Etiology Of Tea-Oil Tree Anthracnose,Analysis Of Genetic Diversity And CaCUTl Gene Function In Colletotrichum Camelliae

As a serious disease of tea-oil tree,anthracnose occurs widely in the production area of Camellia oleifera,causing severe economic losses and posing a huge threat to tea-oil production.However,the studies on tea-oil tree anthracnose were limited.In this study,the pathogen of tea-oil tree anthracnose from the main production areas was analyzed.Based on the morphological investigation and multi-loci gene sequences analysis,the pathogen species of tea-oil tree anthracnose were identified,and the biological characteristics were compared among different species,such as pathogenicity,fungicides sensitivity.The genetic diversity,genetic variation and population structure of C.camelliae were analyzed by using ISSR molecular markers.At the same time,the function of the CaCUT1 gene was demonstrated by using Agrobacterium tumefaciens mediated transformation(ATMT)method.The main results are as follows:A total of 232 strains were isolated from the fruit and leaf samples of tea-oil tree collected from Hubei,Anhui,Hunan,Zhejiang,Fujian,Jiangxi,Guangxi and Guangdong provinces.According to the pathogenic test and the differences of colony morphology,growth rate,conidia and appressorium morphology,the 232 isolates were identified to be five species: C.camelliae,C.fructicola,C.siamense,C.aenigma and C.gloeosporioides.To further confirm the results of morphological identification,the ITS,TUB2,ACT,CAL,GAPDH,and CHSI-encoding genes were used to conduct phylogenetic analyses,and the 232 strains were also clustered into five groups: C.camelliae,C.fructicola,C.siamense,C.aenigma and C.gloeosporioides groups.Among the 232 strains,170 strains were C.camelliae,which accounted for 73.3% and was the dominant species,and widely distributed in Hubei,Hunan,Zhejiang,Anhui,Guangdong,Jiangxi,Fujian and Guangxi.C.camelliae maily damaged to fruits of Ca.oleifera.Fifty four strains were C.fructicola,which accounted for 23.3%,and widely distributed in Hubei,Hunan,Zhejiang,Anhui,Guangdong,Jiangxi and Guangxi.C.fructicola belonged to the dominant species on leaves and mainly damaged to leaves of Ca.oleifera.The occurrence frequency of C.siamense,C.aenigma and C.gloeosporioides was low.In addition,the anthracnose on leaves and fruits of tea-oil tree in the same field or the same tree could be caused by the same or different species.The pathogenicity results showed that there were significant differences among the 38 strains of five species and C.camelliae showed strong virulence to leaves and fruits.The fungicides sensitivity showed that 38 strains of 5 species were most sensitive to prochloraz and resistant to carbendazim to different degrees.Through the analysis of ?-tubulin gene of C.camelliae,the nucleotide GAG changed to GCG at the codon 198 of HR and R strains,leading to the substitution of amino acid from glutamic acid(Glu)to alanine(Ala).Also the nucleotide TTC changed to TAC at the codon 200 of MR strains,leading to the amino acid change from phenylalanine(Phe)to tyrosine(Tyr).ISSR-PCR was used to analyze the genetic diversity of C.camelliae isolated from seven provinces.The result showed that C.camelliae had abundant genetic diversity,the percentage of polymorphic bands(PPB)was 98.99%;Nei's genetic diversity index(H)was 0.28;Shannon information index(I)was 0.43,indicating that genetic variation was accumulated in the long-term evolution process.The results of genetic variation and population structure showed that the genetic differentiation of C.camelliae existed to different degrees in the geographical populations of Hubei,Zhejiang,Jiangxi,Hunan and Guangxi.The diversity among the five populations was higher than that within individual populations.The intra-population diversity in Hubei population was higher than inter-population diversity,while the inter-population diversity was higher than intra-population diversity in Hunan,Zhejiang,Jiangxi and Guangxi populations.The results of cluster analysis showed that there were certain genetic variations among populations and individuals,and the genetic diversity had no correlation with geographical origins.The CaCUT1 gene including a 727 bp open reading frame(ORF)and a 52 bp intron was obtained from C.camelliae using specific primers cutF and cutR.The CaCUT1 showed high similarity with those from other ascomycetes.The predicted molecular weight was 23.4 kDa,the isoelectric point(pI)was 6.58.The CaCUT1 contained the domain protein sequences from 44 to 223 amino acids,the GYSQG motif signature pattern for cutinases,and a peptide sign whose the cleavage site was between the 16 th and 17 th amino acids.The 3D structure of the CaCUT1 protein had ?-helix and ?-strand regions.The CaCUT1 gene was knocked-out and complemented using ATMT.The knockout transformants exhibited significant decreases in cutinase activity and virulence compared with the wild-type strain.The cutinase activity and virulence of complemented transformants showed the same as that of the wild-type stain.These results suggested that the CaCUT1 gene played an important role in the pathogenesis of C.camelliae.