| Fusarium head blight （FHB）, caused by Fusarium graminearum, is one of the most devastating fungal diseases in wheat in China. JS399-19is a novel cyanoacrylate fungicide with unique chemical structure with specific mode of action against Fusarium spp. In vitro, JS399-19-resistant mutants were obtained easily by ultra-violet （UV） irradiating and fungicide training and the fitness of JS399-19resistant mutants was not decreased significantly. Potential resistance risk of F. graminearum to JS399-19can be divided into the level of moderate-high. In order to develop a sound recommendation for the use of a new crop fungicide and detection technique for resistance, the resistance mechanism of F. graminearum to JS399-19should be studied before the resistance appears in the field.Physiological and biochemical characteristics were determined for the JS399-19-sensitive resistant strain and its JS399-19-domesticated resisistant mutants of F. graminearum against five stresses, several different carbon and nitrogen resources, cellular glycerol and exoploysaccharides（EPS）. The result indicated that （1） all the highly JS399-19-resisitant strains grew faster than the midderately JS399-19-resisitant strains and the lowly JS399-19-resisitant strains cultured on the different nitrogen source;（2） mycelial radial gowth of the resistant mutants was more significantly inhibitive than the sensitive strain when stressed in the sorbitol, H2O2, congred, high temperature, pH=11. However, all the resistant mutants grew faster than the sensitive strain when stressed in0.4M LiCl.Tolerance of JS399-19-resisitant strains, except for Y2021A and Y2021C, against sodium dodecyl sulfonate （SDS） were much stronger than the sensitive strain.Adaptation response of low and high resistant strain to sour was better than the sensitive strain; content of intracellular glycerol in the resistant strains was significantly higher than that of the sensitive strain without the stress of the fungicide, and content of intracellular glycerol was un-related to the resistant strains with the different sensitivity against JS399-19. But treated with the fungicide, the tolerance against LiCl, contents of the intracellular glycerol and extracellular polysaccharide of the resistant strains and the sensitive strain varied irregularly. To determine the resistant mechanism of F.graminearum to JS399-19, the6F.graminearum isolates （one sensitive-strain and its JS399-19-domesticated resistant-strains） were used for analysis of RAPD. Based on RAPD analysis, the results indicated that the strains tested were relatively rich in hereditary multiplicity, and that6strains of F. graminearum were divided into the two groups. One group was only one member, i.e. a JS399-19-sensitive strain2021of F. graminearum. The other group included all the tested JS399-19-resistant strains with the different resistant level against JS399-19. Sensitive-strain2021and resistant-strain Y2021X induced by were analyzed by cDNA-RAPD. Some polymorphic bands were obtained between sensitive-strain2021and resistant-strains Y2021X and among resistant-strains Y2021X with different resistance level.These polymorphic bands were grouped into seven different types, which were supposed to correspond to the metabolism-related gene, motility-related gene, control gene, signal transduction-related gene and other genes. The sensitive-strain2021was treated with EC90of JS399-19（0.5μg/mL） for12h,24h and36h respectively and the controls were the sensitive-strain2021treated with nothing. Total mRNA of them were extracted.The study was carried out to detect the differential expression of genes in F.graminearum isolate2021.The results of QPCR showed that the expression patterns of each gene were different.FGSG-06149and FGSG-03716were expressed differently during the sensitive strain2021treated with JS399-19for0-36hours in vitro. To evaluate the role of the two genes in the resistance of F.graminearum against JS399-19, F.graminearum isolate Y2021A (high-resistant to18399-19（JS399-19HR） was used for the analysis of functions of FGSG-06149and FGSG-03716. FGSG-06149and FGSG-03716was knocked out respectively by using a double-joint polymerase chain reaction （PCR）. The hph-hsv fragment was transferred into F. graminearum via homologous double crossover at the site where the FGSG-06149or FGSG-03716genes of F. graminearum are normally located （the FGSG-06149or FGSG-03716gene of F. graminearum had been deleted）. The transformants of deleted mutants and their complementary mutants were constructed and confirmed through PCR and Southern Blotting, and their sensitivity to JS399-19was determined. EC50values to JS399-19of the FGSG-06149-deletion or FGSG-03716-deletion mutants of high-resistant strain Y2021A increased by62～71μg mL-1when compared with their progenitor. Mycelial growth rate, conidia productivity, perithecium productivity and other biological characteristics were evaluated for deleted mutants of FGSG-06149or FGSG-03716. FGSG03716-deleted mutants decreased in ability of perithecia production, tolerance of NaCl, LiCl and acid （pH=4）, and30℃, but increased in conidium production,tolerance of KCl, SDS and alkaline. FGSG06149-deleted mutants could not produce perithecia with the decrease in the tolerance of NaCl, LiCl and20℃and30℃, however, improved in tolerance of KCl, SDS and strong alkaline （pH=11） and acidic environment （pH=4）. |
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