phenamacril was 95.12% compared with conventional method. Bioinformatics analysis revealed that the PCR-RFLP method could also be used to detect the codon mutations of A135T and E420K in F. asiaticum.

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.

Effects of the dinitroaniline fungicide fluazinam on Fusarium fujikuroi and rice.

Fusarium fujikuroi is the primary causal agent of rice bakanae disease. Fluazinam is a protective dinitroaniline fungicide which could interrupt the fungal cell's energy production. Little is known about the effects of fluazinam on F. fujikuroi. In this study, baseline sensitivity of F. fujikuroi to fluazinam was determined using 103 isolates collected from diseased young rice of different fields in Shaoxing of Zhejiang Province and Huaian of Jiangsu Province of China in 2016. The EC50 values of fluazinam on inhibiting mycelial growth against 103 isolates of F. fujikuroi ranged from 0.0621 to 0.5446 µg/mL with the average value of 0.2038 ±â€¯0.0099 µg/mL (mean ±â€¯standard error). The EC50 values of fluazinam on suppressing conidium germination against 103 isolates of F. fujikuroi ranged from 0.1006 to 0.9763 µg/mL with the mean value of 0.3552 ±â€¯0.0181 µg/mL. Treated with fluazinam, hyphae of F. fujikuroi were contorted, offshoot of top mycelia increased, conidial production descreased significantly and exopolysaccharide (EPS) content did not change significantly while peroxidase (POD) activity significantly decreased. Meanwhile, cell membrane permeability increased after treated with fluazinam. The analysis of cell ultrastructure indicated that fluazinam could damage the membrane structure of F. fujikuroi and cause a large number of vacuoles formed. In addition, fluazinam did not affect germination rate, plant height and fresh weight of rice, which indicated that fluazinam was safe to rice. All the results indicated that fluazinam had strong antifungal activity against F. fujikuroi and a potential application in controlling rice bakanae disease. These results will provide useful information for management of rice bakanae disease caused by F. fujikuroi and further increase our understanding about the mode of action of fluazinam against F. fujikuroi and other phytopathogens.

Baseline sensitivity of Bipolaris maydis to the novel succinate dehydrogenase inhibitor benzovindiflupyr and its efficacy.

Benzovindiflupyr is a novel member of succinate dehydrogenase inhibitor (SDHI) fungicides. The filamentous fungus Bipolaris maydis Nisik. et Miyake was the causal agent of southern corn leaf blight (SCLB). Here, baseline sensitivity of B. maydis to benzovindiflupyr was established by mycelial growth and conidium germination methods using 96 B. maydis isolates collected from various places of Jiangsu Province of China, and EC50 values ranged from 0.0321 to 0.9149 µg/ml with the mean value of 0.3446 (±0.2248) µg/ml for mycelial growth, and 0.1864 to 0.964 µg/ml with the mean value of 0.5060 (±0.2094) µg/ml for conidium germination respectively. Treated with benzovindiflupyr, the distribution of nuclei and septum of hyphae did not change, but hyphae of offshoot and conidial production of B. maydis decreased significantly, the cell membrane permeability increased. The result of transmission electron microscope showed that the cross section of hypha was out of shape, the cell wall became thin and sparse, the cell membrane were distinctly damaged, organelles dissolved and vacuolated, and the cell nearly broke up. The results suggested that benzovindiflupyr had strong activity against mycelial growth and conidial production of B. maydis by damaging cell wall, membrane and organelles. The protective and curative activity assays for benzovindiflupyr indicated that benzovindiflupyr exhibited excellent suppression of B. maydis development on detached corn leaves. In protective activity assay with application of benzovindiflupyr at 10 µg/ml, the control efficacy reached to 100%. In curative activity assay with application of benzovindiflupyr at 50 µg/ml, the control efficacy reached to 90.72%. This is the first report of baseline sensitivity of B. maydis to benzovindiflupyr and its biological activity against B. maydis. It is recommended that benzovindiflupyr is a excellent candidate for controlling SCLB.

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.