采用EF-1α序列分析法对苜蓿根腐病病原菌——锐顶镰刀菌的鉴定

doi:10.11686/cyxb2016100

草业学报 ›› 2017, Vol. 26 ›› Issue (2): 61-68.DOI: 10.11686/cyxb2016100

采用EF-1α序列分析法对苜蓿根腐病病原菌——锐顶镰刀菌的鉴定

易铭¹, 梁嘉俊¹, 史建², 李洪建¹, 程积民¹, 焦锋^{1*, *}

1.西北农林科技大学动物科技学院,陕西杨凌 712100;
2.彬县西坡乡动物卫生监督所,陕西彬县 713500

收稿日期:2016-03-09 出版日期:2017-02-20 发布日期:2017-02-20
通讯作者: fjiao@nwsuaf.edu.cn
作者简介:易铭(1990-),男,黑龙江牡丹江人,在读硕士。E-mail:yiminggodlike@163.com
基金资助:
国家公益性行业(农业)科研专项经费项目(201303157)和国家现代农业牧草产业技术体系(CRAS-35)资助

Identification of Fusaruim acuminatum isolated from Medicago sativa root using the EF-1α sequence analysis method

YI Ming¹, LIANG Jia-Jun¹, SHI Jian², LI Hong-Jian², CHENG Ji-Min¹, JIAO Feng^{1, *}

1.College of Animal Science and Technology, North West Agriculture and Forestry University, Yangling 712100, China;
2.Animal Health Inspection of Xipo Country, Binxian 713500, China

Received:2016-03-09 Online:2017-02-20 Published:2017-02-20

摘要/Abstract

摘要： 苜蓿根腐病对苜蓿生长和产量造成严重影响,为了解引起根腐病病原真菌的种类,本文对从陕西省定边县牧草草场紫花苜蓿根部分离得到的根腐病病原菌进行形态学观察、EF-1α序列分析鉴定及接种试验。结果表明:通过对病原菌在PDA培养基中的分离、纯化得到的单菌落形态学观察及显微镜下菌丝、孢子的观察,该病原菌属于引起苜蓿根腐病的镰刀菌属(Fusarium);用CTAB法提取病原菌基因组DNA,并对EF-1α序列进行PCR扩增、回收纯化、克隆测序,将测序结果进行Blast比对,构建系统发育树,分析与Genbank已知近缘种属亲缘关系,结果显示与锐顶镰刀菌(F. acuminatum)亲缘关系最近,可信度达99%;最后通过根部接种试验,接种苜蓿根部发病症状与田间根腐病发病症状一致,鉴定结果显示本试验研究的引起紫花苜蓿根腐病的病原菌为镰刀菌属锐顶镰刀菌(F. acuminatum)。

Abstract: Alfalfa root rot causes serious reductions in alfalfa growth and yield. To identify the species of pathogenic fungus causing root rot disease, the root rot pathogen of Medicago sativa (alfalfa) collected from pasture in Dingbian County, Shaanxi Province, was analyzed by morphological observations, EF-1α sequence analysis, and inoculation tests. The mycelium and spores of a single colony of the pathogen growing on culture medium were observed under a microscope. Based on its morphology, the pathogen was identified as a Fusarium fungus. The genomic DNA was extracted from the pathogen using the cetyl-trimethylammonium bromide method, and the translation elongation factor gene was selected for PCR amplification, gel recycling, purification, cloning, sequencing, and phylogenetic analyses. A phylogenetic tree constructed using MEGA and sequences available in Genbank showed that the pathogen had the closest relationship with Fusarium acuminatum (99%). In the root inoculation test, the disease symptoms of roots inoculated with the isolated pathogen were the same as those characteristic of root rot disease of M. sativa in the field. Together, these results verified that the pathogen causing root rot disease of M. sativa in Shaanxi Province is F. acuminatum.

易铭, 梁嘉俊, 史建, 李洪建, 程积民, 焦锋. 采用EF-1α序列分析法对苜蓿根腐病病原菌——锐顶镰刀菌的鉴定[J]. 草业学报, 2017, 26(2): 61-68.

YI Ming, LIANG Jia-Jun, SHI Jian, LI Hong-Jian, CHENG Ji-Min, JIAO Feng. Identification of Fusaruim acuminatum isolated from Medicago sativa root using the EF-1α sequence analysis method[J]. Acta Prataculturae Sinica, 2017, 26(2): 61-68.

参考文献

[1] Geng H Z, Wu Y F, Cao Z Z. Chinese Alfalfa[M]. Beijing: Chinese Agriculture Press, 1995: 114-130.
耿华珠, 吴永敷, 曹致中. 中国苜蓿[M]. 北京: 中国农业出版社, 1995: 114-130.
[2] Nan Z B. Establishing sustainable management system for disease of pasture crops in China. Acta Prataculturae Sinica, 2000, 9(2): 1-9.
南志标. 建立中国的牧草病害可持续管理体系. 草业学报, 2000, 9(2): 1-9.
[3] Cao L X, Zhao C H, Kong Q Q, et al . Advances in Alfalfa root rot pathogen and control research. Inner Mongolia Agriculture Science and Technology, 2006, (3): 36-37.
曹丽霞, 赵存虎, 孔庆全, 等. 紫花苜蓿根腐病病原及防治研究进展. 内蒙古农业科技, 2006, 3: 36-37.
[4] Xue F X. Grassland Protection[M]. Beijing: Chinese Agriculture Press, 2009.
薛福祥. 草地保护学——牧草病理学分册[M]. 北京: 中国农业出版社, 2009.
[5] Wang X W, Wang C L, Zhou G, et al . Investigation and analysis on Lucerne diseases from Altay newly-established areas in northern Xinjiang. Journal of Xinjiang Agriculture University, 1996, 19(3): 40-44.
王雪薇, 王纯利, 周刚, 等. 新疆阿勒泰新垦区苜蓿病害调查与分析. 新疆农业大学学报, 1996, 19(3): 40-44.
[6] Meng Y, Li M Q. Research progress on alfalfa crown and root rot pathogen and their control. Pratacultural Science, 2005, 22(5): 89-92.
孟嫣, 李敏权. 苜蓿根和根茎腐烂病病原及防治研究进展. 草业科学, 2005, 22(5): 89-92.
[7] Chen Y J, Cui G W. Survey and separation of alfalfa root rot pathogen in Heilongjiang Province. Grassland of China, 2001, 23(3): 78-79.
陈雅君, 崔国文. 黑龙江省紫花苜蓿根腐病调查及病原分离. 中国草地, 2001, 23(3): 78-79.
[8] Zhang B. Identification, Classification, and Phylogenetic Relationships of Pathogenic Fungi Based on Mitochondrial Cytochrome b Gene and Translation Elongation Factor Gene Analysis[D]. Jilin: Basic Medical College of Jilin University, 2007.
张波. 线粒体细胞色素b和翻译延伸因子基因分析用于病原真菌的分离、鉴定和系统发生[D]. 吉林: 吉林大学基础医学院, 2007.
[9] Kristensen R, Gauthier G, Berdal K G. DNA microarray to detect and identify trichothecene and moniliformin-producing Fusarium species. Journal of Applied Microbiology, 2007, 102(4): 1060-1070.
[10] Yergeau E, Filion M, Vujanovic V. A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus. Journal of Microbiological Methods, 2005, 60(12): 143-154.
[11] Fang Z D. The Method on Studies of Plant Pathology[M]. Beijing: Chinese Agriculture Press, 1998: 122-125.
方中达. 植病研究方法[M]. 北京: 中国农业出版社, 1998: 122-125.
[12] Guo L D, Hyde K D, Liew E C Y. Identification of endophytic fungi from Livistona chinensis based on morphology and rDNA sequences. New Phytologist, 2000, 147: 617-630.
[13] O’Donnell K, Kistler H C, Cigelnik E, et al . Multiple evolutionary origins of the fungus causing Panama disease of banana: Concordant evidence from nuclear and mitochondrial gene genealogies. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(5): 2044-2049.
[14] Zhang Z Y, Leng H Q, Zhang Z M, et al . Plant Pathogenic Mycology[M]. Chengdu: Sichuan Science and Technology Press, 1988: 384-386.
张中义, 冷怀琼, 张志铭, 等. 植物病原真菌学[M]. 成都: 四川科学技术出版社, 1988: 384-386
[15] Mahdi Dacari, Anne Dvan Diepeningen, Assadollah Babai-Ahari, et al . Rapid identification of Fusarium grammine species complex using Rolling Circle Amplification(RCA). Journal of Microbiological Methods, 2012, 89(1): 63-70.
[16] O’Donnell K, Humber R A, Geiser D M, et al . Phylogenetic diversity of insecticolous fusaria inferred from multilocus DNA sequence data and their molecular identification via FUSARIUM-ID and Fusarium MLST. Mylologia, 2012, 104(2): 427-445.
[17] Stenglein S A, Rodriguero M S, Chandler E, et al . Phylogenetic relationships of Fusarium poae based on EF-1 alpha and mtSSU sequences. Fungal Biology, 2010, 114(1):96-106.
[18] Grafenhan T, Patrick S K, Roscoe M, et al . Fusarium damage in cereal grains from Western Canada. Journal of Agricultural and Food Chemistry, 2013, 61(23):5425-5437.
[19] Guo Y X, Nan Z B, Wang C Z, et al . Progress in research on root invading fungi of Medicago sativa . Acta Prataculturae Sinica, 2009, 18(5): 243-249.
郭玉霞, 南志标, 王成章, 等. 苜蓿根部入侵真菌研究进展. 草业学报, 2009, 18(5): 243-249.
[20] Chen Y J, Liu X M, Cui G W, et al . Research progress in root rot of alfalfa. Grassland of China, 2000, (1): 51-56.
陈雅君, 刘学敏, 崔国文, 等. 紫花苜蓿根腐病的研究进展. 中国草地, 2000, (1): 51-56.
[21] Zhao Z H, Lu G Z, Jiang Z D, et al . Identification of Fusarium kyushuense , F. sporotrichioides and F. chlamydosporum using EF-1α sequence data and morphological characteristics of conidia in aerial mycelium. Mycosystema, 2011, 30(5): 713-720.
[22] Li M Q. Comparative pathogenicity of isolates of Fusarium spp and cultivars resistance in alfalfa. Grassland of China, 2003, 25(1): 39-43.
李敏权. 苜蓿根和根颈腐烂病病原致病性及品种抗病性研究. 中国草地, 2003, 25(1): 39-43.

采用EF-1α序列分析法对苜蓿根腐病病原菌——锐顶镰刀菌的鉴定

Identification of Fusaruim acuminatum isolated from Medicago sativa root using the EF-1α sequence analysis method

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics