不同寄主大丽轮枝菌培养性状的比较及致病力分化的分析
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摘要
为了明确不同寄主来源大丽轮枝菌培养性状的差异及致病类型的分化,本研究对分离自6个寄主的8个大丽轮枝菌菌株通过PDA培养性状、产孢量和孢子萌发率的测定对其生物学性状进行了比较,对4种碳源和氮源的利用对8个菌株的营养需求进行了分析,并通过致病型和生理小种的分子鉴定及对棉花的致病力,对8个菌株的致病力分化进行了研究。培养性状结果表明:5个菌株形成菌核型菌落,3个菌株形成中间型菌落,且菌株之间的培养性状存在明显差异。产孢量和孢子萌发率的结果显示:红花上的Vnu菌株产孢量和孢子萌发率都明显高于其它菌株,具有最强的繁殖力,而番茄上的Vtr菌株的产孢量最低,马铃薯上的S2菌株的孢子萌发率最低,表明各菌株具有不同的繁殖力。对碳源和氮源的利用表明:各菌株对不同种类的碳源和氮源的利用存在明显差异,其中Vtr菌株对所有供试碳源和氮源的利用能力最好,向日葵上的S1对4种碳源的利用最差,表明菌株之间具有不同的碳氮源需求。致病型和生理小种的分子鉴定表明:Vnu为1号小种,除Vtr之外其他菌株均为2号小种,并且只有鉴定为2号小种的菌株才可以进一步分为落叶型和非落叶型;所有供试菌株均能侵染棉花引起黄萎病,马铃薯上的S2菌株和茄子上的S3菌株对棉花的致病力比棉花黄萎病菌V592强,其他菌株对棉花的致病力明显比V592菌株弱,且发病延迟,表明8个菌株致病力分化明显。
This research aimed to evaluate the cultural characteristics and pathogenicity differentiation among strains of Verticillium dahliae isolated from different host plants. Eight V.dahliae stains isolated from 6 host plants were analyzed including:survey of biological characters of cultural characteristics,conidia production and spore germination; the utilization of four kinds of carbon source and nitrogen sources;identification of pathotype or physiological race by PCR amplification and pathogenicity in the cotton seedling stage. According to the quantity of microsclerotia of the colonial morphology on PDA,five strains were sclerotial type and three were intermediate type. There were significant differences in cultural characteristics among strains. The conidia production and spore germination rate of Vnu strain from safflower were significantly higher than those of other strains,so Vnu strain was regarded as the best reproductive capacity,whereas the Vtr strains from tomato had the least conidia production and S2 from potato had the lowest spore germination rate among the eight strains. The results showed that each strain had different reproductive capacity. There were significant differences in the utilization of different types of carbon source and nitrogen source among different strains. Vtr strain grew best but S1 stain grew worst on four kinds of different carbon and nitrogen media,indicating that these strains had different nutritional requirements. Vnu strain belonged to race 1 and other strains belonged to race 2 except Vtr strain by PCR. Furthermore,only race 2 could be classified as defoliating pathotype and nondefoliating pathotype. The symptoms of cotton diseases caused by potato S2 strain and S3 strain of eggplant were stronger than that of cotton V59,whereas symptoms caused by other stains were obviously weaker than that of strain V592,and the disease was delayed.. Our results suggested that the pathogenicity varies among the strains from different host plant.
This research aimed to evaluate the cultural characteristics and pathogenicity differentiation among strains of Verticillium dahliae isolated from different host plants. Eight V.dahliae stains isolated from 6 host plants were analyzed including:survey of biological characters of cultural characteristics,conidia production and spore germination; the utilization of four kinds of carbon source and nitrogen sources;identification of pathotype or physiological race by PCR amplification and pathogenicity in the cotton seedling stage. According to the quantity of microsclerotia of the colonial morphology on PDA,five strains were sclerotial type and three were intermediate type. There were significant differences in cultural characteristics among strains. The conidia production and spore germination rate of Vnu strain from safflower were significantly higher than those of other strains,so Vnu strain was regarded as the best reproductive capacity,whereas the Vtr strains from tomato had the least conidia production and S2 from potato had the lowest spore germination rate among the eight strains. The results showed that each strain had different reproductive capacity. There were significant differences in the utilization of different types of carbon source and nitrogen source among different strains. Vtr strain grew best but S1 stain grew worst on four kinds of different carbon and nitrogen media,indicating that these strains had different nutritional requirements. Vnu strain belonged to race 1 and other strains belonged to race 2 except Vtr strain by PCR. Furthermore,only race 2 could be classified as defoliating pathotype and nondefoliating pathotype. The symptoms of cotton diseases caused by potato S2 strain and S3 strain of eggplant were stronger than that of cotton V59,whereas symptoms caused by other stains were obviously weaker than that of strain V592,and the disease was delayed.. Our results suggested that the pathogenicity varies among the strains from different host plant.
引文
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[2]夏红飞,陈志荣,宾毅,等.大丽轮枝菌微菌核形成的影响因素研究[J].石河子大学学报(自然科学版),2017,35(5):618-624.Xia H F,Chen Z R,Bin Y,et al.Influence factors on microsclerotial formation in Verticillium dahliae[J].Journal of Shihezi University(Natural Science),2017,35(5):618-624.
[3]王克荣,陆家云.大丽轮枝菌培养性状的变异[J].植物病理学报,1987,17(5):27-33.Wang K R,Lu J Y.Variability in culture characteristic of verticillium dahliae kleb[J].Acta Phytopathologica Sinica,1987,17(5):27-33.
[4]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 H Q,Feng Z L,Yin Z X,et al.Pathogenicity differentiation and ISSR fingerprint analysis of cotton Verticillium dahliae in China[J].Acta Phytopathologica Sinica,2012,42(3):225-235.
[5]邓晟,章如意,林玲,等.苏省大丰市棉花黄萎病菌培养特性及致病力分化[J].江苏农业科学,2012,40(12):133-135.Deng Y,Zhang R Y,Lin L,et al.Culture characteristics and virulence differentiation of Verticillium dahliae in cotton in Dafeng City,Jiangsu Province[J].Jiangsu Agricultural Sciences,2012,40(12):133-135.
[6]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 L,Zhang R Y,Zhang X,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.
[7]姚耀文,傅翠真,王文录,等.棉花黄萎病菌生理型鉴定的初步研究[J].植物保护学报,1982,9(3):145-148.Yao Y W,Fu C Z,Wang W L,et al.Preliminary studies on physiological forms of cotton verticillium wilt fungus[J].Journal of Plant Protection,1982,9(3):145-148.
[8] Pérez-artés E,García-pedrajas M D,Bejarano-alcázar J,et al.Differentiation of cotton-defoliating and nondefoliating pathotypes of Verticillium dahliae by rapd and specific PCR analyses[J].European Journal of Plant Pathology,2000,106(6):507-517.
[9] Fradin E F,Zhang Z,Juarez Ayala J C,et al.Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1[J].Plant Physiology,2009,150(5):320-332
[10] De Jonge R,Van Esse H P,Maruthachalam K,et al.Tomato immune receptor 1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(13):5110-5115.
[11] White T J,Bruns T D,Lee S B,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for Phylogenetics[J].Genetics Selection Evolution,1990,38:315-322.
[12]张婷,张博森,刘启,等.不同致病力棉花黄萎病菌乙烯含量及相关基因表达量的测定[J].石河子大学学报(自然科学版),2016,34(5):632-637.Zhang T,Zhang B S,Liu Q,et al.Determination of ethylene and transcript levels of genes involved in ethylene biosynthesis by different virulent Verticillim dahliae from cotton[J].Journal of Shihezi University(Natural Science),2016,34(5):632-637.
[13] Jiménez-díaz R M,Cirulli M,Bubici G,et al.Verticillium Wilt,a major threat to olive production:current Status and future prospects for its management[J].Plant Disease,2012,96(96):304-329.
[14] Usami T,Shu I,Takashina H,et al.Cloning of DNA fragments specific to the pathotype and race of Verticillium Dahliae[J].Journal of General Plant Pathology,2007,73(2):89-95.
[15] Short D P,Gurung S,Maruthachalam K,et al.Verticillium dahliae race 2-specific PCR reveals a high frequency of race 2 strains in commercial spinach seed lots and delineates race structure[J].Plant Pathology,2014,104(7):779.
[16] Gao F,Zhou B J,Li G Y,et al.A glutamic acid-rich protein identified in Verticillium Dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity[J].Plos One,2010,12(5):e15319
[17]石磊岩,王波,文学.我国棉花黄萎病菌类型分化及培养特性研究[J].植物保护学报,1993,20(3):247-252.Shi L Y,Wang B,Wen X.Study on type differentiation and cultural characteristics of cotton Verticillium wilt in China[J].Journal of Plant Protection,1993,20(3):247-252.
[18]喻秀秀,王卿,张昕,等.江苏省大丽轮枝菌的培养、遗传及致病特性分析[J].植物病理学报,2018,48(3):378-388.Yu X X,Wang Q,Zhang X,et al.Cultural,genetic and phytopathogenic characterizations of a Verticillium dahliae population from Jiangsu province[J].Acta Phytopathologica Sinica,2018,48(3):378-388.
[19]黄薇,袁斌,万鹏,等.不同钾钠比的查氏培养基对大丽轮枝菌生物学性状及致病力的影响[J].湖北农业科学,2016,55(22):5815-5820.Huang W,Yuan B,Wan P,et al.The Influence of Different Ratios of K+/Na+in Czapek's Medium to Biological Characters and Virulence of Verticcilium dahilae[J].Hubei Agricultural Sciences,2016,55(22):5815-5820.
[20]刘琳琳,张文文,周易,等.棉花与番茄抗棉花黄萎病不依赖于Ve1[J].中国科学:生命科学,2014,44(8):803-814.Liu L L,Zhang W W,Zhou Y,et al.Resistance of Cotton and Tomato to Verticillium dahliae from Cotton is Independent on Ve1[J].Scientia Sinica Vitae,2014,44(8):803-814.
[2]夏红飞,陈志荣,宾毅,等.大丽轮枝菌微菌核形成的影响因素研究[J].石河子大学学报(自然科学版),2017,35(5):618-624.Xia H F,Chen Z R,Bin Y,et al.Influence factors on microsclerotial formation in Verticillium dahliae[J].Journal of Shihezi University(Natural Science),2017,35(5):618-624.
[3]王克荣,陆家云.大丽轮枝菌培养性状的变异[J].植物病理学报,1987,17(5):27-33.Wang K R,Lu J Y.Variability in culture characteristic of verticillium dahliae kleb[J].Acta Phytopathologica Sinica,1987,17(5):27-33.
[4]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 H Q,Feng Z L,Yin Z X,et al.Pathogenicity differentiation and ISSR fingerprint analysis of cotton Verticillium dahliae in China[J].Acta Phytopathologica Sinica,2012,42(3):225-235.
[5]邓晟,章如意,林玲,等.苏省大丰市棉花黄萎病菌培养特性及致病力分化[J].江苏农业科学,2012,40(12):133-135.Deng Y,Zhang R Y,Lin L,et al.Culture characteristics and virulence differentiation of Verticillium dahliae in cotton in Dafeng City,Jiangsu Province[J].Jiangsu Agricultural Sciences,2012,40(12):133-135.
[6]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 L,Zhang R Y,Zhang X,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.
[7]姚耀文,傅翠真,王文录,等.棉花黄萎病菌生理型鉴定的初步研究[J].植物保护学报,1982,9(3):145-148.Yao Y W,Fu C Z,Wang W L,et al.Preliminary studies on physiological forms of cotton verticillium wilt fungus[J].Journal of Plant Protection,1982,9(3):145-148.
[8] Pérez-artés E,García-pedrajas M D,Bejarano-alcázar J,et al.Differentiation of cotton-defoliating and nondefoliating pathotypes of Verticillium dahliae by rapd and specific PCR analyses[J].European Journal of Plant Pathology,2000,106(6):507-517.
[9] Fradin E F,Zhang Z,Juarez Ayala J C,et al.Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1[J].Plant Physiology,2009,150(5):320-332
[10] De Jonge R,Van Esse H P,Maruthachalam K,et al.Tomato immune receptor 1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(13):5110-5115.
[11] White T J,Bruns T D,Lee S B,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for Phylogenetics[J].Genetics Selection Evolution,1990,38:315-322.
[12]张婷,张博森,刘启,等.不同致病力棉花黄萎病菌乙烯含量及相关基因表达量的测定[J].石河子大学学报(自然科学版),2016,34(5):632-637.Zhang T,Zhang B S,Liu Q,et al.Determination of ethylene and transcript levels of genes involved in ethylene biosynthesis by different virulent Verticillim dahliae from cotton[J].Journal of Shihezi University(Natural Science),2016,34(5):632-637.
[13] Jiménez-díaz R M,Cirulli M,Bubici G,et al.Verticillium Wilt,a major threat to olive production:current Status and future prospects for its management[J].Plant Disease,2012,96(96):304-329.
[14] Usami T,Shu I,Takashina H,et al.Cloning of DNA fragments specific to the pathotype and race of Verticillium Dahliae[J].Journal of General Plant Pathology,2007,73(2):89-95.
[15] Short D P,Gurung S,Maruthachalam K,et al.Verticillium dahliae race 2-specific PCR reveals a high frequency of race 2 strains in commercial spinach seed lots and delineates race structure[J].Plant Pathology,2014,104(7):779.
[16] Gao F,Zhou B J,Li G Y,et al.A glutamic acid-rich protein identified in Verticillium Dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity[J].Plos One,2010,12(5):e15319
[17]石磊岩,王波,文学.我国棉花黄萎病菌类型分化及培养特性研究[J].植物保护学报,1993,20(3):247-252.Shi L Y,Wang B,Wen X.Study on type differentiation and cultural characteristics of cotton Verticillium wilt in China[J].Journal of Plant Protection,1993,20(3):247-252.
[18]喻秀秀,王卿,张昕,等.江苏省大丽轮枝菌的培养、遗传及致病特性分析[J].植物病理学报,2018,48(3):378-388.Yu X X,Wang Q,Zhang X,et al.Cultural,genetic and phytopathogenic characterizations of a Verticillium dahliae population from Jiangsu province[J].Acta Phytopathologica Sinica,2018,48(3):378-388.
[19]黄薇,袁斌,万鹏,等.不同钾钠比的查氏培养基对大丽轮枝菌生物学性状及致病力的影响[J].湖北农业科学,2016,55(22):5815-5820.Huang W,Yuan B,Wan P,et al.The Influence of Different Ratios of K+/Na+in Czapek's Medium to Biological Characters and Virulence of Verticcilium dahilae[J].Hubei Agricultural Sciences,2016,55(22):5815-5820.
[20]刘琳琳,张文文,周易,等.棉花与番茄抗棉花黄萎病不依赖于Ve1[J].中国科学:生命科学,2014,44(8):803-814.Liu L L,Zhang W W,Zhou Y,et al.Resistance of Cotton and Tomato to Verticillium dahliae from Cotton is Independent on Ve1[J].Scientia Sinica Vitae,2014,44(8):803-814.