东北地区茄子黄萎病菌致病力分化与ISSR遗传变异分析
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摘要
为了揭示东北地区茄子黄萎病菌的遗传变异,从东北地区4个省49个县(市)的茄子主产区采集黄萎病菌株,从中分离、鉴定并选取80个单孢菌株,对其培养特性、致病力和ISSR(Inter-simple sequencerepeat)遗传分化进行了研究。菌株培养性状研究表明,在PDA培养基上存在4种不同的培养类型,其中菌核型菌株最多,占供试菌株的56.25%,菌丝型菌株最少,仅占供试菌株的7.5%。通过鉴别寄主接种试验,对供试菌株进行致病力分化测定,明确了东北地区茄子主产区存在黄萎病菌强(Ⅰ型)、中(Ⅱ型)、弱(Ⅲ型)3种致病力类型,分别占供试菌株的26.25%,31.25%和42.50%。特异性引物PCR检测结果显示,在供试菌株中落叶型菌株占31.25%,以中、强致病力菌株为主;非落叶型菌株为68.75%,多为弱致病力类型菌株。ISSR的聚类分析结果表明,东北地区茄子黄萎病菌的遗传分化较小,并且遗传分化与地理来源存在明显的相关性。
This study revealed the genetic variation of eggplant Verticilium dahlia in Northeast China by studying the characteristics,pathogenicity and ISSR(Inter-simple sequence repeat)genetic variation of 80 representative eggplant V. dahliae strains collected from 49 major eggplant-growing counties in four northeastern provinces. The results showed that 80 tested strains were divided into 4 different culture types(A to D)according to cultural characteristics on PDA medium. Among them,sclerotia type isolates were the most(56.25% of the tested strains)and mycelium type isolates were the least(7.5% of the tested strains). The pathogenicity test showed that 80 isolates had obvious pathogenicity differentiation and could be classified into three pathogenic types,i.e. strong(Ⅰ),moderate(Ⅱ)and weak(Ⅲ),which occupied for 26.25%,31.25% and 42.50% of the total tested strains,respectively. According to the results of PCR,the deciduous isolates which accounted for 31.25% were mainly moderate and strong pathogenic strains;the non-deciduous strains which accounted for 68.75% were mostly weak pathogenic strains. ISSR analysis indicated that the genetic variation of 80 isolates was small and related to geographic region.
This study revealed the genetic variation of eggplant Verticilium dahlia in Northeast China by studying the characteristics,pathogenicity and ISSR(Inter-simple sequence repeat)genetic variation of 80 representative eggplant V. dahliae strains collected from 49 major eggplant-growing counties in four northeastern provinces. The results showed that 80 tested strains were divided into 4 different culture types(A to D)according to cultural characteristics on PDA medium. Among them,sclerotia type isolates were the most(56.25% of the tested strains)and mycelium type isolates were the least(7.5% of the tested strains). The pathogenicity test showed that 80 isolates had obvious pathogenicity differentiation and could be classified into three pathogenic types,i.e. strong(Ⅰ),moderate(Ⅱ)and weak(Ⅲ),which occupied for 26.25%,31.25% and 42.50% of the total tested strains,respectively. According to the results of PCR,the deciduous isolates which accounted for 31.25% were mainly moderate and strong pathogenic strains;the non-deciduous strains which accounted for 68.75% were mostly weak pathogenic strains. ISSR analysis indicated that the genetic variation of 80 isolates was small and related to geographic region.
引文
Hui Hui,Guo Xiao-ping,Zhu Long-fu,Zhang Xian-long.2013.Pathogenicity differentiation of defoliation strains of Veticillium dahliae in the cotton.Acta Phytophylacica Sinica,40(5):445-449.(in Chinese)惠慧，郭小平，朱龙付，张献龙.2013.落叶型棉花黄萎病菌的致病力分化.植物保护学报，40(5):445-449.
Li Hai-lian.2005.The characterization and genomic DNA ISSR fingerprinting of Verticillium dahliae of eggplant isolates[M.D.Dissertation].Nanjing:Nanjing Agricultural University.(in Chinese)李海莲.2005.茄子黄萎病病原菌鉴定及其ISSR分子指纹分析[硕士论文].南京：南京农业大学.
Lin Mi,Zhang Pei-zhi,Guan Zhong-yan,Lu Shu-wen,Niu Bai-zhong,Zhang Jun-min,Qu Hong-yun,Liu Jing-lan,Zhang Hui.2008.Research on characteristics and pathogenicity differentiation of Verticillium dahliae strains in Heilongjiang.China Vegetables,(7):27-28.(in Chinese)林密，张佩芝，关钟燕，卢淑雯，牛柏忠，张军民，曲红云，柳景兰，张慧.2008.黑龙江省茄子黄萎病病原菌及其致病力的分化.中国蔬菜，(7):27-28.
Mercado-Blanco J,Rodríguez-Jurado D,Parrilla-Araujo S,Jiménez-D??az R M.2003.Simultaneous detection of the defoliating and nondefoliating Verticillium dahliae pathotypes in infected olive plants by duplex,nested polymerase chain reaction.Plant Disease,87:1487-1494.
Pérez-Artés E,García-Pedrajas M D,Bejarano-Alcàzar J,Jiménez-Díaz R M.2000.Differentiation of cotton-defoliating and nondefoliating pathotypes of Verticillium dahliae by RAPD and specific PCR analyses.European Journal of Plant Pathology,106:507-517.
Qiao Yan-yan,Yang Zhao-guang,Li Jie,Zhang Xing-hua.2015.Research on cultural characteristics and pathogenicity differentiation of Verticillium dahliae strains in Jiangxi.Acta Agriculturae Jiangxi,27(1):47-49.(in Chinese)乔艳艳，杨兆光，李捷，张兴华.2015.江西棉花黄萎病菌培养特性及致病力分化研究.江西农业学报，27(1):47-49.
Raeder U,Broda P.1985.Rapid preparation of DNA from filamentous fungi.Lett Appl Microbiol,1:17-20.
Ran Hong-chang,Xiao Yan-nong,Jiang Dao-hong,Li Guo-qing.2005.Pathogenicity differentiation of Verticillium dahliae strains and polymerphisms based on RAPD analysis in Hubei Province.Journal of Huazhong Agricultural University,24(5):442-447.(in Chinese)冉鸿昌，肖炎农，姜道宏，李国庆.2005.湖北省棉花黄萎病菌致病力分化和遗传多样性分析.华中农业大学学报，24(5):442-447.
Schnathorst W C,Mathre D E.1966.Host range and differentiation of a severe form Verticillium alboatrum in cotton.Phytopathology,56:1156-1161.
Shi Bo,Fang Rong,Hu Jian-kun,Zhou Kun-hua,Yuan Xin-jie,Wu Yin,Huang Rui-rong,Chen Xue-jun.2016.Research advance in integrated control techniques of eggplant Verticillium wilt.Acta Agriculturae Jiangxi,28(12):70-74.(in Chinese)石博，方荣，胡建坤，周坤华，袁欣捷，吴茵，黄瑞荣，陈学军.2016.茄子黄萎病综合防治技术研究进展.江西农业学报，28(12):70-74.
Song Xiao-xuan,Zhu He-qin,Guo Jin-cheng.1997.Study on pathogenicity differentiation for Verticillium dahliae Kleb.Anyang strains in cotton.Scientia Agricultura Sinica,30(1):13-18.(in Chinese)宋晓轩，朱荷琴，郭金城.1997.棉花黄萎病（Verticillium dahliae Kleb.）安阳菌系致病力分化研究.中国农业科学，30(1):13-18.
Verhalen L M,Brinkerhoff L A,Fun K C,Morrison W C.1971.A quantitative genetic study of Verticillium wilt resistance among selected lines of upland cotton.Crop Science,11(3):407-412.
Wang Guo-ning,Zhao Gui-yuan,Yue Xiao-wei,Li Zhi-kun,Zhang Yan,Zhang Gui-yin,Wu Li-qiang,Wang Xing-fen,Ma Zhi-ying.2012.Pathogenicity and ISSR genetic differentiation of Verticillium dahliae isolates from cotton growing areas of Hebei Province.Cotton Science,24(4):348-357.(in Chinese)王国宁，赵贵元，岳晓伟，李志坤，张艳，张桂寅，吴立强，王省芬，马峙英.2012.河北省棉花黄萎病菌致病性与ISSR遗传分化.棉花学报，24(4):348-357.
Xu F,Yang L,Zhang J,Guo X P.2012.Prevalence of the defoliating pathotype of Verticillium dahliae on cotton in central China and virulence on selected cotton cultivars.Journal of Phytopathology,160:369-376.
Xu Fei.2012.Characterization of the Verticillium dahliae defoliating pathotypeon cotton in central China and factors associated with prevalence of this severe pathotype[Ph.D.Dissertation].Wuhan:Huazhong Agricultural University.(in Chinese)徐飞.2012.落叶型棉花黄萎病菌在华中棉区广泛分布及其原因分析[博士论文].武汉：华中农业大学.
Yu Chun,Li Shu-ying,Liu Fang-zhi,Zhu Jia-bao,Cheng Fu-ru.2015.Analysis of characters and pathogenicity of cotton Verticillium dahliae cultivation in Anhui Province.Cotton Sciences,37(1):19-23.(in Chinese)於春，李淑英，刘方志，朱加保，程福如.2015.安徽省棉花黄萎病菌培养性状与致病性分析.棉花科学，37(1):19-23.
Yu Miao.2013.Identification of the pathogens of Verticillium wilt on eggplant and screening of resistant germplasm[M.D.Dissertation].Baoding:Agricultural University of Hebei.(in Chinese)于淼.2013.河北省茄子黄萎病菌的分离鉴定及抗性资源筛选[硕士论文].保定：河北农业大学.
Yuan Hong-bo,Ai Ni-jiang,Zhao Jian-jun,Chen Tian-zi,Yang Yu-wen,Liu Ai-min,Zhang Bao-long.2013.Pathogenicity differentiation and ISSRgenetic variation analysis of Verticillium dahliae isolates from cotton in Shihezi,Xinjiang.Acta Agriculturae Boreali-sinica,28(5):84-89.(in Chinese)袁洪波，艾尼江，赵建军，陈天子，杨郁文，刘蔼民，张保龙.2013.棉花黄萎病菌致病力分化与ISSR遗传变异分析.华北农学报，28(5):84-89.
Zhang Wu-jun,Li Ning,Luo Na,Wen Cheng-jing.2006.Comparison on biological characteristics of different pathogenicity strains of the Verticillium wilt pathogen of eggplant.Journal of Sichuan Agricultural University,24(3):281-287.(in Chinese)张武军，李宁，罗娜，文成敬.2006.茄子黄萎病菌不同致病力菌株生物学特性的比较.四川农业大学学报，24(3):281-287.
Zhao Ming.2010.The cultural characteristics and pathogenic differentiation of Verticillium dahliae from cotton in Shandong Province[M.D.Dissertation].Tai’an:Shandong Agricultural University.(in Chinese)赵鸣.2010.山东省主要棉区棉花黄萎病菌致病力分化及其生物学特性的研究[硕士论文].泰安：山东农业大学.
Zhu He-qin,Feng Zi-li,Yin Zhi-xin,Li Zhi-fang,Zhao Li-hong,Shi Yong-qiang,Yang Jia-rong.2012.Pathogenicity differentiation and ISSRfingerprint analysis of cotton Verticillium dahliae in China.Acta Phytopathologica Sinica,42(3):225-235.(in Chinese)朱荷琴，冯自力，尹志新，李志芳，赵丽红，师勇强，杨家荣.2012.我国棉花黄萎病菌致病力分化及ISSR指纹分析.植物病理学报，42(3):225-235.
Zhu Jun,Ye Hua-zhi.2006.Studies on species and pathogenicity of Verticillium wilt of eggplant in mountain area of western Sichuan.Southwest China Journal of Agricultural Sciences,19(5):897-899.(in Chinese)朱军，叶华智.2006.四川西部山区茄子黄萎病菌种类鉴定及致病性分化研究.西南农业学报，19(5):897-899.
Li Hai-lian.2005.The characterization and genomic DNA ISSR fingerprinting of Verticillium dahliae of eggplant isolates[M.D.Dissertation].Nanjing:Nanjing Agricultural University.(in Chinese)李海莲.2005.茄子黄萎病病原菌鉴定及其ISSR分子指纹分析[硕士论文].南京：南京农业大学.
Lin Mi,Zhang Pei-zhi,Guan Zhong-yan,Lu Shu-wen,Niu Bai-zhong,Zhang Jun-min,Qu Hong-yun,Liu Jing-lan,Zhang Hui.2008.Research on characteristics and pathogenicity differentiation of Verticillium dahliae strains in Heilongjiang.China Vegetables,(7):27-28.(in Chinese)林密，张佩芝，关钟燕，卢淑雯，牛柏忠，张军民，曲红云，柳景兰，张慧.2008.黑龙江省茄子黄萎病病原菌及其致病力的分化.中国蔬菜，(7):27-28.
Mercado-Blanco J,Rodríguez-Jurado D,Parrilla-Araujo S,Jiménez-D??az R M.2003.Simultaneous detection of the defoliating and nondefoliating Verticillium dahliae pathotypes in infected olive plants by duplex,nested polymerase chain reaction.Plant Disease,87:1487-1494.
Pérez-Artés E,García-Pedrajas M D,Bejarano-Alcàzar J,Jiménez-Díaz R M.2000.Differentiation of cotton-defoliating and nondefoliating pathotypes of Verticillium dahliae by RAPD and specific PCR analyses.European Journal of Plant Pathology,106:507-517.
Qiao Yan-yan,Yang Zhao-guang,Li Jie,Zhang Xing-hua.2015.Research on cultural characteristics and pathogenicity differentiation of Verticillium dahliae strains in Jiangxi.Acta Agriculturae Jiangxi,27(1):47-49.(in Chinese)乔艳艳，杨兆光，李捷，张兴华.2015.江西棉花黄萎病菌培养特性及致病力分化研究.江西农业学报，27(1):47-49.
Raeder U,Broda P.1985.Rapid preparation of DNA from filamentous fungi.Lett Appl Microbiol,1:17-20.
Ran Hong-chang,Xiao Yan-nong,Jiang Dao-hong,Li Guo-qing.2005.Pathogenicity differentiation of Verticillium dahliae strains and polymerphisms based on RAPD analysis in Hubei Province.Journal of Huazhong Agricultural University,24(5):442-447.(in Chinese)冉鸿昌，肖炎农，姜道宏，李国庆.2005.湖北省棉花黄萎病菌致病力分化和遗传多样性分析.华中农业大学学报，24(5):442-447.
Schnathorst W C,Mathre D E.1966.Host range and differentiation of a severe form Verticillium alboatrum in cotton.Phytopathology,56:1156-1161.
Shi Bo,Fang Rong,Hu Jian-kun,Zhou Kun-hua,Yuan Xin-jie,Wu Yin,Huang Rui-rong,Chen Xue-jun.2016.Research advance in integrated control techniques of eggplant Verticillium wilt.Acta Agriculturae Jiangxi,28(12):70-74.(in Chinese)石博，方荣，胡建坤，周坤华，袁欣捷，吴茵，黄瑞荣，陈学军.2016.茄子黄萎病综合防治技术研究进展.江西农业学报，28(12):70-74.
Song Xiao-xuan,Zhu He-qin,Guo Jin-cheng.1997.Study on pathogenicity differentiation for Verticillium dahliae Kleb.Anyang strains in cotton.Scientia Agricultura Sinica,30(1):13-18.(in Chinese)宋晓轩，朱荷琴，郭金城.1997.棉花黄萎病（Verticillium dahliae Kleb.）安阳菌系致病力分化研究.中国农业科学，30(1):13-18.
Verhalen L M,Brinkerhoff L A,Fun K C,Morrison W C.1971.A quantitative genetic study of Verticillium wilt resistance among selected lines of upland cotton.Crop Science,11(3):407-412.
Wang Guo-ning,Zhao Gui-yuan,Yue Xiao-wei,Li Zhi-kun,Zhang Yan,Zhang Gui-yin,Wu Li-qiang,Wang Xing-fen,Ma Zhi-ying.2012.Pathogenicity and ISSR genetic differentiation of Verticillium dahliae isolates from cotton growing areas of Hebei Province.Cotton Science,24(4):348-357.(in Chinese)王国宁，赵贵元，岳晓伟，李志坤，张艳，张桂寅，吴立强，王省芬，马峙英.2012.河北省棉花黄萎病菌致病性与ISSR遗传分化.棉花学报，24(4):348-357.
Xu F,Yang L,Zhang J,Guo X P.2012.Prevalence of the defoliating pathotype of Verticillium dahliae on cotton in central China and virulence on selected cotton cultivars.Journal of Phytopathology,160:369-376.
Xu Fei.2012.Characterization of the Verticillium dahliae defoliating pathotypeon cotton in central China and factors associated with prevalence of this severe pathotype[Ph.D.Dissertation].Wuhan:Huazhong Agricultural University.(in Chinese)徐飞.2012.落叶型棉花黄萎病菌在华中棉区广泛分布及其原因分析[博士论文].武汉：华中农业大学.
Yu Chun,Li Shu-ying,Liu Fang-zhi,Zhu Jia-bao,Cheng Fu-ru.2015.Analysis of characters and pathogenicity of cotton Verticillium dahliae cultivation in Anhui Province.Cotton Sciences,37(1):19-23.(in Chinese)於春，李淑英，刘方志，朱加保，程福如.2015.安徽省棉花黄萎病菌培养性状与致病性分析.棉花科学，37(1):19-23.
Yu Miao.2013.Identification of the pathogens of Verticillium wilt on eggplant and screening of resistant germplasm[M.D.Dissertation].Baoding:Agricultural University of Hebei.(in Chinese)于淼.2013.河北省茄子黄萎病菌的分离鉴定及抗性资源筛选[硕士论文].保定：河北农业大学.
Yuan Hong-bo,Ai Ni-jiang,Zhao Jian-jun,Chen Tian-zi,Yang Yu-wen,Liu Ai-min,Zhang Bao-long.2013.Pathogenicity differentiation and ISSRgenetic variation analysis of Verticillium dahliae isolates from cotton in Shihezi,Xinjiang.Acta Agriculturae Boreali-sinica,28(5):84-89.(in Chinese)袁洪波，艾尼江，赵建军，陈天子，杨郁文，刘蔼民，张保龙.2013.棉花黄萎病菌致病力分化与ISSR遗传变异分析.华北农学报，28(5):84-89.
Zhang Wu-jun,Li Ning,Luo Na,Wen Cheng-jing.2006.Comparison on biological characteristics of different pathogenicity strains of the Verticillium wilt pathogen of eggplant.Journal of Sichuan Agricultural University,24(3):281-287.(in Chinese)张武军，李宁，罗娜，文成敬.2006.茄子黄萎病菌不同致病力菌株生物学特性的比较.四川农业大学学报，24(3):281-287.
Zhao Ming.2010.The cultural characteristics and pathogenic differentiation of Verticillium dahliae from cotton in Shandong Province[M.D.Dissertation].Tai’an:Shandong Agricultural University.(in Chinese)赵鸣.2010.山东省主要棉区棉花黄萎病菌致病力分化及其生物学特性的研究[硕士论文].泰安：山东农业大学.
Zhu He-qin,Feng Zi-li,Yin Zhi-xin,Li Zhi-fang,Zhao Li-hong,Shi Yong-qiang,Yang Jia-rong.2012.Pathogenicity differentiation and ISSRfingerprint analysis of cotton Verticillium dahliae in China.Acta Phytopathologica Sinica,42(3):225-235.(in Chinese)朱荷琴，冯自力，尹志新，李志芳，赵丽红，师勇强，杨家荣.2012.我国棉花黄萎病菌致病力分化及ISSR指纹分析.植物病理学报，42(3):225-235.
Zhu Jun,Ye Hua-zhi.2006.Studies on species and pathogenicity of Verticillium wilt of eggplant in mountain area of western Sichuan.Southwest China Journal of Agricultural Sciences,19(5):897-899.(in Chinese)朱军，叶华智.2006.四川西部山区茄子黄萎病菌种类鉴定及致病性分化研究.西南农业学报，19(5):897-899.