| Wheat is one of the most important food crops in China and around the world.Fusarium pseudograminearum is the dominant pathogen causing Crown rot of wheat in the Yellow and Huaihe River wheat region,but there are few studies on the molecular pathogenesis of Fusarium pseudograminearum.In this study,272 Fusarium pseudograminearum strains isolated from wheat stem base rot strains in 13 prefectures and cities in Henan Province in 2020 were used as test materials for indoor seedling stem base inoculation.The results of measuring the pathogenicity of stem basals at the indoor seedling stage showed that there were 109,137 and 26 high strains with medium pathogenicity and low pathogenicity according to the disease index.A total of 51 strains with strong,medium and weak pathogenicity at the stem base of wheat seedlings were selected,and the pathogenicity was determined by inoculation of field panicle monoflower instillation.The results of the determination of pathogenicity of monoflower infusion inoculation in field panicles showed that 51 strains could cause disease and cause different degrees of white spike symptoms,and there were 18,21 and 12 high,medium and low pathogenic strains,respectively.Further analysis and comparison of the virulence of wheat stem base and panicle showed that the same strain had no obvious correlation in the pathogenicity of wheat stem base and panicle part.According to the results of wheat stem basal virulence determination,a total of 60 strains with strong,medium and weak virulence were selected for multiple virulence measurements at the stem base,whole genome sequencing was performed to obtain genotype data,genome-wide association analysis was used to mine genetic loci related to pathogenicity traits at the base of wheat stem,genome annotation analysis was performed on the obtained significant SNP sites,and 7 candidate genes related to pathogenicity traits were identified.The deletion and complement mutant strains of gene deletion and complement mutant were constructed for five genes,and the functions of these genes in the growth,development,spore production and pathogenicity of Fusarium pseudograminus were analyzed.In order to further explore the key pathogenic genes of Fusarium pseudograminaris,this paper lays a foundation for the prevention and control of wheat stem base diseases.The results of the study are as follows:A total of 137 GB of genotypic data were obtained from whole genome resequencing of 60 Fusarium pseudograminearum strains,with an average sequencing depth of 57.51 x.The genome resequencing yielded an association population of 8.79 million SNP loci,and the GWAS method of wheat stem base pathogenicity trait using ECMLM and Farm CPU models was used to mine for SNP loci significantly associated with this trait,and a total of one A total of one significant SNP was identified（Chr4425970,P < 8.45 ×10-7）,and genome annotation analysis yielded a total of seven candidate genes related to pathogenicity,zinc finger transcription factors（Fpg08853,Fpg08852）,tannin acyl hydrolase（Fpg08851）,Cupin superfamily（Fpg08850）,alginate lyase（Fpg08849）,heptanoyl-ACP methyl ester carboxylase（Fpg08848）,and（Fpg08847）are not functionally annotated at this time.The homologous recombination principle was used to obtain（35）Fpg08853,（35）Fpg08852,（35）Fpg08851,（35）Fpg08850,（35）Fpg08849,The deletion mutant strains of five genes of were constructed.Fpg08853,Fpg08852,Fpg08851,Fpg08850,Fpg08849 gene back-complement vectors were constructed respectively,and the backcomplement transformants of these five genes were obtained by PEG-mediated protoplast transformation into the gene deletion mutant strains.The functional validation results showed that the four deletion mutants,Fpg08853,Fpg08851,Fpg08850 and Fpg08849,did not differ significantly from the wild type in basic phenotypes such as colony morphology,growth rate and sporulation,and the indoor wheat seedling pathogenicity assay.The Fpg08852 gene deletion mutant did not affect growth and sporulation,had enhanced susceptibility in PDA plates spiked with hydrogen peroxide,participated in the redox reaction of the strain,and significantly reduced the disease index in the indoor wheat seedling pathogenicity assay compared with the wild type,which may be the effector gene of this significant SNP locus.In this study,the pathogenicity traits of Fusarium pseudograminearum were analyzed by a mixed linear model,and candidate genes related to pathogenicity were screened and functionally validated based on the genes annotated to the SNP loci.The theoretical basis was laid for revealing the molecular pathogenicity mechanism of Fusarium pseudograminearum. |
