Yiping Hou

Activity of a novel strobilurin fungicide benzothiostrobin against Sclerotinia sclerotiorum.

Benzothiostrobin is a novel strobilurin fungicide. In this study, baseline sensitivity of Sclerotinia sclerotiorum (Lib.) de Bary to benzothiostrobin was determined using 100 strains collected during 2012 and 2013 from different geographical regions in Jiangsu Province of China, and the average EC50 value was 0.0218 (± 0.0111)μg/mL for mycelial growth. After benzothiostrobin treatment, hyphae were contorted with offshoot of top increasing and cell membrane permeability increased markedly, while sclerotial production and oxalic acid content significantly decreased. Benzothiostrobin strongly inhibited mycelial respiration within 12h and the oxygen consumption of the mycelia could not be inhibited after 24h. On detached rapeseed leaves, the protective and curative activity test of benzothiostrobin suggested that benzothiostrobin had good control efficiency against S. sclerotiorum, and protective activity was better than curative activity. These results will contribute to us evaluating the potential of the new strobilurin fungicide benzothiostrobin for management of diseases caused by S. sclerotiorum and understanding the mode of action of benzothiostrobin against S. sclerotiorum.

FgFim, a key protein regulating resistance to the fungicide JS399-19, asexual and sexual development, stress responses and virulence in Fusarium graminearum

Fimbrin is an actin-bundling protein found in intestinal microvilli, hair cell stereocilia and fibroblast filopodia. Its homologue Sac6p has been shown to play a critical role in endocytosis and diverse cellular processes in Saccharomyces cerevisiae. FgFim from the wheat scab pathogenic fungus Fusarium graminearum strain Y2021A, which is highly resistant to the fungicide JS399-19, was identified by screening a mutant library generated by HPH-HSV-tk cassette-mediated integration. The functions of FgFim were evaluated by constructing a deletion mutant of FgFim, designated ΔFgFim-15. The deletion mutant exhibited a reduced rate of mycelial growth, reduced conidiation, delayed conidium germination, irregularly shaped hyphae, a lack of sexual reproduction on autoclaved wheat kernels and a dramatic decrease in resistance to JS399-19. ΔFgFim-15 also exhibited increased sensitivity to diverse metal cations, to agents that induce osmotic stress and oxidative stress, and to agents that damage the cell membrane and cell wall. Pathogenicity assays showed that the virulence of the FgFim deletion mutant on flowering wheat heads was impaired, which was consistent with its reduced production of the toxin deoxynivalenol in host tissue. All of these defects were restored by genetic complementation of the mutant with the parental FgFim gene. Quantitative real-time polymerase chain reaction (PCR) assays showed that the basal expression of three Cyp51 genes, which encode sterol 14α-demethylase, was significantly lower in the mutant than in the parental strain. The results of this study indicate that FgFim plays a critical role in the regulation of resistance to JS399-19 and in various cellular processes in F. graminearum.

Whole-genome sequencing reveals that mutations in myosin-5 confer resistance to the fungicide phenamacril in Fusarium graminearum

To determine the mechanism of resistance to the fungicide phenamacril (JS399-19) in Fusarium graminearum, the causal agent of Fusarium head blight, we sequenced and annotated the genome of the resistant strain YP-1 (generated by treating the F. graminearum reference strain PH-1 with phenamacril). Of 1.4 million total reads from an Illumina-based paired-end sequencing assay, 92.80% were aligned to the F. graminearum reference genome. Compared with strain PH-1, strain YP-1 contained 1,989 single-nucleotide polymorphisms that led to amino acid mutations in 132 genes. We sequenced 22 functional annotated genes of another F. graminearum sensitive strain (strain 2021) and corresponding resistant strains. The only mutation common to all of the resistant mutants occurred in the gene encoding myosin-5 (point mutations at codon 216, 217, 418, 420, or 786). To confirm whether the mutations in myosin-5 confer resistance to phenamacril, we exchanged the myosin-5 locus between the sensitive strain 2021 and the resistant strain Y2021A by homologous double exchange. The transformed mutants with a copy of the resistant fragment exhibited resistance to phenamacril, and the transformed mutant with a copy of the sensitive fragment exhibited sensitivity to phenamacril. These results indicate that mutations in myosin-5 confers resistance to phenamacril in F. graminearum.

Proteomic analysis of Fusarium graminearum treated by the fungicide JS399-19.

JS399-19 (2-cyano-3-amino-3-phenylancryic acetate), a novel cyanoacrylate fungicide, has powerful inhibition against Fusarium species, especially to Fusarium graminearum. Treated with JS399-19, mycelium of F. graminearum was distorted and swelled. The embranchment increased. In order to investigate the effect of JS399-19 on protein expression of F. graminearum, total protein of F. graminearum cultured in normal condition and that treated with 0.5 μg/mL (EC90 value) JS399-19 were extracted respectively and proteomic analysis was performed using two-dimensional gel electrophoresis. The expression levels of 38 proteins varied quantitatively at least twofold. 33 proteins out of the 38 were successfully identified by MALDI-TOF-MS/MS and MASCOT. According to the classification of physiological functions from Conserved Domain Database analysis, 19, 5, 2, 3, 2 and 2 proteins were respectively associated with metabolism, regulation, motility, defense, signal transduction, and unknown function, which indicated that energy metabolism, the synthesis and transport of proteins and DNA of F. graminearum were inhibited by JS399-19 in different degrees. The expression levels of the genes were further confirmed by quantitative real-time PCR analyses. This study represents the first proteomic analysis of F. graminearum treated by JS399-19 and will provide some useful information to find the mode of action of the fungicide against F. graminearum.

Application of tetra primer ARMS-PCR approach for detection of Fusarium graminearum genotypes with resistance to carbendazim

Tetra-primer ARMS-PCR (amplification refractory mutation system-PCR) was developed to detect isolates of Fusarium graminearum with resistance to carbendazim, a methyl benzimidazole carbamate-group fungicide. Resistance to carbendazim in F. graminearum is caused by point mutations at codon 167, 198, and 200 of the β2-tubulin gene (FGSG_06611.3). According to single nucleotide polymorphisms on codon 167, 198, 200 of the resistance gene β2-tubulin gene, two pairs of primer designed on each codon respectively, were used for amplification. SNP genotyping was made successfully based on the different patterns in agarose gels between point mutations and wild type strain. Tetra-primer ARMS-PCR protocol developed in this study is a simple and useful technique to identify genotypes and has a potential application in management of carbendazim resistance in F. graminearum. It will help researchers and growers select the best antifungal strategy to use in the field.

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.

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.

Effects of the Novel Fungicide Benzothiostrobin on Sclerotinia sclerotiorum in the Laboratory and on Sclerotinia Stem Rot in Rape Fields

We determined the effects and efficacy of benzothiostrobin, a new strobilurin-derived fungicide, against the plant-pathogenic fungus Sclerotinia sclerotiorum (the causal agent of Sclerotinia stem rot). Mycelial growth and sclerotial germination in vitro were strongly inhibited by benzothiostrobin in the presence of salicylhydroxamic acid. On detached rapeseed leaves, benzothiostrobin at 40 μg/ml reduced lesion development by 87%. No cross-resistance was detected between benzothiostrobin and carbendazim, iprodione, fludioxonil, or boscalid. A formulated mixture of benzothiostrobin and fluazinam at 1:1 had synergistic activity against S. sclerotiorum in vitro. In field trials, benzothiostrobin alone or formulated with fluazinam at 1:1 (150 g a.i. ha-1) was significantly (P < 0.05) superior to iprodione in controlling Sclerotinia stem rot of rapeseed. These results suggest that benzothiostrobin has substantial potential for the control of Sclerotinia stem rot.