Temperature-responded Biological Fitness Of β₂-tubulin Mutants F167Y,E198K And E198l Of Fusarium Graminearum

Fusarium head blight（FHB）caused predominately by F.graminearum is a destructive fungal disease of wheat.Its occurrence,development and epidemic are closely related to meteorological conditions.Infection by F.graminearum in cereal crops may lead to huge yield losses in severe pidemic years.Except for economic losses,the pathogen can bring serious impacts on human and animal health by contaminating wheat and maize with DON,zearalmone and aurofusarin.Due to the lack of effective disease-resistant varieties for a long time,the main measurement of control this disease is to spray with fungicides in the wheat flowering period.Benzimidazole fungicides such as carbendazim have been used to control FHB in China for more than 40 years.Under long-term selection pressure,carbendazim-resistance population withβ₂-tubulin mutation has been formed in F.gramineum,and the mutant F167Y accounted for more than 95%of the resistant strains in field.In order to understand the rules of development and key influencing factors in the population of carbendazim-resistance pathogens,we evaluated the effects of various temperatures on biological fitness of wild-type sensitive strain 2021 and carbendazim-resistance mutants conferringβ₂-tubulin substitutions F167Y,E198K and E198L.The results showed that mycelial growth and conidiation of four strains increased with the increase of the temperature between 10℃and 25℃.The conidiation of mutant E198K was significantly lower than that of the other three strains.While at 10℃,only the mutant F167Y still had a small number of conidiation and its conidia germination rate was significantly higher than that of wild-type strain 2021 at low temperature,which indicated that the conidia of F167Y displayed strong adaptability to low temperature.The virulence of F.graminearum directly affects the damage degree of F.graminearum to wheat,so we studied the virulence of four strains at different temperatures.The results showed tha the virulence of the four strains was largely similar at the same temperature,and the virulence was positively correlated with the increasing temperature between 10℃and 25℃.This means that higher temperatures help increase the virulence of F.graminearum.The growth and development of cells were inhibited by low temperature.At 10℃,there were more autophagosomes in the cells,and internal tissue degradation dispersed,and vacuolization occurred and the cell wall was concaved.We determined the sensitivities of the four strains at different temperatures to ten fungicides,including carbendazim,benomyl,thiabendazole,phenamacril,pydiflumetofen pyraclostrobin,prochloraz,tebuconazole,metconazole and prothioconazole.It was found that the sensitivity of wild-type sensitive strain and carbendazim-resistance mutants to phenamacril,prochloraz,metconazole and prothioconazole decreased with the temperature increasing,indicating that these fungicides are more active when used at lower temperatures.There was positive cross-resistance between carbendazim,benomyl and thiabendazole,while there was no cross-resistance between carbendazim and pydiflumetofen,pyraclostrobin and tebuconazole.These results would direct us to use fungicide preventing the infection of F.graminearum with changeable atmospheric temperature at wheat flower stage.Temperature could affect not only the growth and development of F.graminearum but also the content of metabolites in F.graminearum.We found that DON contents of the four strains showed an increasing trend from 15℃to 25℃,and the DON contents of the mutants were higher than those of the wild-type strain.We subsequently measured the expression of Tri5,a DON biosynthesis-related gene and content of pyruvate,an important precursor of DON biosynthesis.The results showed that at the same temperature,the expression of Tri5 and the content of pyruvate in the mutants were significantly higher than those in the wild-type strain,which further printed that the DON content of the mutants was higher than that of the wild-type strain.Aurofusarin is also an important secondary metabolite produced by F.graminearum,and we found that at 10℃,four strains produced less aurofusarin,and the production of aurofusarin increased with increasing temperature,among which the mutants F167Y and E198K had higher aurofusarin contents than the wild-type strain 2021 and the mutant E198L,which showed stronger stress resistance.In summary,the fitness ofβ₂-tubulin resistance mutants of F.graminearum varies with temperature.We can choose reasonable control methods according to the temperature of wheat flower stage to reduce the occurrence frequency of resistance mutants in the field,so as to reduce the occurrence and spread of FHB.