湖南省棉花黄萎病菌致病力分化及致病型分布研究
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摘要
【目的】研究湖南主产棉区黄萎病菌致病性变异情况,为湖南棉花抗病品种选育推广及棉花黄萎病综合防控提供理论依据。【方法】对湖南省各主产棉县(区)分离的77个黄萎病菌单孢进行培养特性观察,测定其中31个菌株生长速率、产孢量和致病力,并以SAS 9.1.3统计分析软件对供试菌株致病力聚类。以特异性引物(D-1/D-2和ND-1/ND-2)经聚合酶链式反应检测24个菌株的致病类型。【结果】根据微菌核产量及在马铃薯葡萄糖琼脂培养基上培养的菌落特性,菌株可划分为菌核型、菌丝型和中间型3种培养类型,分别占总菌株数的14.28%、42.86%和42.86%。供试菌株生长速率为1.22~2.54 mm·d~(-1),产孢量为5.3×10~6~40.6×10~6 mL~(-1),各菌株之间存在明显差异。根据平均病情指数聚类结果将供试菌株划分为致病力强的Ⅰ型、致病力弱的Ⅱ型和致病力中等的Ⅲ型,分别占3.2%、12.9%和83.9%。【结论】湖南省棉花黄萎病菌以菌丝型和中间型为主要培养类型,且致病力存在明显分化;致病型检测结果表明目前湖南主产棉区黄萎病菌以落叶型为主。
[Objective] The study of pathogenicity differentiation of Verticillium dahliae in the cotton growing areas in Hunan province could provide theories for breeding and promoting resistant varieties as well as integrated control of Verticillium wilt of cotton. [Method] Seventy seven strains of V. dahliae from major cotton growing areas in Hunan province were evaluated their cultural characteristics. The growth rate, sporulation quantity and pathogenicity of 31 strains among them were tested and SAS9.1.3 statistical analysis software was used to cluster the pathogenicity of the tested strains. The special primer(D-1/D-2 and ND-1/ND-2) for polymerase chain reaction assay was used to test pathotype of 24 strains. [Result] According to their colony morphology on potato dextrose agar media and culture characteristics, the strains were classified into the sclerotium type, the hyphal type and the intermediate type accounted for 14.28%, 42.86% and 42.86%, respectively. The growth rate and sporulation quantity of the strains were 1.22–2.54 mm·d~(-1) and 5.3×10~6–40.6×10~6 spore·mL~(-1), which showed significant difference. In addition, based on the clustering results of average disease index,the strains were clustered into three groups which showed high(Type Ⅰ), low(Type Ⅱ) and moderate(Type Ⅲ) virulence to cotton with the proportion of 3.2%, 12.9% and 83.9%,respectively. [Conclusion] It is conclude that the main culture types of Ⅴ. dahliae in Hunan province are the hyphal type and the intermediate type, and the strains showed pathogenicity differentiation. The result showed that defoliating pathotype of V.dahliae was dominant in the cotton growing areas of Hunan province.
[Objective] The study of pathogenicity differentiation of Verticillium dahliae in the cotton growing areas in Hunan province could provide theories for breeding and promoting resistant varieties as well as integrated control of Verticillium wilt of cotton. [Method] Seventy seven strains of V. dahliae from major cotton growing areas in Hunan province were evaluated their cultural characteristics. The growth rate, sporulation quantity and pathogenicity of 31 strains among them were tested and SAS9.1.3 statistical analysis software was used to cluster the pathogenicity of the tested strains. The special primer(D-1/D-2 and ND-1/ND-2) for polymerase chain reaction assay was used to test pathotype of 24 strains. [Result] According to their colony morphology on potato dextrose agar media and culture characteristics, the strains were classified into the sclerotium type, the hyphal type and the intermediate type accounted for 14.28%, 42.86% and 42.86%, respectively. The growth rate and sporulation quantity of the strains were 1.22–2.54 mm·d~(-1) and 5.3×10~6–40.6×10~6 spore·mL~(-1), which showed significant difference. In addition, based on the clustering results of average disease index,the strains were clustered into three groups which showed high(Type Ⅰ), low(Type Ⅱ) and moderate(Type Ⅲ) virulence to cotton with the proportion of 3.2%, 12.9% and 83.9%,respectively. [Conclusion] It is conclude that the main culture types of Ⅴ. dahliae in Hunan province are the hyphal type and the intermediate type, and the strains showed pathogenicity differentiation. The result showed that defoliating pathotype of V.dahliae was dominant in the cotton growing areas of Hunan province.
引文
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[6]杨家荣,赵小明.棉花黄萎病菌的遗传变异[J].西北农业大学学报,1999,27(3):101-105.Yang Jiarong,Zhao Xiaoming.Genetic variation of cotton Verticillium wilt[J].Acta Universitatis Agriculturalis Boreali-Occidentalis,1999,27(3):101-105.
[7]朱荷琴,冯自力,尹志新,等.我国棉花黄萎病菌致病力分化及ISSR指纹分析[J].植物病理学报,2012,42(3):225-235.Zhu Heqin,Feng Zili,Yin Zhixin,et al.Pathogenicity differentiation and ISSR fingerprint analysis of cotton Verticillium dahliae in China[J].Acta Phytopathologica Sinica,2012,42(3):225-235.
[8]林玲,章如意,张昕,等.江苏省棉花黄萎病菌的培养特性及致病力分化监测[J].棉花学报,2012,24(3):199-206.Lin Ling,Zhang Ruyi,Zhang Xin,et al.Cultural characteristics and pathogenicity differentiation among strains of Verticillium dahliae from cotton in Jiangsu province[J].Cotton Science,2012,24(3):199-206.
[9]Pérez-Artés E,García-Pedrajas M,Bejarano-Alcázar J,et al.Differentiation of cotton-defoliating and non-defoliating pathotypes of Verticillium dahliae by RAPD and specific PCR analysis[J].European Journal of Plant Pathology,2000,106:507-517.
[10]李彩红,王永波,赵瑞元,等.湖南常德地区棉花黄萎病菌培养特性及致病力分化研究[J].中国棉花,2015,42(7):18-21.Li Caihong,Wang Yongbo,Zhao Ruiyuan,et al.Study on the cultural characteristics and pathogenic differentiation of Veticillium dahliae from cotton in Changde,Hunan province[J].China Cotton,2015,42(7):18-21.
[11]杨凤祥,桑茜,马奇祥,等.河南省棉花黄萎病菌培养特性与致病力分化研究[J].河南农业科学,2009(6):85-89.Yang Fengxiang,Sang Qian,Ma Qixiang,et al.Study on the cultural characteristics and pathogenic differentiation of Veticillium dahliae from cotton in Henan[J].Journal of Henan Agricultural Sciences,2009(6):85-89.
[12]刘廷利,惠慧,阚家亮,等.新疆北部棉花黄萎病菌培养特性、致病型、致病性分化及ISSR遗传变异研究[J].棉花学报,2017,29(6):541-549.Liu Tingli,Hui Hui,Kan Jialiang,et al.The monitoring of cultural characteristics,pathotype,pathogenicity differentiation and ISSR genetic variation of Verticillium dahliae isolates from cotton in northern of Xinjiang[J].Cotton Science,2017,29(6):541-549.
[13]王国宁,赵贵元,岳晓伟,等.河北省棉花黄萎病菌致病性与ISSR遗传分化[J].棉花学报,2012,24(4):348-357.Wang Guoning,Zhao Guiyuan,Yue Xiaowei,et al.Pathogenicity and ISSR genetic differentiation of Verticillium dahliae isolates from cotton growing areas of Hebei province[J].Cotton Science,2012,24(4):348-357.
[14]惠慧.不同棉区落叶型黄萎病菌培养特性与致病力分化研究[D].武汉:华中农业大学,2012.Xi Hui.Study on cultural characteristics and pathogenicity differention of defoliating pathotype strains of Verticillium dahliae in different cotton-producing provinces[D].Wuhan:Huazhong Agricultural University,2012.
[15]乔艳艳,杨兆光,李捷,等.江西棉花黄萎病菌培养特性及致病力分化研究[J].江西农业学报,2015,27(1):47-49.Qiao Yanyan,Yang Zhaoguang,Li Jie,et al.Research on cultural characteristics and pathogenicity differentiation of Verticillium dahliae strains in Jiangxi[J].Acta Agriculturae Jiangxi,2015,27(1):47-49.
[16]袁媛,冯自力,李志芳,等.棉花黄萎病菌致病力测定及评价方法研究[J].植物病理学报,2018,48(2):248-255.Yuan Yuan,Feng Zili,Li Zhifang,et al.Pathogenicity assay and evaluation of Verticillium dahliae on cotton[J].Acta Phytopathologica Sinica,2018,48(2):248-255.
[17]朱荷琴,宋晓轩,简桂良.棉花黄萎病菌致病力变异生理机制的初步研究[J].棉花学报,2004,16(5):275-279.Zhu Heqin,Song Xiaoxuan,Jian Guiliang.The preliminary study of variation mechanism of pathogenic type of Verticillium dahliae Kleb.[J].Cotton Science,2004,16(5):275-279.
[18]李艳,杨家荣.土壤棉花黄萎病菌致病力分化的初步研究[J].棉花学报,2007,19(4):291-295.Li Yan,Yang Jiarong.Preliminary study on pathogenicity of Verticillium dahliae in soil[J].Cotton Science,2007,19(4):291-295.
[19]林玲,张爱香,陈志石,等.江苏省棉花黄萎病菌培养特性与致病力的相关性研究[J].江苏农业科学,2005,2:49-51.Lin Ling,Zhang Aixiang,Chen Zhishi,et al.Study on the correlation between the cultural characteristics and pathogenicity of Verticillium dahliae strains in Jiangsu province[J].Jiangsu Agricultural Sciences,2005,2:49-51.
[20]Li Zhifang,Liu Yijie,Feng Zili,et al.VdCYC8,encoding CYC8 glucose repression mediator protein is required for microsclerotia formation and full virulence in Verticillium dahlia[J/OL].PLoS ONE,2015,10(12):e0144020(2015-12-03)[2019-01-01].https://doi.org/10.1371/journal.pone.0144020
[21]Zhang Yalin,Li Zhifang,Feng Zili,et al.Isolation and functional analysis of the pathogenicity-related gene VdPR3 from Verticillium dahliae on cotton[J].Current Genetics,2015,4(61):555-566.
[22]Zhang Yalin,Li Zhifang,Feng Zili,et al.Functional analysis of the pathogenicity-related gene VdPR1 in the vascular wilt fungus Verticillium dahliae[J/OL].PLoS ONE,2016,11(11):e0166000(2016-11-15)[2019-01-01].https://doi.org/10.1371/journal.pone.0166000
[23]Lengeler K B,Davidson R C,Dsouza C,et al.Signal transduction cascades regulating fungal development and virulence[J].Microbiolgy and Molecular Biology Reviews,2000,64(4):746-785.
[24]朱荷琴,李志芳,冯自力,等.我国棉花黄萎病研究十年回顾及展望[J].棉花学报,2017,29(增刊):37-50.Zhu Heqin,Li Zhifang,Feng Zili,et al.Overview of cotton Verticillium wilt research over the past decade in China and its prospect in future[J]Cotton Science,2017,29(Suppl):37-50.
[25]王彦,鹿秀云,郭庆港,等.河北省棉花黄萎菌落叶型和非落叶型菌系初步鉴定[J].华北农学报,2010,25(4):196-200.Wang Yan,Lu Xiuyun,Guo Qinggang,et al.Molecular identification of defoliatiny pathotype and non-defoliating pathotype of Verticillium dahliae from cotton in Hebei province[J].Acta Agriculturae Boreali-Sinica,2010,25(4):196-200.
[26]徐飞.落叶型棉花黄萎病菌在华中棉区广泛分布及其原因分析[D].武汉:华中农业大学,2012.Xu Fei.Characterization of the Verticillium dahliae defoliating pathotype on cotton in central and factors associated with prevalence of this severe pathotype[D].Wuhan:Huazhong A-gricultural University,2012.
[2]简桂良,卢美光,仇家山,等.棉花黄萎病防治策略[J].中国植保导刊,2004,24(4):30-31.Jian Guiliang,Lu Meiguang,Qiu Jiashan,et al.The control strategy of cotton Verticillium wilt[J].China Plant Protection,2004,24(4):30-31.
[3]简桂良,邹亚飞,马存.棉花黄萎病连年流行的原因及对策[J].中国棉花,2003,30(3):13-14.Jian Guiliang,Zou Yafei,Ma Cun.The outbreak reason and integral control of cotton Verticillium wilt[J].China Cotton,2003,30(3):13-14.
[4]马存.棉花枯萎病和黄萎病的研究[M].北京:中国农业出版社,2007:61-63.Ma Cun.Study on cotton Fusarium wilt and Verticillium wilt[M].Beijing:China Agricultural Press,2007:61-63.
[5]张绪振,张树琴,陈吉棣,等.我国棉花黄萎病菌“种”的鉴定[J].植物病理学报,1981,11(3):149-156.Zhang Xuzhen,Zhang Shuqin,Chen Jidi,et al.Identification of"species"of cotton Verticillium wilt in China[J].Acta Phytopathologica Sinca,1981,11(3):149-156.
[6]杨家荣,赵小明.棉花黄萎病菌的遗传变异[J].西北农业大学学报,1999,27(3):101-105.Yang Jiarong,Zhao Xiaoming.Genetic variation of cotton Verticillium wilt[J].Acta Universitatis Agriculturalis Boreali-Occidentalis,1999,27(3):101-105.
[7]朱荷琴,冯自力,尹志新,等.我国棉花黄萎病菌致病力分化及ISSR指纹分析[J].植物病理学报,2012,42(3):225-235.Zhu Heqin,Feng Zili,Yin Zhixin,et al.Pathogenicity differentiation and ISSR fingerprint analysis of cotton Verticillium dahliae in China[J].Acta Phytopathologica Sinica,2012,42(3):225-235.
[8]林玲,章如意,张昕,等.江苏省棉花黄萎病菌的培养特性及致病力分化监测[J].棉花学报,2012,24(3):199-206.Lin Ling,Zhang Ruyi,Zhang Xin,et al.Cultural characteristics and pathogenicity differentiation among strains of Verticillium dahliae from cotton in Jiangsu province[J].Cotton Science,2012,24(3):199-206.
[9]Pérez-Artés E,García-Pedrajas M,Bejarano-Alcázar J,et al.Differentiation of cotton-defoliating and non-defoliating pathotypes of Verticillium dahliae by RAPD and specific PCR analysis[J].European Journal of Plant Pathology,2000,106:507-517.
[10]李彩红,王永波,赵瑞元,等.湖南常德地区棉花黄萎病菌培养特性及致病力分化研究[J].中国棉花,2015,42(7):18-21.Li Caihong,Wang Yongbo,Zhao Ruiyuan,et al.Study on the cultural characteristics and pathogenic differentiation of Veticillium dahliae from cotton in Changde,Hunan province[J].China Cotton,2015,42(7):18-21.
[11]杨凤祥,桑茜,马奇祥,等.河南省棉花黄萎病菌培养特性与致病力分化研究[J].河南农业科学,2009(6):85-89.Yang Fengxiang,Sang Qian,Ma Qixiang,et al.Study on the cultural characteristics and pathogenic differentiation of Veticillium dahliae from cotton in Henan[J].Journal of Henan Agricultural Sciences,2009(6):85-89.
[12]刘廷利,惠慧,阚家亮,等.新疆北部棉花黄萎病菌培养特性、致病型、致病性分化及ISSR遗传变异研究[J].棉花学报,2017,29(6):541-549.Liu Tingli,Hui Hui,Kan Jialiang,et al.The monitoring of cultural characteristics,pathotype,pathogenicity differentiation and ISSR genetic variation of Verticillium dahliae isolates from cotton in northern of Xinjiang[J].Cotton Science,2017,29(6):541-549.
[13]王国宁,赵贵元,岳晓伟,等.河北省棉花黄萎病菌致病性与ISSR遗传分化[J].棉花学报,2012,24(4):348-357.Wang Guoning,Zhao Guiyuan,Yue Xiaowei,et al.Pathogenicity and ISSR genetic differentiation of Verticillium dahliae isolates from cotton growing areas of Hebei province[J].Cotton Science,2012,24(4):348-357.
[14]惠慧.不同棉区落叶型黄萎病菌培养特性与致病力分化研究[D].武汉:华中农业大学,2012.Xi Hui.Study on cultural characteristics and pathogenicity differention of defoliating pathotype strains of Verticillium dahliae in different cotton-producing provinces[D].Wuhan:Huazhong Agricultural University,2012.
[15]乔艳艳,杨兆光,李捷,等.江西棉花黄萎病菌培养特性及致病力分化研究[J].江西农业学报,2015,27(1):47-49.Qiao Yanyan,Yang Zhaoguang,Li Jie,et al.Research on cultural characteristics and pathogenicity differentiation of Verticillium dahliae strains in Jiangxi[J].Acta Agriculturae Jiangxi,2015,27(1):47-49.
[16]袁媛,冯自力,李志芳,等.棉花黄萎病菌致病力测定及评价方法研究[J].植物病理学报,2018,48(2):248-255.Yuan Yuan,Feng Zili,Li Zhifang,et al.Pathogenicity assay and evaluation of Verticillium dahliae on cotton[J].Acta Phytopathologica Sinica,2018,48(2):248-255.
[17]朱荷琴,宋晓轩,简桂良.棉花黄萎病菌致病力变异生理机制的初步研究[J].棉花学报,2004,16(5):275-279.Zhu Heqin,Song Xiaoxuan,Jian Guiliang.The preliminary study of variation mechanism of pathogenic type of Verticillium dahliae Kleb.[J].Cotton Science,2004,16(5):275-279.
[18]李艳,杨家荣.土壤棉花黄萎病菌致病力分化的初步研究[J].棉花学报,2007,19(4):291-295.Li Yan,Yang Jiarong.Preliminary study on pathogenicity of Verticillium dahliae in soil[J].Cotton Science,2007,19(4):291-295.
[19]林玲,张爱香,陈志石,等.江苏省棉花黄萎病菌培养特性与致病力的相关性研究[J].江苏农业科学,2005,2:49-51.Lin Ling,Zhang Aixiang,Chen Zhishi,et al.Study on the correlation between the cultural characteristics and pathogenicity of Verticillium dahliae strains in Jiangsu province[J].Jiangsu Agricultural Sciences,2005,2:49-51.
[20]Li Zhifang,Liu Yijie,Feng Zili,et al.VdCYC8,encoding CYC8 glucose repression mediator protein is required for microsclerotia formation and full virulence in Verticillium dahlia[J/OL].PLoS ONE,2015,10(12):e0144020(2015-12-03)[2019-01-01].https://doi.org/10.1371/journal.pone.0144020
[21]Zhang Yalin,Li Zhifang,Feng Zili,et al.Isolation and functional analysis of the pathogenicity-related gene VdPR3 from Verticillium dahliae on cotton[J].Current Genetics,2015,4(61):555-566.
[22]Zhang Yalin,Li Zhifang,Feng Zili,et al.Functional analysis of the pathogenicity-related gene VdPR1 in the vascular wilt fungus Verticillium dahliae[J/OL].PLoS ONE,2016,11(11):e0166000(2016-11-15)[2019-01-01].https://doi.org/10.1371/journal.pone.0166000
[23]Lengeler K B,Davidson R C,Dsouza C,et al.Signal transduction cascades regulating fungal development and virulence[J].Microbiolgy and Molecular Biology Reviews,2000,64(4):746-785.
[24]朱荷琴,李志芳,冯自力,等.我国棉花黄萎病研究十年回顾及展望[J].棉花学报,2017,29(增刊):37-50.Zhu Heqin,Li Zhifang,Feng Zili,et al.Overview of cotton Verticillium wilt research over the past decade in China and its prospect in future[J]Cotton Science,2017,29(Suppl):37-50.
[25]王彦,鹿秀云,郭庆港,等.河北省棉花黄萎菌落叶型和非落叶型菌系初步鉴定[J].华北农学报,2010,25(4):196-200.Wang Yan,Lu Xiuyun,Guo Qinggang,et al.Molecular identification of defoliatiny pathotype and non-defoliating pathotype of Verticillium dahliae from cotton in Hebei province[J].Acta Agriculturae Boreali-Sinica,2010,25(4):196-200.
[26]徐飞.落叶型棉花黄萎病菌在华中棉区广泛分布及其原因分析[D].武汉:华中农业大学,2012.Xu Fei.Characterization of the Verticillium dahliae defoliating pathotype on cotton in central and factors associated with prevalence of this severe pathotype[D].Wuhan:Huazhong A-gricultural University,2012.