Colletotrichum Species Associated With Anthracnose Of Watermelon And Analysis Of Pathogenicity Related Genes In Colletotrichum Magnum

With the increase of watermelon acreage in China,the occurrence and damage of watermelon anthracnose in various watermelon-cultivation areas are increasing,not only causing serious fruit rot,but also affecting the preservation and transportation of watermelon.In this study,samples of watermelon anthracnose were collected from different cultivation areas in China,and the pathogens were isolated by tissue separation and purification to demonstrate the genetic diversity and pathogenicity diversity of Colletotrichum spp.The results provided effective information for diagnosis of watermelon anthracnose and fulfilled the database of Colletotrichum classification.Moreover,the C.magnum was analyzed,based on the genome and transcriptome analyses and a mutant library of C.magnum was constructed with Agrobacterium tumefaciensmediated transformation(ATMT).This work provided foundations for further exploration of mechanisms of pathogenicity of C.magnum and the development of effective management strategies for watermelon anthracnose control.The main results are as follows:1.Diversity of Colletotrichum spp.associated with anthracnose of watermelon in China.In this study,a total of 224 samples with symptoms of watermelon anthracnose were collected from 8 main production areas in China.A total of 526 strains were isolated by separation and purification.Phylogenetic analyses using seven loci(ITS,Gadph,Chs1,His3,Act,Tub2,and Gs)coupled with morphology of 146 representative isolates showed that they belonged to 12 known species of Colletotrichum,including C.aenigma,C.chlorophyti,C.fructicola,C.jiangxiense,C.karstii,C.magnum,C.nymphaeae,C.nigrum,C.orbiculare,C.plurivorum,C.sojae,and C.truncatum and three new species,here described as C.citrulli,C.kaifengense,and C.qilinense.Colletotrichum orbiculare was the dominant species.Pathogenicity tests showed that all isolates of 15 Colletotrichum species described above were pathogenic,with C.magnum and C.kaifengense being the most aggressive to leaves and fruits,respectively.This is the first report of C.aenigma,C.chlorophyti,C.fructicola,C.jiangxiense,C.nymphaeae,C.nigrum,C.plurivorum,and C.sojae on watermelon.2.Genes related to conidia germination and appressorium formation of C.magnum.In ordre to reveal the regulatory mechanisms involved in fungal growth and pathogenicity,the C.magnum genome of the representative isolate CAASZK4 was sequenced and assembled by high-throughput whole genome sequencing.A total of 4.59 Gb high-quality genomic sequences were obtained by PacBio RS Ⅱ sequencing.After processing the sequence data to filter out low-quality reads,genomic sequences of 61.87 Mb were generated by hierarchical genome-assembly process.A total of 13,943 genes were predicted,including 1,853 genes encoding proteins with signal peptides,3,177 transmembrane protein encoding genes,1,411 secretory protein encoding genes and 269 effector protein encoding genes.Through transcriptomic analysis at different conidia germination stages of C.magnum,we identified 13,103 differentially expressed genes(DEGs).Among them,8426 were up regulated and 4677 were down regulated.Altogether,116 DEGs(encoding 10 chitinase,27 mitogen activated protein kinase(MAPK),34 ABC transporters,1 autophagy protein,13 fatty acid degradation,6 peroxidases,1 calcium-transport P-type ATPase,2 allantoin,22 melanin synthesis)were detected.This study provides a useful foundation for further insights into molecular mechanisms of pathogenicity of C.magnum,and to develop more effective management strategies of watermelon anthracnose.3.Identification of pathogenicity associated genes of C.magnum by random insertion mutagenesis.To explore the molecular mechanisms underlying the pathogenicity of C.magnum,we developed an Agrobacterium tumefaciens-mediated transformation(ATMT)system for the genetic transformation of C.magnum.The efficiency of ATMT was 130-360 transformants per 108 conidia.Southern blot analysis showed that approximately 75%of the transformants contained single copy of T-DNA.Pathogenicity test revealed that three transformants completely lost the pathogenicity.The T-DNA integration sites(TISs)was rescued by thermal asymmetric interlaced PCR(TAIL-PCR)and whole genome re-sequencing.In one transformant,the T-DNA integrated into the intron region of the gene which encoded a protein containing AP-2 complex subunit σ,and simultaneously gene deletions were observed.Two deleted genes encoded the transcription initiation protein SPT3 and a hypothetical protein,respectively.In the second transformant,the T-DNA had integrated into the 5’-flanking regions of a gene with similarity to the MYO5 encoding Myosin I of Pyricularia oryzae(78%).In the third transformant,the T-DNA integrated into the exon regions of two adjacent genes.One was 5’-3’ exoribonuclease 1 encoding gene(XRN1)and the other was RBBP4,which encoded a WD-repeat protein retinoblastoma binding protein 4,the homolog of the negative regulator MSl1 of the Ras-cyclic AMP pathway in the yeast Saccharomyces cerevisiae.