Study On Mechanism Of Sclerotia Development In Villosiclava Virens And Its Transcriptome Analysis

In the past 40 years,the occurrence scale and severity of rice false smut have been increasing in all major rice cultivation areas in the world,and it has gradually developed into a major rice disease.Among them,Zhejiang Province has become a hardest-hit area of rice false smut in China.The recent studies in our laboratory showed that in the years with low temperature in autumn,the number of sclerotia on the diseased rice with late maturity was more.In this study,the effects of low temperature on the mechanism of molecular regulation were preliminarily explored by using high-throughput sequencing of transcriptome and gene knockout of filamentous fungi and achieved the following results.1.According to the field survey,the autumn temperature in Zhejiang Province was relatively high in 2017,and only the self-growing rice seedlings with slow growth had the occurrence of rice false smut,while the later the self-growing rice seedlings were,the more serious the disease was and the more sclerotia were produced.The results showed that it was more conductive to the occurrence of rice false smut when the temperature was low after autumn.Furthermore,120 rice false smut balls were randomly selected and sliced,16 balls with implied sclerotia were found,the proportion was about 13.56%.Further anatomical observation showed that the sclerotia differentiated from the inside of the rice false smut balls indeed,but also show that the sclerotia were produced in the middle and later stages of the development,and the low temperature was more conducive to the sclerotia differentiation.2.Through the germination experiment showed that only the germination of fresh chlamydospores could play a role in the disease transmission in that year.Compared with the chlamydospores preserved at low temperature for one year or cultivated in the sand simulating the winter environment in the field,it could not germinate normally.And the implied sclerotia could not germinate to form a fruiting body.It can be concluded that in the next year,only the mature sclerotia is the main primary source of infection.3.In order to understand the molecular formation mechanism of the sclerotia,a total of 4845 differentially expressed genes were obtained from 59.78G sequencing data,of which 2504 genes were significantly up-regulated and 2341 genes were significantly down-regulated using transcriptome sequencing technology.GO analytic results showed that the oxidoreductase genes were significantly up-regulated in the low-temperature treatment of Villosiclava virens in the classification of molecular functions.In combination with the KEGG pathway,the differential genes were mainly involved in the biosynthesis of secondary metabolites,starch and sucrose metabolism and other nutrient metabolism and energy metabolism pathways.Further excavation of differential genes showed that the formation of sclerotia might be caused by the induction of low-temperature environment stress,which made the fungi in an oxidative stress state.Through the amplification of signal transduction pathway,the expression of genes such as transmembrane transport,cell morphology and biosynthesis was up-regulated,which made the protein expression active in the process of sclerotia formation,reaching the peak of tissue and substance synthesis,and then promoting the formation of sclerotia.4.Through the above transcriptome data analysis,9 genes that might be related to the sclerotia formation were screened out.Then,PEG-mediated protoplast transformation technology was used to knock out the target gene and analyze the pathogenicity of the mutant.Two mutant strains were obtained.The analysis of the biological characteristics of the mutants showed that all the mutants not only grew slowly in the early stage of growth,but also decreased the sporulation ability and the growth rate of germ tube during germination.In addition,the mutant strain was very sensitive to H₂O₂.Artificial inoculation test showed that the mutant lost pathogenicity and did not infect and produce sclerotia successfully.However,the pathogenicity of wild strains was high,and 67%of balls produced sclerotia.The results showed that the gene was closely related to pathogenicity and sclerotia formation.