黄淮海地区夏玉米弯孢叶斑病菌遗传多样性分析
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摘要
【目的】针对黄淮海地区发生的玉米弯孢叶斑病,通过分子生物学技术,明确其致病菌新月弯孢(Curvularia lunata)在不同地区和年份间的遗传差异及亲缘关系,为研究该病害的发生和流行提供数据资料。【方法】对2013、2016和2017年采集自黄淮海地区5省(河南、河北、山东、安徽、江苏)的病样进行分离,并采用形态学和分子生物学(ITS和EF-1α序列分析)对分离到的菌株进行鉴定,共获得175个新月弯孢菌株。从哥伦比亚大学开发的通用引物中筛选出13条多态性高、重复性好的引物,利用筛选出的引物对175个新月弯孢菌株进行ISSR-PCR扩增,利用Popgen32软件计算多态性比率、Shannon’s信息指数、群体间的遗传距离和遗传相似性,使用NTsys2.10e软件进行UPGMA聚类分析和基于遗传相似系数的主坐标分析,构建聚类分析图和散点图。【结果】利用筛选出的引物对175个菌株进行PCR扩增,共获得105条多态性条带,多态性比率为100%。在群体平均水平上,基因多样性水平(H)为0.3867,Shannon’s的信息指数(I)为0.5682,表明玉米弯孢叶斑病菌具有丰富的遗传多样性;不同地理种群间的遗传多样性存在一定差异,河南和安徽种群遗传多样性最高,江苏种群较低;年度间相同地理来源的菌群亲缘关系较远,相同年份不同地理来源菌群亲缘关系较近。聚类分析显示所有菌株相似系数为0.51—0.93,在相似系数为0.59水平上,175个菌株被划分为2群5个亚群,亚群间表现出年度间的差异,不同地理种群病菌间存在基因交流,遗传相关性较高;主坐标分析结果与聚类分析结果一致,同一年份的菌株明显聚集在一起。【结论】引起黄淮海地区玉米弯孢叶斑病的病原菌群体存在较高的遗传变异,地域相邻的病菌遗传关系较近;同一地区的菌株在年度间表现出一定遗传距离,而同一年份不同地理来源的菌株遗传距离较近。引起该地区玉米弯孢叶斑病的新月弯孢菌株不是以本地菌源为主,其主要菌源可能来自南方水稻和草坪草或东南亚玉米生产区,但也存在少量存活于地表病残体上的菌株可作翌年的初侵染源。
【Objective】The objective of this study is to identify the genetic diversity and relationship of Curvularia lunata, the causal agent of maize Curvularia leaf spot occurred in different regions and years in Huang-Huai-Hai region via molecular techniques, and to provide scientific data for studying the occurrence and prevalence of the disease. 【Method】 The samples collected from five provinces(Henan, Hebei, Shandong, Anhui and Jiangsu) in Huang-Huai-Hai region in 2013, 2016 and 2017 were separated and identified by morphology and molecular biology(ITS and EF-1α sequence analysis). A total of 175 C. lunata strains were obtained. Thirteen highly polymorphic and reproducible ISSR primers were screened out from universal primers developed by Columbia University and were used to amplify the 175 C. lunata strains. Popgen32 software was used to calculate the polymorphism ratio, Shannon's information index, genetic distance and genetic similarity among populations. UPGMA cluster analysis and principal coordinate analysis based on genetic similarity coefficient were carried out by NTsys 2.10 e software, and cluster analysis map and scatter plot were constructed. 【Result】 A total of 105 polymorphic bands were obtained with a polymorphism ratio of 100%. At the group average level, the level of genetic diversity(H) was 0.3867, and Shannon's information index(I) was 0.5682, indicating that the C. lunata had abundant genetic diversity. The genetic diversity among different geographical populations was different. The genetic diversity of Henan and Anhui populations was the highest, and that of Jiangsu population was low. The flora of the same geographical origin was far away from each other during the year, and the flora from different geographical origins in the same year was relatively close. Cluster analysis showed that the similarity coefficients of all strains were 0.51-0.93. At the similarity coefficient of 0.59 level, 175 strains were divided into 2 groups and 5 subgroups. There were annual differences among the subgroups, gene exchanges among the different geographical populations, and the genetic correlation was high. The results of principal component analysis were consistent with those of cluster analysis, and the strains in the same year were obviously clustered together.【Conclusion】There was a high genetic variation in the pathogen populations causing maize Curvularia leaf spot disease in Huang-Huai-Hai region, and the genetic relationship of the adjacent pathogens was close. The strains from the same area showed a certain genetic distance in different years, while the genetic distance of strains of different geographical origins in the same year was close. The results of the analysis indicated that the main pathogen causing the leaf spot disease of C. lunata was not mainly from local source, they probably came from the southern rice and turf grass or maize production areas of Southeast Asia, but occasionally a few strains that survived on the ground surface could be the initial source of infection in the following year.
【Objective】The objective of this study is to identify the genetic diversity and relationship of Curvularia lunata, the causal agent of maize Curvularia leaf spot occurred in different regions and years in Huang-Huai-Hai region via molecular techniques, and to provide scientific data for studying the occurrence and prevalence of the disease. 【Method】 The samples collected from five provinces(Henan, Hebei, Shandong, Anhui and Jiangsu) in Huang-Huai-Hai region in 2013, 2016 and 2017 were separated and identified by morphology and molecular biology(ITS and EF-1α sequence analysis). A total of 175 C. lunata strains were obtained. Thirteen highly polymorphic and reproducible ISSR primers were screened out from universal primers developed by Columbia University and were used to amplify the 175 C. lunata strains. Popgen32 software was used to calculate the polymorphism ratio, Shannon's information index, genetic distance and genetic similarity among populations. UPGMA cluster analysis and principal coordinate analysis based on genetic similarity coefficient were carried out by NTsys 2.10 e software, and cluster analysis map and scatter plot were constructed. 【Result】 A total of 105 polymorphic bands were obtained with a polymorphism ratio of 100%. At the group average level, the level of genetic diversity(H) was 0.3867, and Shannon's information index(I) was 0.5682, indicating that the C. lunata had abundant genetic diversity. The genetic diversity among different geographical populations was different. The genetic diversity of Henan and Anhui populations was the highest, and that of Jiangsu population was low. The flora of the same geographical origin was far away from each other during the year, and the flora from different geographical origins in the same year was relatively close. Cluster analysis showed that the similarity coefficients of all strains were 0.51-0.93. At the similarity coefficient of 0.59 level, 175 strains were divided into 2 groups and 5 subgroups. There were annual differences among the subgroups, gene exchanges among the different geographical populations, and the genetic correlation was high. The results of principal component analysis were consistent with those of cluster analysis, and the strains in the same year were obviously clustered together.【Conclusion】There was a high genetic variation in the pathogen populations causing maize Curvularia leaf spot disease in Huang-Huai-Hai region, and the genetic relationship of the adjacent pathogens was close. The strains from the same area showed a certain genetic distance in different years, while the genetic distance of strains of different geographical origins in the same year was close. The results of the analysis indicated that the main pathogen causing the leaf spot disease of C. lunata was not mainly from local source, they probably came from the southern rice and turf grass or maize production areas of Southeast Asia, but occasionally a few strains that survived on the ground surface could be the initial source of infection in the following year.
引文
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