Analysis Of The Overwintering Potential Of Villosiclava Virens Sclerotia And Its Biocontrol Fungi

Rice false smut is a rice disease caused by fungus Villosiclava virens,which specifically penetrates rice spikelets from the stamen filaments.As the large-scale cultivation of super rice and hybrid rice,it has gradually developed from a minor disease to a major rice disease.Rice false smut not only causes a severe yield loss,but also threatens food safety due to its production of mycotoxins.In recent years,the sclerotia of V.virens has been proved to be an important source of initial infection of rice false smut.Therefore,the study of sclerotia overwintering behavior will play a guiding role in the control of rice false smut.Therefore,further clarifying the overwintering behavior of sclerotia and screening biocontrol fungi that can degrade it will be of great value to the comprehensive prevention and control of rice false smut.The techniques and methods for gene biofunction analysis related to sclerotium formation of V.virens were also tried here.The results are as follows:1.The sclerotia overwintering simulation experiment found that after the sclerotia overwintering in May of the following year,20%of the sclerotia on the soil surface disintegrated,and 70%of the sclerotia buried 2 cm below the soil surface disintegrated..The sclerotia intact after overwintering were germinated and cultured,the velvety yellow hyphae began to germinate after 10 days.After 10-20 days,the yellow hyphae elongated upward to form the primordium of the fruit body.After 20-40 days,the fruiting bodies continue to elongate and begin to form capitate pedestals.After 40-60days,the fruit body grows to 2-4 cm,and the color changes from yellow to yellow-green.After 90 days,the fruit body was completely blackened.The germination results showed that about 30%of the overwintering sclerotium on the soil surface germinated to produce fruiting bodies,approximately 6.67%of overwintering sclerotia germinated and produced fruit bodies at 2 cm below the soil surface.Therefore,the successful overwintering rate of sclerotia on the soil surface is 30%,the successful overwintering rate of sclerotia 2 cm below the soil surface is 6.67%.The results showed that the sclerotia can survive the winter safely in the field as a source of initial infection.2.By separating the saprophytic fungi from the surface of ergot sclerotia and sclerotia of Villosiclava virens,10 candidate strains were screened.The strain JH-5isolated from the surface of sclerotia of Villosiclava virens was rapidly colonized within10 days,the sclerotium was completely softened within 30 days,and the sclerotium rotted and lost its germination activity within 30-60 days.Although other candidate strains can be colonized on the surface of sclerotia,they have no obvious degradation effect.rDNA-ITS sequence alignment and morphological observation of the strains showed that strain JH-5 is Talaromyces funiculosus.Transmission electron microscope observation revealed that the mechanism of action of the biocontrol fungi JH-5 is similar to that of heavy parasitism,and its hyphae can enter the sclerotium tissue cells to destroy the integrity of the sclerotium tissue.3.In the transcriptome data of low temperature-induced sclerotia formation,the difference in gene expression levels between the control group and the treatment group was analyzed by multiples,and six genes with significant differences were initially screened out in the up-regulated data.They are Uv8b-1535(protein kinase domain-containing protein),Uv8b-5068(beta-galactosidase),Uv8b-6995(eliciting plant response-like protein),Uv8b-7484(glutathione s-transferase),Uv8b-7491(FAD dependent oxidoreductase),Uv8b-7023(regulatory P domain-containing protein).Combined with homologous recombination technology,CRISPR vector is introduced to knock out related genes.By PEG-mediated transformation of protoplasts,the concentration of prepared protoplasts is 10~7/m L.As a result,a total of 45 transformants were obtained,40 of which were translocation transformants,and no knockout mutants were obtained.Transplantation of the grown transformants revealed that the sporulation and hypha density of some translocation insertion mutants increased significantly.