早竹丛枝病的调查及病原菌的分子鉴定
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摘要
[目的]对早竹丛枝病进行调查及病原菌分子鉴定,为早竹丛枝病的病害诊断和防治提供科学依据。[方法]采用单株水平和单枝盘水平的2种病害分级标准对早竹丛枝病进行调查。使用植原体16S rDNA和真菌rDNA-ITS序列的特异性PCR引物,对早竹丛枝病的病原菌进行分子鉴定。[结果]调查的6块样地早竹丛枝病的平均发病率为18.59%,平均病情指数为6.67。感病的早竹DNA样品能够扩增出真菌的rDNA-ITS序列,而不能够扩增出植原体的16S rDNA序列;扩增出的序列与报道的竹针孢座囊菌的序列同源性达到99.00%,与其它真菌的序列同源性最高仅为94.00%。[结论]浙江省德清县早竹丛枝病的病原菌为竹针孢座囊菌。
[Objective]In this study,the witches' broom disease of Phyllostachys praecox was assessed,and the pathogen of the disease was identified through molecular biology techniques. [Method]The disease severity was assessed by grading witches' broom disease at individual level and branch level. The pathogen of witches' broom disease of Ph. praecox was identified by specific PCR primers of phytoplasmal 16 S r DNA and fungal r DNA-ITS sequences. [Result]Disease assessment data showed that the average incidence of the 6 plots was 18. 59%,and the average disease index was 6. 67. PCR results indicated that all the samples infected were able to amplify r DNA-ITS sequence of fungus,and could not amplify 16 S r DNA of phytoplasma. Comparative BLAST analysis determined that the amplicons shared 99. 00% similarity with sequences from Aciculosporium take,and the highest similarity with other fungi was only 94. 00%. [Conclusion]A. take was the causal agent of witches' broom disease of Ph. praecox of Deqing County,Zhejiang Province.
[Objective]In this study,the witches' broom disease of Phyllostachys praecox was assessed,and the pathogen of the disease was identified through molecular biology techniques. [Method]The disease severity was assessed by grading witches' broom disease at individual level and branch level. The pathogen of witches' broom disease of Ph. praecox was identified by specific PCR primers of phytoplasmal 16 S r DNA and fungal r DNA-ITS sequences. [Result]Disease assessment data showed that the average incidence of the 6 plots was 18. 59%,and the average disease index was 6. 67. PCR results indicated that all the samples infected were able to amplify r DNA-ITS sequence of fungus,and could not amplify 16 S r DNA of phytoplasma. Comparative BLAST analysis determined that the amplicons shared 99. 00% similarity with sequences from Aciculosporium take,and the highest similarity with other fungi was only 94. 00%. [Conclusion]A. take was the causal agent of witches' broom disease of Ph. praecox of Deqing County,Zhejiang Province.
引文
[1]易同培,史军义,马丽莎,等.中国竹类图志[M].北京:科学出版社,2008.
[2]莫润宏,汤富彬,丁明,等.氨基酸分析仪法测定竹笋中游离氨基酸[J].化学通报,2012,75(12):1126-1131.
[3]丁兴萃.覆盖栽培早竹开花的激素机制研究[J].林业科学,2007,43(7):10-15.
[4]盛常顺.几种笋用竹对竹丛枝病的抗性试验研究[J].安徽林业科技,2004,122(3):23-24.
[5]陈建寅,陈晓岚.安吉竹种园竹丛枝病调查报告[J].浙江林业科技,1992,12(2):47-50.
[6]Kao C W,Leu L S.Finding perfect stage of Aciculosporium take Miyake,the causal organism of bamboo witches’broom disease and its conidial germination[J].Plant Protection Bulletin,1976,18(3):276-285.
[7]江泽慧.世界竹藤[M].沈阳:辽宁科学技术出版社,2002.
[8]Tanaka E,Tanaka C,Shibata S.Bamboo witches’broom in Japan[J].Nippon Kingakukai Kaiho,2009,50:56-60.
[9]朱熙樵.竹类几种丛枝病的特征[J].中国森林病虫,1985,4(2):42-44.
[10]杨永刚,吴小芹.竹丛枝病病原研究进展[J].浙江农林大学学报,2011,28(1):144-148.
[11]朱熙樵,黄金生.关于类菌原体引起竹丛枝病的探讨[J].竹子研究汇刊,1992,11(1):4-9.
[12]Tanaka E.Mechanisms of bamboo witches’broom symptom development caused by endophytic/epiphytic fungi[J].Plant Signaling&Behavior,2010,5(4):415-418.
[13]Tanaka E.Specific in situ visualization of the pathogenic endophytic fungus Aciculosporium take,the cause of witches’broom in bamboo[J].Applied and Environmental Microbiology,2009,75(14):4829-4834.
[14]Duduk B,Paltrinieri S,Lee I M,et al.Nested PCR and RFLP analysis based on the 16S rRNA gene[M]//Dickinson M,Hodgetts J.Phytoplasma:Methods and Protocols.Dordrecht:Springer Science and Business Media,2013:159-171.
[15]Gardes M,Bruns T D.ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts[J].Molecular Ecology,1993,2(2):113-118.
[16]Bellemain E,Carlsen T,Brochmann C,et al.ITS as an environmental DNA barcode for fungi:an in silico approach reveals potential PCR biases[J].BMC Microbiology,2010,10(1):189.
[17]Mohanan C.Little leaf disease of bamboo in Kerala,India[J].BIC-India Bulletin,1994,4(1):30-37.
[18]Jung H Y,Chang M U,Lee J T,et al.Detection of“Candidatus Phytoplasma asteris”associated with henon bamboo witches'broom in Korea[J].Journal of General Plant Pathology,2006,72(4):261-263.
[19]邱并生,李横虹,史春霖,等.从我国20种感病植物中扩增植原体16S r DNA片段及其RFLP分析[J].林业科学,1998,34(6):67-74.
[20]徐梅卿,戴玉成,范少辉,等.中国竹类病害记述及其病原物分类地位(上)[J].林业科学研究,2006,19(6):692-699.
[21]Schoch C L,Seifert K A,Huhndorf S,et al.Nuclear ribosomal internal transcribed spacer(ITS)region as a universal DNA barcode marker for fungi[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(16):6241-6246.
[22]赵杰.ITS序列分析及其在植物真菌病害分子检测中的应用[J].陕西农业科学,2004,(4):35-37.
[23]朱熙樵,黄焕华.竹丛枝病的研究III.病原菌的侵染特点和防治试验[J].南京林业大学学报,1989,13(2):46-51.
[24]Tanaka E,Tanaka C,Tsuda M.Transmission and intraspecific variation of Aciculosporium take,the causal agent of witches’broom of bamboo[J].Forest Research,Kyoto,2002,74:13-20.
[25]刘军,周云娥,许岳冲,等.笋用竹病虫害调查与研究[J].竹子研究汇刊,2001,20(2):72-79.
[2]莫润宏,汤富彬,丁明,等.氨基酸分析仪法测定竹笋中游离氨基酸[J].化学通报,2012,75(12):1126-1131.
[3]丁兴萃.覆盖栽培早竹开花的激素机制研究[J].林业科学,2007,43(7):10-15.
[4]盛常顺.几种笋用竹对竹丛枝病的抗性试验研究[J].安徽林业科技,2004,122(3):23-24.
[5]陈建寅,陈晓岚.安吉竹种园竹丛枝病调查报告[J].浙江林业科技,1992,12(2):47-50.
[6]Kao C W,Leu L S.Finding perfect stage of Aciculosporium take Miyake,the causal organism of bamboo witches’broom disease and its conidial germination[J].Plant Protection Bulletin,1976,18(3):276-285.
[7]江泽慧.世界竹藤[M].沈阳:辽宁科学技术出版社,2002.
[8]Tanaka E,Tanaka C,Shibata S.Bamboo witches’broom in Japan[J].Nippon Kingakukai Kaiho,2009,50:56-60.
[9]朱熙樵.竹类几种丛枝病的特征[J].中国森林病虫,1985,4(2):42-44.
[10]杨永刚,吴小芹.竹丛枝病病原研究进展[J].浙江农林大学学报,2011,28(1):144-148.
[11]朱熙樵,黄金生.关于类菌原体引起竹丛枝病的探讨[J].竹子研究汇刊,1992,11(1):4-9.
[12]Tanaka E.Mechanisms of bamboo witches’broom symptom development caused by endophytic/epiphytic fungi[J].Plant Signaling&Behavior,2010,5(4):415-418.
[13]Tanaka E.Specific in situ visualization of the pathogenic endophytic fungus Aciculosporium take,the cause of witches’broom in bamboo[J].Applied and Environmental Microbiology,2009,75(14):4829-4834.
[14]Duduk B,Paltrinieri S,Lee I M,et al.Nested PCR and RFLP analysis based on the 16S rRNA gene[M]//Dickinson M,Hodgetts J.Phytoplasma:Methods and Protocols.Dordrecht:Springer Science and Business Media,2013:159-171.
[15]Gardes M,Bruns T D.ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts[J].Molecular Ecology,1993,2(2):113-118.
[16]Bellemain E,Carlsen T,Brochmann C,et al.ITS as an environmental DNA barcode for fungi:an in silico approach reveals potential PCR biases[J].BMC Microbiology,2010,10(1):189.
[17]Mohanan C.Little leaf disease of bamboo in Kerala,India[J].BIC-India Bulletin,1994,4(1):30-37.
[18]Jung H Y,Chang M U,Lee J T,et al.Detection of“Candidatus Phytoplasma asteris”associated with henon bamboo witches'broom in Korea[J].Journal of General Plant Pathology,2006,72(4):261-263.
[19]邱并生,李横虹,史春霖,等.从我国20种感病植物中扩增植原体16S r DNA片段及其RFLP分析[J].林业科学,1998,34(6):67-74.
[20]徐梅卿,戴玉成,范少辉,等.中国竹类病害记述及其病原物分类地位(上)[J].林业科学研究,2006,19(6):692-699.
[21]Schoch C L,Seifert K A,Huhndorf S,et al.Nuclear ribosomal internal transcribed spacer(ITS)region as a universal DNA barcode marker for fungi[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(16):6241-6246.
[22]赵杰.ITS序列分析及其在植物真菌病害分子检测中的应用[J].陕西农业科学,2004,(4):35-37.
[23]朱熙樵,黄焕华.竹丛枝病的研究III.病原菌的侵染特点和防治试验[J].南京林业大学学报,1989,13(2):46-51.
[24]Tanaka E,Tanaka C,Tsuda M.Transmission and intraspecific variation of Aciculosporium take,the causal agent of witches’broom of bamboo[J].Forest Research,Kyoto,2002,74:13-20.
[25]刘军,周云娥,许岳冲,等.笋用竹病虫害调查与研究[J].竹子研究汇刊,2001,20(2):72-79.
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