Population Genetic Analyses Of The Fungal Pathogen Colletotrichum On Tea-Oil Trees In China And Characterization Of A MAPK Gene CfPMK1 In The Pathogen

Camellia oleifera is native to China and is cultivated in many parts of southern China.Colletotrichum fungi can infect C.oleifera trees and result in huge economic losses.In this paper,filamentous fungus Colletotrichum spp were collected from 10 provinces in south China:Hunan,Jiangxi,Guangxi,Hubei,Fujian,Hainan,Jiangsu,Chongqing,Yunnan and Guizhou.We identified the pathogen causing stem and leaf anthracnose on oil-tea in China.Population genetics and epidemiology of Colletotrichum was studied.The results provide a theoretical basis for controlling anthracnose of C.oleifera.The main results were as follows:(1)Identification of pathogen species of C.oleifera in China.A total of 406 Colletotrichum strains were isolated from oil tea plants in 10 provinces.Pathogenicity tests showed that all of the isolates could infect oil-tea leaves and fruit.However,there were differences among strains in virulence properties such as the time of spot appearance.For each isolate,gene fragments at rDNA-ITS,CAL,GAPDH,ACT,GS and TUB region were amplified and sequenced.Based on the morphology and multiple gene sequences,the 406 isolates were identified as C.fructicola,C.siamense,C.gloeosporioides,C.horii and C.camelliae.C.fructicola was the most frequently isolated accounting for 71%.The conidia production of the five pathogens was significantly different(P<0.01).(2)Population genetics and epidemiology of Colletotrichum fructicola.Here we analyzed 167 isolates of C.fructicola obtained from the leaves of tea-oil tree C.oleifera at 15 plantations in seven Chinese provinces.Multilocus sequence typing was conducted for all isolates based on DNA sequences at fragments of four genes:the internal transcribed spacers of the nuclear ribosomal RNA gene cluster(539 bp),calmodulin(633 bp),glutamine synthetase(711 bp),and glyceraldehyde-3-phosphate dehydrogenase(190 bp),yielding 3.52%,0.63%,8.44%,and 7.89%of single nucleotide polymorphic sites and resulting in 15,5,12 and 11 alleles respectively at the four gene fragments in the total sample.The combined allelic information from all four loci identified 53 multilocus genotypes with the most frequent represented by 21 isolates distributed in eight tea-oil plantations in three provinces,consistent with long-distance clonal dispersal.However,despite evidence for clonal dispersal,statistically significant genetic differentiation among geographic populations was detected.In addition,while no evidence of recombination was found within any of the four gene fragments,signatures of recombination were found among the four gene fragments in most geographic populations,consistent with sexual mating of this species in nature.(3)The molecular genetic variation of C.fructicola for different host plants.In this study,a total of 108 strains were isolated from Hainan,18 haplotypes were identified based on sequence anlyses at three gene fragments.The most common sequence type,haplotype 11,was found in 70 samples and distributed across every geographic and host population.AMOVA test showed that 3%of genetic differentiation occurred among populations and 97%within population.The Fst and Nm values showed there is a very low genetic divergence among the populations and the high level of gene flow was among populations.There are genetic recombination among some populations of C.fructicola from Oil-tea camellia and the other host plants.(4)Characterization of a MAPK gene CfPMKl in the pathogen of C.fructicola.We used Fus3 sequence of Saccharomyces cerevisiae do Blast analysis in genome database of C.fructicola.We obtained a homologous gene and named CfPMK1.Disruption of CfPMKl in C.fructicola resulted in a mutant with reduced mycelial growth and conidia formation.Conidia and mycelia of Cfpmk1 mutant can't form appressorium and appressorium-like structure formation.Cfpmk1 mutant was sensitive to cell wall inhibitors,can't undergo sexual reproduction,and was avirulent on host plants.Overall,the results indicate that the Fus3/Kssl-related MAPK gene has a conserved role in infection structure formation and pathogenicity in phytopathogenic fungi.Overall,isolates of Colletotrichum spp.on C.oleifera were grouped into six species including one new taxon in China.And C.fructicola was the predominant pathogen.There are two ways of breeding,asexual reproduction and sexual reproduction.Genetic differentiation among geographic populations was detected.And there is a very low genetic divergence among the populations and the high level of gene flow was among populations.The CfPMK1 gene plays important roles in growth,conidiation,appressorium and pathogenicity in C.oleifera.