Genetic Diversity Analysis And Potential Pathogenic Factors Identification Of Stagonosporopsis Cucurbitacearum In Seed Pumpkin

Seed pumpkin is a cultivated species of cucurbita genus,which was one of the main cash crops in Heilongjiang province in China.Gummy stem blight(GSB)is formed by the infection of fungus,which belongs to Didymella genus,has a rapid spread and wide range.In recent years,a large area of GSB has occurred in Heilongjiang province due to the continuous cultivation of seed pumpkin year after year that seriously reduced both pumpkin yields and quality.However,the dominant population and disease caused genes of GSB of seed pumpkin are not clear uptonow which seriously affect the control effect.In view of this,we have performed a field study on the main producing areas of seed pumpkin in Heilongjiang province from 2012 to 2016,and collected sick leaves of GSB.Combined with morphology and ITS sequence for genetic diversity analysis of seed pumpkin GSB on the basis of pathogen isolation,and high-throughput sequencing was used to further obtain the assembly information of whole genome,and the differential expression analysis of transcripts before and after the fungi infection of the host was conducted for mining the disease-caused genes.The results are as follows:To elucidate the genetic diversity and dominant population of GSB in Heilongjiang province,30 marked different GSB isolates were identified and purified based on the characteristics of colony morphology,growth rate and hyphae diameter from across northeast China,which were all Stagonosporopsis cucurbitacearum(Sc.)from subjected to internal transcribed spacer(ITS)sequencing analysis.So we concluded that Sc.was the main dominant and widely distributed fungal species in this region.Comprehensive colony color,shape,growth rate,as well as differences,seed pumpkin GSB were divided into 7 types of morphology,respectively named type?-type?.In addition,to fully understand the Sc.and epidemic regularity,the effect of external conditions on the growth of mycelium of seed pumpkin was studied,the final optimal conditions were: glucose as the optimum carbon source,peptone as the best nitrogen source,the p H value of 6.0 and growth temperature was 25?.The isolated strains were divided into two genotypes by multilocus phylogenetic analysis of genome sequence.By analyzing ITS conserved sequences of these phenotypically diverse groups,we found that Cucurbita GSB pathogens broadly shared two motifs that contained unique sequence variations of two groups in addition to common identical motifs.Ultimately,this study provided useful data for rapid and accurate identification of Sc.and diagnosis in early symptoms of Cucurbita GSB.To promote the study of growth development and pathogenicity molecular regulation mechanism of GBS,we performed whole-genome high-throughput sequencing and assembly of Stagonosporopsis cucurbitacearum(Sc.)by constructing the c DNA library of Sc.representative strain zq-1,Pac Bio RS II sequencing was performed to obtain 5.24 Gb of high-quality genome data,after filtering the original data and quality control data were assembled.The whole genome sequence of Sc.was obtained with the genome size of 35.28 Mb,and 9844 genes were predicted.Among them,237 non-coding RNAs,1024 proteins containing signal peptides,2066 transmembrane proteins and 756 secretory proteins from 82 families were identified based on the Rfam database.Three proprietary databases CAZyme,TCDB and PHI were also used to predict secreted proteins with the number of 605,130 and 2869,respectively.30,833 of 6-m A methylation sites and 1,228,069 of 4-m C DNA methylation sites were identified.Genome assembly and annotation information of Sc.providing basis for comprehensive and in-depth analysis of genomic information and further pathogenic exploration.Meanwhile,we found that Sc.and Leptosphaeria maculans(Lm.)were relatively similar in terms of genome length(35.28 Mb vs.45.12 Mb)and protein-coding genes(9844 vs.12,469).It was speculated that these two straits were closely related in the evolutionary terms.The similar genes of Sc.and Lm.were found in collinearity analysis laid the foundations for further revealing the potential gene function of Sc.,which were elucidating the evolutionary relationship of species and exploring the internal structure of genome.In the present study,transcriptome analyses was performed to identify differentially expressed genes(DEGs)relevant to GSB infection.RNA-seq was perforned on six samples,44.62 Gb clean datas with a Q30 base percentage of 92.38% or higher were obtained.All the single genes were annotated according to the expression amount of genes in different samples,341 differentially expressed genes were identified,including 170 up-regulated genes and 171 downregulated genes.GO functional annotation and KEGG pathway analysis were performed on these DEGs,and 52 genes were predicted to be pathogenic genes,in which 34 genes were involved in multiple molecular function such as hydrolysis enzyme,protein kinase,phosphatase and cellulase,and 15 genes were involved in biological process.Finally,three genes were related to mitogen-activated protein kinase(MAPK)pathway,ABC transporter and endocytosis,which were likely performed an important role in the growth and pathogenesis of GSB.Identification of DEGs should enhance our understanding of pathogenic genes involved in pumpkin infection by Sc.and should facilitate the systematic investigation of pathogenic gene expression patterns and host-pathogen interactions.