从混合线虫样品和植物组织中直接检测香蕉穿孔线虫的ITS-PCR方法

doi:10.3864/j.issn.0578-1752.2011.19.008

摘要/Abstract

摘要： 【目的】建立直接从多种线虫混合样品以及香蕉和红掌根组织中检测鉴定香蕉穿孔线虫的PCR方法。【方法】使用Primer Premier 5.0在香蕉穿孔线虫的ITS区设计1对特异性引物，运用PCR技术对目的线虫DNA进行特异性检测。【结果】通过对17种24个种群线虫的检测表明，所设计的引物只能从香蕉穿孔线虫种群中特异扩增出rDNA-ITS片段，产物大小为518 bp。利用该特异引物以及建立的DNA提取方法和PCR体系，可以直接从香蕉穿孔线虫与短体线虫、螺旋线虫、肾状线虫、根结线虫、茎线虫、矮化线虫、纽带线虫、丝尾垫刃线虫、滑刃线虫和小杆线虫混合样品中特异扩增出目的线虫的rDNA-ITS片段，并可以分别从混合有不少于3条香蕉穿孔线虫的2 cm香蕉或红掌根组织（约0.1 g）中特异检测出目的线虫的DNA片段。【结论】本研究设计的特异性引物以及建立的DNA提取方法和PCR体系可直接从多种线虫混合的样品以及香蕉和红掌根组织中快速检测鉴定出香蕉穿孔线虫。

关键词:

香蕉穿孔线虫, ITS区特异引物, 混合线虫样品, 植物组织, PCR检测

Abstract: 【Objective】 The objective of this study is to build an ITS-PCR (internal transcribed spacer-polymerase chain reaction) method for detecting Radopholus similis (Cobb) Thorne from mixtures of different nematodes, or tissues of Musa spp. and Anthurium andraeanum. 【Method】 A specific primer pair was designed according to ITS region of rDNA from R. similis using Primer Premier 5.0. A single-step PCR method was used to specially detect the target ITS-rDNA of R. similis. 【Result】 The specificity of the designed primers was confirmed that a 518 bp PCR product was amplified from 7 populations of R. similis, not from the other 16 species including 17 populations. Using the established DNA extraction method and PCR system by the specific primers, the target ITS-rDNA fragment was specially amplified from DNA templates of the mixture of individuals of R. similis with species of Pratylenchus, Helicotylenchus, Rotylenchulus, Meloidogyne, Ditylenchus, Tylenchorhynchus, Hoplolaimus, Filenchus, Aphelenchoides, and Rhabditida, and also from the DNA mixtures of at least 3 individuals of R. similis with root tissues (2 cm long, about 0.1 g) of banana and Anthurium andraeanum. 【Conclusion】 The ITS-PCR method established in this study was proven to be a reliable and useful technique for fast and accurate detecting of R. similis from mixed nematode samples and mixtures of root tissues of banana or A.andraeanum.

Key words:

Radopholus similis, rDNA-ITS region specific primers, nematodes mixture, plant tissues, PCR detection

刘一帆, 徐春玲, 张超, 苏秀敏, 谢辉. 从混合线虫样品和植物组织中直接检测香蕉穿孔线虫的ITS-PCR方法[J]. 中国农业科学, 2011, 44(19): 3991-3998.

LIU Yi-Fan, XU Chun-Ling, ZHANG Chao, SU Xiu-Min, XIE Hui. Direct PCR Detection of Radopholus similis from Plant Tissues and Mixed Sample of Nematodes by ITS Specific Primers[J]. Scientia Agricultura Sinica, 2011, 44(19): 3991-3998.

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参考文献

[1]O’Bannon J H. Worldwide dissemination of Radopholus similis and its importance in crop production. Journal of Nematology, 1977, 9(1): 16-25.

[2]Griffith R, Koshy P K. Nemtodes parasite of coconuts and other palms //Luc M, Sikora R T, Bridge J. Plant Parasitic Nematodes in Subtropical and Tropical Agriculture. UK: CAB International, 1990: 363-386.

[3]Richardson P N, Grewal P S. Nematode pests of glasshouse crops and mushrooms//Evans K, Trudgill D L, Webster J M. Plant Parasitic Nematodes in Temperate Agriculture. UK: CAB International, 1993: 501-544.

[4]Uchida J Y, Sipes B S, Kadooka C Y. Burrowing nematode on Anthurium: recognizing symptoms, understanding the pathogen and preventing disease. Plant Disease, 2003, 4, PD-24: 1-11.

[5]谢辉. 香蕉穿孔线虫及其检测和防疫控制. 植物检疫, 2006, 20(5): 321-324.

Xie H. Detection and management of Radopholus similis. Plant Quarantine, 2006, 20(5): 321-324. (in Chinese)

[6]Cotton J, Van Riel H. Quarantine: problems and solutions//Evans K, Trudgill D L, Webster J M. Plant Parasitic Nematodes in Temperate Agriculture. UK: CAB International, 1993: 593-608.

[7]OEPP/EPPO. Radopholus similis. Bulletin OEPP/EPPO Bulletin, 2008, 38: 374-378.

[8]中华人民共和国农业部. 中华人民共和国进境植物检疫性有害生物名录. 中华人民共和国农业部公告第862号, 2007.

Ministry of Agriculture of the People’s Republic of China. Entry plant quarantine pest list of the People's Republic of China. Bulletin No. 862 of the Ministry of Agriculture of the People’s Republic of China, 2007. (in Chinese)

[9]Gowen S, Queneherve P. Nematode parasites of bananas, plantains, and abaca // Luc M, Sikora R A, Bridge J St. Albans. Plant Parasitic Nematodes in Subtropical and Tropical Agriculture. UK: CAB International, 1990: 431-460.

[10]李迅东, 翟留香, 孙茂林. 云南省香蕉线虫发生分布和种群密度调查. 西南农业学报, 1999, 12(1): 92-95.

Li X D, Zhai L X, Sun M L. Survey of occurrence, distribution and population density of banana nematodes in Yunnan. Southwest China Journal of Agricultural Sciences, 1999, 12(1): 92-95. (in Chinese)

[11]周春娜, 谢辉, 徐春玲, 陈淳, 蔚应俊, 黄德超. 矮化属线虫2个中国新纪录种记述. 华中农业大学学报, 2004, 23(5): 507-509.

Zhou C N, Xie H, Xu C L, Chen C, Wei Y J, Huang D C. Description of two new record species of the genus Tylenchorhynchus in China. Journal of Huazhong Agricultural University, 2004, 23(5): 507-509. (in Chinese)

[12]周春娜, 谢辉, 徐春玲, 陈淳, 吴仕豪, 黄德超. 中国花卉植物寄生线虫新记录种记述. 华中农业大学学报, 2005, 24(1): 10-14.

Zhou C N, Xie H, Xu C L, Chen C, Wu S H, Huang D C. Description of new record species from flowers plant parasitic nematode in China. Journal of Huazhong Agricultural University, 2005, 24(1): 10-14. (in Chinese)

[13]周春娜, 谢辉, 吴仕豪, 曹俐, 李梅辉, 武目涛. 广东省花卉线虫新记录种. 华南农业大学学报, 2005, 26(1): 93-95.

Zhou C N, Xie H, Wu S H, Cao L, Li M H, Wu M T. The new record of flowers parasitic nematodes in Guangdong Province. Journal of South China Agricultural University, 2005, 26(1): 93-95. (in Chinese)

[14]周春娜, 徐春玲, 陈淳, 谢辉. 观赏植物上两种纽带亚科线虫记述. 中国线虫学研究 (第三卷), 2010, 3: 32-36.

Zhou C N, Xu C L, Chen C, Xie H. Description of two species of the subfamily Hoplolaiminae from ornamental plants in China. Nematology Research in China (vol.3), 2010, 3: 32-36. (in Chinese)

[15]鄢小宁, 郑服丛, 林茂松. 两广地区香蕉根际寄生线虫的调查与鉴定. 热带农业科学, 2005, 25(6): 4-8.

Yan X N, Zheng F C, Lin M S. Investigation and identification of nematode parasites of banana in Guangdong and Guangxi. Chinese Journal of Tropical Agriculture, 2005, 25(6): 4-8. (in Chinese)

[16]Elbadri G A A, Geraert E, Moens M. Morphological differences among Radopholus similis (Cobb, 1893) Thorne, 1949 populations. Russian Journal of Nematology, 1999, 7(2): 139-153.

[17]Huettel R N, Yaegashi T. Morphological differences between Radopholus citrophilus and R. similis. Journal of Nematology, 1988, 20(1): 150-157.

[18]Kaplan D T, Opperman C H. Genome similarity implies that citrus-parasitic burrowing nematodes do not represent a unique species. Journal of Nematology, 1997, 29(4): 430-440.

[19]Kaplan D T, Thomas W K, Frisse L M, Sarah J L, Stanton J M, Speijer P R, Marin D H, Opperman C H. Phylogenetic analysis of geographically diverse Radopholus similis via rDNA sequence reveals a monomorphic motif. Journal of Nematology, 2000, 32(2): 134-142.

[20]Koshy P K, Jasy T, Mathew J, Nampoothiri C K. Morphometric variability in Radopholus similis. Indian Journal of Nematology, 1991, 21(1): 24-38.

[21]Tarté R, Pinochet J, Gabrielli C, Ventura O. Differences in population increase, host preferences and frequency of morphological variants among isolates of the banana race of Radopholus similis. Nematropica, 1981, 11(1): 43-52.

[22]Valette C, Mounport D, Nicole M, Sarah J L, Baujard P. Scanning electron microscope study of two African populations of Radopholus similis (Nematoda: Pratylenchidae) and proposual of R. citrophilus as a junior synonym of R. similis. Fundamental and Applied Nematology, 1998, 21(2): 139-146.

[23]Elbadri G A A, Ley P D, Waeyenberge L, Vierstraete A, Moens M, Vanfleteren J. Intraspecific variation in Radopholus similis isolates assessed with restriction fragment length polymorphism and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA cistron. International Journal for Parasitology, 2002, 32(2): 199-205.

[24]Fallas G A, Hahn M L, Fargette M, Burrows P R, Sarah J L. Molecular and biochemical diversity among isolates of Radopholus spp. from different areas of the world. Journal of Nematology, 1996, 28(4): 422-430.

[25]龙海, 李芳荣, 彭德良, 李一农. 香蕉穿孔线虫（Radopholus similis）的普通PCR和实时荧光PCR诊断. 中国线虫学研究 (第二卷), 2008, 2: 70-76.

Long H, Li F R, Peng D L, Li Y N. Development of a PCR and real-time fluorescent PCR diagnostic for the burrowing nematode Radopholus similis. Nematology Reaserch in China, 2008, 2: 70-76. (in Chinese)

[26]周春娜, 谢辉, 马以桂, 王金成, 李云, 徐春玲. 相似穿孔线虫PCR检测鉴定的技术与方法. 华中农业大学学报, 2006, 25(1): 40-42.

Zhou C N, Xie H, Ma Y G, Wang J C, Li Y, Xu C L. PCR identification of Radopholus similis Throne. Journal of Huazhong Agricultural University, 2006, 25(1): 40-42. (in Chinese)

[27]葛建军, 曹爱新, 周国梁, 赵国柱, 谢丙炎. 香蕉穿孔线虫双重PCR快速检测技术研究. 植物病理学报, 2007, 37(5): 472-478.

Ge J J, Cao A X, Zhou G L, Zhao G Z, Xie B J. Development of duplex PCR assay for detection of borrowing nematode, Radopholus similis. Acta Phytopathologica Sinica, 2007, 37(5): 472-478. (in Chinese)

[28]谢辉. 植物线虫分类学. 第二版. 北京: 高等教育出版社, 2005: 38-84.

Xie H. Taxonomy of Plant Nematodes. 2nd ed. Beijing: China Higher Education Press, 2005: 38-84. (in Chinese)

[29]Rahman S A S A, Mohamed Z, Othman R Y, Swennen R, Panis B, Waele D D, Remy S, Carpentier S C. In planta PCR-based detection of early infection of plant-parasitic nematodes in the roots: a step towards the understanding of infection and plant defence. European Journal of Plant Pathology, 2010, 128: 343-351.