尖孢镰刀菌分子检测技术的建立与应用

doi:10.11686/cyxb2015335

草业学报 ›› 2016, Vol. 25 ›› Issue (5): 109-115.DOI: 10.11686/cyxb2015335

尖孢镰刀菌分子检测技术的建立与应用

伍文宪^{1, 2}, 刘勇^{1, 2*, *}, 黄小琴^{1, 2}, 张蕾^{1, 2}, 周西全^{1, 2}, 刘红雨¹

1.四川省农业科学院植物保护研究所,四川成都 610066;
2.农业部西南作物有害生物综合治理重点实验室,四川成都 610066

收稿日期:2015-07-07 出版日期:2016-05-20 发布日期:2016-05-20
通讯作者: *通信作者Corresponding author. E-mail: liuyongdr@163.com
作者简介:作者简介:伍文宪(1988-),男,江西安福人,研究实习员,硕士。E-mail: wuwenxian07640134@163.com
基金资助:
农业公益性行业计划和草地病害防治技术研究与示范(201303057)资助

Establishment and application of rapid molecular detection for Fusarium oxysporum

WU Wen-Xian^{1, 2}, LIU Yong^{1, 2, *}, HUANG Xiao-Qin^{1, 2}, ZHANG Lei^{1, 2}, ZHOU Xi-Quan^{1, 2}, LIU Hong-Yu¹

1.Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China;
2.Key Laboratory of Integrated Pest Management in Southwest Agriculture Crops of Ministry of Agriculture, Chengdu 610066, China

Received:2015-07-07 Online:2016-05-20 Published:2016-05-20

摘要/Abstract

摘要： 尖孢镰刀菌从形态学和分子生物学上与属内多种菌难于区分,为准确鉴定豆科牧草上的尖孢镰刀菌,本研究基于尖孢镰刀菌与其他镰刀菌的核糖体DNA基因间间隔区IGS(intergenic spacer)序列间的差异,设计了一对特异性引物P₁/P₂,检测体系可以特异地从尖孢镰刀菌中扩增出一条1081 bp的条带;引物P₁/P₂对尖孢镰刀菌基因组DNA的检测阈值为100 pg,对土壤中尖孢镰刀菌分生孢子的检测灵敏度为100个孢子/g土;同时,引物P₁/P₂也可以对发病牧草根部组织中尖孢镰刀菌的存在进行特异性检测。检测体系可不经过室内病原菌的分离纯化即可有效的从土壤和病株中提取的DNA进行PCR检测鉴定,是一种快速有效的豆科牧草根腐病病原菌分子检测技术。

Abstract: It is difficult to distinguish Fusarium oxysporum from other Fusarium species using traditional morphological observations or molecular analysis based on rDNA-ITS sequencing. In order to accurately identify F. oxysporumin legumes, a pair of primers named P₁/P₂ was designed based on differences in ribosomal DNA intergenic spacer (rDNA-IGS) sequences of the Fusarium genus, which can be used to amplify DNA from F. oxysporum by conventional PCR. More than 23 species of root rot pathogens were used to verify the specificity of the primers. P₁/P₂ could amplify a unique 1081 bp sequence from different biotypes of F. oxysporum while it could not amplify from other root rot pathogens. The sensitivity of P₁/P₂ was 100 pg for genomic DNA and 100 conidia/g soil for the root rot pathogens. Furthermore, this pair of primers could directly amplify sequences from the genomic DNA of F. oxysporum diseased plant samples without pathogen isolation, indicating that this is a rapid and effective legume root rot pathogen detection technology.

伍文宪, 刘勇, 黄小琴, 张蕾, 周西全, 刘红雨. 尖孢镰刀菌分子检测技术的建立与应用[J]. 草业学报, 2016, 25(5): 109-115.

WU Wen-Xian, LIU Yong, HUANG Xiao-Qin, ZHANG Lei, ZHOU Xi-Quan, LIU Hong-Yu. Establishment and application of rapid molecular detection for Fusarium oxysporum[J]. Acta Prataculturae Sinica, 2016, 25(5): 109-115.

参考文献

[1] Nan Z B. Leguminous forage root rot. Grassland and Turf, 1991, (2): 5-11.
[2] Li M Q. Studies on Pathogens and Resistance to Crown and Root Rot in Alfalfa[D]. Lanzhou: Gansu Agricultural University, 2002.
[3] Li X L, Li Y Z. Research advances in biological control of soil-borne disease. Acta Prataculturae Sinica, 2015, 24(3): 204-212.
[4] Wang J S, Wang J M, Li X, et al . Rapid molecular detection of Fusarium oxysporum by PCR. Acta Phytopathologica Sinica, 2013, 43(3): 318-322.
[5] Zhang S M, Li J, Wang Y X, et al . Advances in molecular diagnosis of plant fungal pathogens. Journal of Anhui Agricultural Science, 2010, 38(2): 597-599.
[6] Bruns T D, Taylor J W. Fungal molecular Systematics. Annual Review of Ecology & Systematics, 2003, 22(1): 525-564.
[7] Jacques G, Jacqueline J, Jean F L, et al . Phylogeny of some Fusarium species, as determined by large-subunit rRNA sequence comparison. Molecular Biology & Evolution, 1989, 6(3): 227-242.
[8] Bai L Y. Study on Pathogen Identification and Molecular Detection Technology of Soybean Root Rot[D]. Urumqi: Xinjiang Agricultural University, 2009.
[9] Zhao J. Application of rDNA ITS sequences in molecular detection plant fungous disease. Journal of Shanxi Agricultural Science, 2004, (4): 35-37.
[10] Innis M A. PCR Protocols: a Guide to Methods and Applications[M]. PCR Protocols: A Guide to Methods & Applications. Academic Press, 1990.
[11] Tooley P W, Carras M M, Falkenstein K F. Relationships among group IV phytophthora species inferred by restriction analysis of the ITS2 region. Journal of Phytopathology, 1996, 144(7-8): 363-369.
[12] Liang H, Zhang G Z, Chen W Q, et al . Sequence analysis of rDNA-ITS of Tilletia controversa and its related species. Acta Phytopathologica Sinica, 2005, 35(6): 181-183.
[13] Tang J H, Wang W, Wang Y C. Molecular detection of Colletotrichum orbiculare . Scientia Agricultura Sinica, 2006, 39(10): 2028-2035.
[14] Yi J P, Peng J H. The advances in research methods of Tilletia indica Mitra. Plant Quarantine, 2002, 16(2): 95-97.
[15] Wang Y Q, Fan R H, Liu B, et al . Preliminary analysis of rDNA-IGS of Ustilaginoidea virens isolates from different geographical regions in China. Acta Phytopathologica Sinica, 2010, 40(2): 214-216.
[16] Liang H, Peng Y L, Zhang G Z, et al . Amplification and sequence analysis of the rDNA intergenic spacer(rDNA-IGS) from three Tilletia species. Acta Phytopathologica Sinica, 2006, 36(5): 407-412.
[17] Mitchell J I, Roberts P J, Moss S T. Sequence or structure? A short review on the application of nucleic acid sequence information to fungal taxonomy. Mycologist, 1995, 9: 67-75.
[18] Sugita T, Ikeda R, Shinoda T. Diversity among strains of Cryptococcus neoformans var. gattii as revealed by a sequence analysis of multiple genes and a chemotype analysis of capsular polysaccharide. Microbiology & Immunology, 2001, 45(11):757-768.
[19] Takeshi S, Norio T, Takao S. Characterization of subrepeat regions within rDNA intergenic spacers of the edible basidiomycete Lentinula edodes. Bioscience Biotechnology & Biochemistry, 2002, 66(10): 2125-2133.
[20] Wang J S. Rapid Molecular Detection and Genetic Diversity of Fusarium oxysporum and Identification of Resistance to Soybean Fusarium Wilt[D]. Nanjing: Nanjing Agricultural University, 2012.
[21] Wang C Y, Guo B L. The characteristics of the frequently used nuclear ribosome gene spacers and their utilization in phylogenetic study of plants. Journal of Wuhan Botanical Research, 2008, 26(4): 417-423.
[22] Vander S J, Marant S, Volckaert G. Molecular characterization and phylogenetic utility of the rDNA external transcribed spacer region in Stylosanthes (Fabaceae). Theoretical & Applied Genetics, 2003, 107(2): 291-298.
[23] Acevedo-Rosas R, Cameron K, Sosa V, et al . A molecular phylogenetic study of Graptopetalum (Crassulaceae) based on ETS, ITS, RPL16, and TRNL-F nucleotide sequences. American Journal of Botany, 2004, 91(7): 1099-1104.
[24] Markos S, Baldwin B G. Higher-level relationships and major lineages of Lessingia (Compositae, Astereae) based on nuclear rDNA internal and external transcribed Spacer (ITS and ETS) sequences. Willdenowia, 2009, 26(1):168-183.
[25] Hoggard G D, Kores P J, Molvray M, et al . The phylogeny of Gaura (Onagraceae) based on ITS, ETS, and trnL-F sequence data. American Journal of Botany, 2004, 91(1): 139-148.
[26] Linder C R. The complete external transcribed spacer of 18S-26S rDNA: amplification and phylogenetic utility at low taxonomic levels in asteraceae and closely allied families. Molecular Phylogenetics & Evolution, 2000, 14(2): 285-303.
[1] 南志标. 豆科牧草根腐病. 国外畜牧学-草原与草坪, 1991, (2): 5-11.
[2] 李敏权. 苜蓿根和根颈腐烂病的病原及种质抗病性研究[D]. 兰州: 甘肃农业大学, 2002.
[3] 李兴龙, 李彦忠. 土传病害生物防治研究进展. 草业学报, 2015, 24(3): 204-212.
[4] 王健生, 王佳妹, 李潇, 等. 尖镰孢菌( Fusarium oxysporum )的快速分子检测. 植物病理学报, 2013, 43(3): 318-322.
[5] 张淑梅, 李晶, 王玉霞, 等. 植物病原真菌分子诊断技术研究进展. 安徽农业科学, 2010, 38(2): 597-599.
[8] 白丽艳. 大豆根腐病病原种类鉴定及分子检测技术研究[D]. 乌鲁木齐: 新疆农业大学, 2009.
[9] 赵杰. ITS序列分析及其在植物真菌病害分子检测中的应用. 陕西农业科学, 2004, (4): 35-37.
[12] 梁宏,张国珍,陈万权,等. 小麦矮腥黑穗病菌与其近缘种的rDNA-ITS序列分析. 植物病理学报, 2005, 35(6): 181-183.
[13] 唐建辉, 王伟, 王源超. 西瓜炭疽病菌 Colletotrichum orbiculare 的分子检测. 中国农业科学, 2006, 39(10): 2028-2035.
[14] 易建平, 彭金火. 小麦印度腥黑穗病菌鉴定方法的新进展. 植物检疫, 2002, 16(2): 95-97.
[15] 王永强, 樊荣辉, 刘兵,等. 中国不同地理来源稻曲病菌rDNA-IGS的初步分析. 植物病理学报, 2010, 40(2): 214-216.
[16] 梁宏, 彭友良, 张国珍, 等. 腥黑粉菌属3种检疫性真菌rDNA-IGS区的扩增及其序列分析. 植物病理学报, 2006, 36(5): 407-412.
[20] 王健生. 大豆尖镰孢菌的快速分子检测与遗传多样性分析及品种抗性研究[D]. 南京: 南京农业大学, 2012.
[21] 王川易, 郭宝林. 植物核基因组核糖体基因间隔区序列的结构特点及其在系统发育研究中的应用. 武汉植物学研究, 2008, 26(4): 417-423.

尖孢镰刀菌分子检测技术的建立与应用

Establishment and application of rapid molecular detection for Fusarium oxysporum

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