The Acquisition And Biological Characteristics Of Fusarium Graminearum Heterokaryon

Fusarium head blight（FHB）is one of the main diseases of wheat,barley and other major grain crops.It causes the decrease in wheat yield and deterioration in grain quality.In addition,diseased wheat grains produce various mycotoxins,the most common ones are deoxynivalenol（DON）and zearalenone（ZEN）.The main means of controling FHB is the use of resistant varieties and chemicals.The loss of disease resistance and the improvement of drug resistance have increasingly become the limiting factors for stable and high yield of crops.Studies have revealed that sexual hybridization,heterokaryotic effect,quasi-sexual reproduction and mutants of plant pathogenic fungi are closely related to pathogen variation.Therefore,the study on heterokaryotic phenomenon of Fusarium graminearum and the quasi-sexual reproduction and resistance evolution is the theoretical basis for controling the disease.In this study,the fluorescent proteins GFP and RFP were used to label the histones H₂A.Z and H₂B of carbendazim-resistant and carbendazim-sensitive strains of Fusarium graminearum,respectively.The two fluorescent protein-labeled strains were fused by protoplasts to produce heterokaryons.PCR verification,phenotypic observation,and the stability of the heterokaryons during the continuous transformation of mycelia were carried out,and the pathogenicity of the two parents was compared.Thus,the inheritance stability of heterokaryons generated by protoplast fusion of F.graminearum and other homologous mating fungi and the genetic variation of asexual reproduction were expounded.The above results will be more intuitive to clarify the genetic variation of heterokaryotes of F.graminearum and other homologous mating fungi,and lay a theoretical foundation for the study of pathogenic variation,genetic evolution of fungicide resistance and genetic breeding of beneficial fungi.The main research results are as follows:1.Obtaining of green fluorescence-labeled and red fluorescence-labeled F.graminearum strains:Fusion PCR was used to construct the fusion gene fragment of GFP inserted into the end of histone H₂A.Z gene coding region of carbendazim-resistant strains and the fusion gene fragment of RFP inserted into the end of histone H₂B gene coding region of carbendazim-sensitive strains.Fusion mutant strains were obtained through PEG-mediated protoplast transformation and hygromycin or hereditarymycin resistance screening.Through PCR verification,fluorescence observation and DAPI staining,it was proved that green fluorescent protein and red fluorescent protein successfully labeled the nucleus.At the same time,in order to test the effect of the introduction of exogenous genes GFP and RFP on the phenotype of transformants,the biological determination of transformants were carried out.The results showed that there was no significant difference in the mycelial growth rate,mycelial top morphology,colony morphology and conidial yield morphology between the transformants and the parent strains,indicating that the fusion of GFP and RFP had no effect on the biological functions of H₂A.Z and H₂B,which was aimed to provide a basis for the acquisition of heterokaryote strains.2.Obtaining of heterokaryons of F.graminearum:The protoplasts of green fluorescent protein and red fluorescent protein-labeled strains were fused by protoplast fusion technology,and then heterokaryons strains were obtained.The phenotypic results of antibiotics and carbendazim indicated that heterokaryotic strains had hygromycin,geneticin and carbendazim resistance.PCR verification of heterokaryotic strains showed that the green fluorescent protein gene,red fluorescent protein gene,hygromycin resistance gene and genomycin resistance gene had been successfully inserted into the heterokaryotic genome.Fluorescence observation of heterokaryotic hyphae and conidia showed that green fluorescent nuclei were observed in heterokaryotic hyphae,but no red fluorescent nuclei were observed.At the same time,the single conidia were observed after asexual sporulation,and it was found that the fluorescence phenotypes of nuclei in each cell of conidia were consistent,that is,there was only one fluorescence phenotype of nuclei in the same conidia.The proportions of the two fluorescent labeled nuclei in conidia were statistically analyzed.The results showed that the proportions of the two nuclei were extremely unbalanced.The number of green fluorescent nuclei was much more than that of red fluorescent nuclei,and the proportion of red fluorescent nuclei was less than 1%,indicating that the carbendazim-resistant nuclei were dominant in heterokaryote strains.3.Determination of the biological characteristics of the heterokaryons of F.graminearum:The subculture stability of the heterokaryons strains was determined.The results showed that the heterokaryons strains from the first generation to the fifth generation could grow on the plates containing hygromycin and geneticin,carbendazim and geneticin,which proved that the heterokaryons had good genetic stability and carbendazim resistance.The biological characteristics of heterokaryotic strains were determined and compared with the two parental strains.The results showed that the heterokaryotic strain was slightly lower than the two parental strains in mycelial growth rate.In the hyphal tip morphology,most of the heterokaryotic strains were branched,similar to the two parental strains.In colony morphology,the heterokaryotic strains produced dense white aerial mycelia,which was not significantly different from the parent strains.At the same time,the heterokaryotic strains produced purple and yellow pigments during mycelial growth,and the production of pigments was similar to that of carbendazim-resistant strains,while carbendazim-sensitive strains only produced purple pigments.In the morphology of conidia,the conidia of heterokaryotic strains were Fusarium-like,with more septums,most of which were 3,and there was no significant difference between the two parental strains.In terms of conidial production,the conidial production of heterokaryotic strains was（2.72±0.14）×10⁶/mL,that of carbendazim-sensitive strains was（0.34±0.11）×10⁶/mL,and that of carbendazim-resistant strains was（0.73±0.16）×10⁶/mL.The conidial production of heterokaryotic strains was significantly higher than that of the two parental strains.The above results showed that the total viability of heterokaryotic strains was stronger than that of the two parental strains.At the same time,a large number of asexual spores were coated on plates containing hygromycin and genomycin or carbendazim and genomycin.The results showed that the single spore progeny of asexual recombination could not be screened,indicating that the probability of parasexual reproduction of heterokaryons was low under the experimental conditions.