【作者】 王义勋；

【导师】 黄俊斌；

【作者基本信息】 华中农业大学 ， 植物病理学， 2019， 博士

【摘要】 炭疽病是我国油茶产区油茶上重要病害之一,严重影响了油茶产业健康发展。目前,关于油茶炭疽病的系统性研究报道较少。本文研究了我国油茶主产区油茶炭疽病的病原,根据形态学和多基因序列分析进行病原菌种级鉴定,比较了不同种间炭疽病菌的致病力、药剂敏感性等生物学特性差异;以优势种山茶炭疽菌为材料,采用ISSR分子标记技术研究了山茶炭疽菌的遗传多样性;采用ATMT介导转化进行了角质酶CaCUT1基因的敲除和互补,研究了该基因在致病过程中的作用。主要研究结果如下:(1)从湖北、安徽、湖南、浙江、福建、江西、广西和广东8个省(自治区)20个油茶园发病的油茶果实和叶片上分离获得232个菌株。在致病性测定的基础上,根据菌落形态、生长速率、分生孢子和附着胞形态等特征的差异,从232个菌株中鉴定出山茶炭疽菌(C.camelliae)、果生炭疽菌(C.fructicola)、暹罗炭疽菌(C.siamense)、隐秘炭疽菌(C.aenigma)和胶孢炭疽菌(C.gloeosporioides)5种病原菌。采用ITS、ACT、TUB2、CAL、CHS1和GAPDH基因序列联合分析进行分子鉴定,232个菌株被聚类成5个组,即C.camelliae聚类组、C.fructicola聚类组、C.siamense聚类组、C.aenigma聚类组和C.gloeosporioides聚类组,进一步佐证了形态学鉴定结果。232个炭疽菌中,山茶炭疽菌有170个菌株,占73.3%,属于优势种群,在湖北、湖南、浙江、安徽、广东、江西、福建和广西等8省(自治区)油茶上广泛分布;该病菌以危害果实为主。果生炭疽菌有54株,占总菌株数的23.3%,在湖北、湖南、浙江、安徽、广东、江西和广西等7省(自治区)广泛分布,属于叶片上炭疽菌优势种群,以危害叶片为主。暹罗炭疽菌、隐秘炭疽菌和胶孢炭疽菌的菌株数出现频率较低。此外,还发现在同一田块或同一棵油茶树叶片和果实的炭疽病可以由相同种或不同种的炭疽菌引起。5个种的38个代表性菌株的致病力测定结果表明,炭疽菌种间致病力差异显著,其中,山茶炭疽菌对油茶叶片和果实的致病力最强。不同种的炭疽菌对杀菌剂敏感性也存在显著差异,对咪鲜胺表现最为敏感,而对多菌灵表现出不同程度的抗性。通过β-tubulin基因分析,山茶炭疽菌高抗菌株和抗性菌株的基因密码子第198位由GAG变为GCG,相应氨基酸由谷氨酸(Glu)变为丙氨酸(Ala);中等抗性菌株的基因密码子第200位由TTC变为TAC,相应氨基酸由苯丙氨酸(Phe)变为酪氨酸(Tyr)。(2)应用ISSR-PCR分子标记技术对山茶炭疽菌的遗传多样性的分析结果表明,在物种水平上,7个省16个地理种群的山茶炭疽菌的多态性条带的百分比(PPB)为98.99%,Nei?s基因多样性指数(H)为0.28,Shannon信息指数(I)为0.43,山茶炭疽菌具有丰富的遗传多样性,表明山茶炭疽菌在长期进化过程中积累了较高的遗传变异。遗传变异和种群结构分析结果表明,山茶炭疽菌在湖北省、浙江省、江西省、湖南省和广西壮族自治区的地理种群内存在不同程度的遗传分化,5个省之间的种群间多样性高于种群内多样性;在同一个省内,湖北地理种群间多样性高于种群内多样性,而浙江、江西、湖南和广西的地理种群内多样性高于种群间多样性。从菌株的群体和个体聚类分析结果表明,菌株群体之间和个体之间都存在一定的遗传变异,且菌株的遗传多样性与其地理来源无明显相关性。(3)用特异性引物cutF和cutR扩增山茶炭疽菌基因组DNA获得角质酶基因CaCUT1,该基因开放阅读框序列长度为727bp,含有52bp内含子,与其它子囊菌门真菌的角质酶基因具有较高的相似性;CaCUT1基因蛋白分子量为23.4 kDa;等电位点(pI)为6.58;蛋白序列的结构域为从44个氨基酸到223个氨基酸。CaCUT1基因含有角质酶基因所含有的-GYSQG-保守序列模块,并且存在一个肽信号,剪切位点位于第16个氨基酸与第17个氨基酸之间;含有α-螺旋和β-折叠片,且β-折叠片被α-螺旋包围。利用农杆菌介导转化(ATMT)基因同源重组技术,获得山茶炭疽菌CaCUT1基因敲除转化子和互补转化子,敲除转化子的角质酶活性和致病力都有显著降低;而互补转化子的产酶活性和致病性与野生型菌株无显著差异,表明角质酶基因CaCUT1在致病中起着重要作用。更多还原

【Abstract】 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 β-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 α-helix and β-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.更多还原