Genomic Analysis Of Sclerotium Rolfsii Causing The Southern Blight And Germplasm Resistance Identification In Pepper (Capsicum Annuum L)

Pepper southern blight is a destructive soil-borne fungal disease,which leads to a large area of pepper crop failure and has a great impact on agricultural production.Since the wide host range of the pathogen and its sclerotia can survive in the soil for many years,it is very difficult to manage this disease.Breeding and planting resistant varieties is the most economical and feasible way to control pepper blight.At present,the research on resistance to pepper southern blight is still lacking in China.This study was carried out including pathogen isolation,identification and biological characteristic test of seven different pepper disease sample,and the strong virulence strains was send to the company for whole genome sequencing analysis.Large-scale identification and screening of pepper germplasm resources were carried out,and the materials of middle resistant to southern blight disease were obtained for the first time.The main findings are as follows:1.After using plant pathology and molecular biology methods,the pathogenic fungus isolated from seven different regions were identified as Sclerotium rolfsii Sacc,causing the pepper southern blight.However,evolutionary analysis showed that there were evolutionary differences among the strains from different regions.The bands of ITS sequences were different in size and geographical distribution resulted in differences in virulence.CSR1 was a highly virulent strain,which was most suitable for growth at 30℃,pH 5.9 and relative humidity above 90%.2.The whole genome of CSR1 strain was sequenced using Nanopore third-generation sequencing technology combined with RNA-seq sequencing.And the obtained fungal genome size was 65.16 Mb.According to GO database comparison,4908 coding protein sequences were annotated in molecular function related items,1110 coding protein sequences were annotated in cell components,and 1556 genes were annotated in biological process.KOG database comparison showed that10,196 proteins were annotated.Compared with KEGG database,249 genes were annotated in the cell process coding proteins for their transport,regulation of cell growth and synthesis,indicating that the cell growth and protein synthesis were more active in fungal genome composition.A comparison of the CAZY database revealed 950 carbohydrate-related enzymes,of which the glycoside hydrolase family was the most common,accounting for up to 46.45%of all carbohydrate enzymes.Through further analysis and prediction of fungal effector proteins from secreted proteins,a total of 24 proteins were obtained.After comparison with PHI database,they were noted to be functionally related to attenuated virulence,mixed type and effector factor,respectively,laying a foundation for further study on the interaction between pathogenic fungi and plants.3.The GFP gene was successfully introduced into the CSR1 strain using hygromycin as the screening marker.After seven generations of continuous culture,the expression of green fluorescent protein gene was stable,and there was no significant difference between the virulence and the wild-type strain.Thus,PEG-CaCl₂-mediated protoplast transformation was successfully constructed,which provided technical support for the further study on the function of pathogenicity related genes of pepper.4.The identification standard of disease resistant to pepper southern blight was established for the first time.The optimal inoculation method and the classification method of disease investigation after inoculation were studied,and the evaluation standard of disease classification and the evaluation method of population disease resistance were established.In order to determine the effectiveness and stability of the proposed method,this study adopted the membership function,cluster analysis and principal component analysis method to30 pepper materials with different sense of resistance phenotype of physiological and biochemical index determination results of comprehensive evaluation,the result was consistent with the phenotypic evaluation method,thus the evaluation technology system of resistance to pepper southern blight was established.5.Based on the established indoor identification system of resistance to pepper southern blight,more than 600 pepper germplasm resources with different resistance to soil-borne diseases such as Phytophthora root rot and bacterial wilt were identified by the pepper research group of Hunan Vegetable Research Institute,and 215 pepper core germplasm resources of different types were screened out.The215 materials were evaluated and analyzed for three times,and 14 intermediate resistant varieties were screened out.This is the first time to screen the pepper germplasm with resistance or above to southern blight pathogen,which provides a material basis for further analysis of the molecular mechanism of resistance to southern blight disease in pepper,the creation of resistant resources and selection of resistant varieties.