Hypovirulence And Hyovirulent Mechanism Of Mycovirus Fusarium Oxysporum Ourmia-like Virus 1

Mycoviruses are a group of viruses that infect and replicate in fungi.The infection of most mycoviruses does not cause phenotypic changes in the host,but some mycoviruses can reduce the pathogenicity of phytopathogenic fungi,that is,hypovirulence.It is of great scientific significance to study the mechanism by which mycoviruses reduce the virulence of pathogenic fungus for the application of mycoviruses to plant disease control and to study the pathogenic mechanism of pathogenic fungi.In this study,the weak pathogenic strain Hu N8(carrying mycoviruses)of Fusarium oxysporum f.sp.momordicae Sun & Huang(FOM)was used as the material,and cloning of viral genome sequences carried by strain Hu N8.The horizontal transmission of the virus with the same specialized and different specialized types was carried out using hyphal fusion,and the pathogenicity of the obtained strains was tested.Determination of the control effect of weak pathogenic strain,and determination and analysis of rhizosphere soil microbiome.Gene expression differences and related pathway changes between SD-V strain(carrying mycoviruses)and wild-type strain SD-1 using transcriptome assay and analysis.The gene knockout technology was used to verify the function of the significantly down-regulated haloacid dehalogenase type II gene and catalase gene in SD-V.This study provides a theoretical basis for the research on the pathogenic mechanism of using mycovirus to control Fusarium wilt.The results of the study are as follows:1.A ourmia-like mycovirus was discovered from FOM,named Fusarium oxysporum ourmia-like virus 1(Fo Ou LV1).The virus is classified in the family Botourmiaviridae,genus Magoulivirus,and its genome contains two nucleotide sequences,L-segment and S-segment.The full-length sequence of the L-segment is 2712 nt and the GC content is 55.5%.It contains an open reading frame(OFR)and encodes an RNA-dependent RNA polymerase(Rd Rp);The full-length sequence of the S-segment is 1173 nt,the GC content is 43.8%,and it does not contain ORF.2.The mycovirus Fo Ou LV1 can significantly reduce the pathogenicity of FOM,and can be horizontally transmitted between different strains of FOM through hyphal fusion.At the same time,Fo Ou LV1 was able to horizontally spread to the Fusarium oxysporum f.sp.cucumerinum,weakening its pathogenicity.3.The weak pathogenic strain SD-V can effectively control the Fusarium wilt of bitter gourd,and the control effect in the field reaches 80%.The effect of strain SD-V on the rhizosphere and root fungal community of bitter gourd plants was analyzed by microbiome sequencing technology.The results showed that there was no significant difference in the relative abundance of Fusarium in the rhizosphere between healthy and diseased plants in the treatment group SD-V and the control,while the relative abundance of Fusarium in the rhizosphere of the healthy and diseased plants in the control group was higher than that in the SD-V treatment group.SD-V treatment mainly affected the number of Fusarium in the rhizosphere of bitter gourd plants.4.FoOuLV1 affected the infectivity of FOM,manifested as delayed infection,and reduced hyphae’s ability to expand in host plant tissues.The results of transcriptome analysis showed that after the Fo Ou LV1 infection,the related functional genes were differentially expressed in FOM,of which 1422 genes were down-regulated and 1078 genes were up-regulated.The results of GO enrichment analysis of differentially expressed genes showed that most of the significantly enriched pathways were related to allergic reactions(peroxidase reaction and activity,etc.),cell wall components(glucan metabolism,and polysaccharide metabolism),and transmembrane transport.KEGG enrichment analysis of differentially expressed genes showed that there were only 4 significant differential pathways,namely starch and sucrose metabolism,glycine,serine,and threonine metabolism,steroid biosynthesis,and pentose and glucuronic acid interconversion.5.The results of functional verification of the catalase gene Focat1 showed that compared with the wild type,the growth rate,sporulation and hyphal tip morphology of the knockout mutant on PDA medium had no significant changes,but the knockout mutant has reduced resistance to external oxidation and the pathogenicity was reduced.DAB staining results showed that the accumulation of hydrogen peroxide in the leaves inoculated with the knockout mutant was higher than that in the leaves inoculated with the wild-type strain.It indicated that the Focat1 gene plays an important role in the pathogenic process of FOM,and can degrade the reactive oxygen species produced by the host plant at the infection site during the infection process.6.The results of functional verification of the gene FoHAD-type II showed that the growth rate and spore production of the Fo HAD-typeⅡ gene knockout mutant on the PDA medium were not significantly different from the wild-type strain,and there were no significant differences in the morphology of hyphal tips and spores.However,the vegetative growth of the knockout mutant was defective,manifested as different colony morphology,reduced aerial hyphae,and significantly reduced pathogenicity,increased sensitivity to osmotic stress.The pathogenicity of the complemented mutant was consistent with that of the wild-type strain.Therefore,it is speculated that the Fo HAD-typeⅡ gene is not involved in the growth and development of FOM,but plays an important role in the pathogenic process of FOM,which may be related to the decline of pathogenicity of FOM caused by mycovirus.