RFLP analysis of the ribosomal DNA ITS regions of four physiological races of Fusarium oxysporum f. sp. Cubense
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摘要:目的
明确香蕉枯萎病菌Fusarium oxysporum f. sp. cubense 4个生理小种的系统发育关系。
方法利用PCR扩增该病菌4个生理小种的核糖体DNA内转录间隔区(Internal transcribed spacer, ITS)，利用AIu Ⅰ、Hpa Ⅱ和Taq Ⅰ 3种限制性内切酶对这些小种的ITS产物进行RFLP分析。
结果PCR扩增结果表明：来自4个生理小种的8个菌株均可扩增得到568 bp的ITS单一片段，但这些生理小种间的片段没有明显的多态性；对这些生理小种ITS产物进行RFLP分析发现，不同生理小种之间的差异很小。
结论PCR-RFLP技术不适用香蕉枯萎病菌生理小种的分析鉴定。
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关键词:
- 香蕉枯萎病菌 /
- 生理小种 /
- 内部转录间隔区 /
- 聚合酶链式反应 /
- 限制性片段长度多态性
Abstract:ObjectiveTo clarify the phylogenetic relationship of four physiological races of Fusarium oxysporum f. sp. cubense (Foc), the causal agent of fusarial wilt of banana.
MethodThe ribosomal DNA internal transcribed spacer (ITS) regions of the four physiological races of Foc were first amplified using PCR technique, and then RFLP analysis of the ITS products of these races were performed using three restriction endonucleases including AIu Ⅰ, Hpa Ⅱ and Taq Ⅰ.
ResultA single 568 bp ITS fragment was amplified specifically from eight strains belonging to four races, respectively, but there was no obvious polymorphism among these races. RFLP analysis of the ITS products from these races showed that the differences among races were very small.
ConclusionPCR-RFLP technique was unsuitable to be used as the identification basis for the physiological races of Foc.
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图 1 香蕉枯萎病菌4个生理小种的ITS-PCR扩增产物
M:DL2000 DNA Marker；1:WG02；2:WG03；3:WG04；4:WG05；5:Z13；6:Z14；7:Z15;8:Z16；9:ddH2O。
Figure 1. ITS-PCR products of four physiological races of Fusarium oxysporum f. sp. cubense
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图 2 香蕉枯萎病菌4个生理小种的ITS序列比对
WG02、WG03、WG04、WG05分别为国外的1~4号生理小种菌株；Z13和Z16为国内的4号生理小种菌株；Z14和Z15为国内的1号生理小种菌株。
Figure 2. ITS sequences alignment of four physiological races of Fusarium oxysporum f. sp. cubense
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[1] GHAG S B, SHEKHAWAT U K S, GANAPATHI T R. Fusarium wilt of banana: Biology, epidemiology and management[J]. Int J Pest Manage, 2015, 61(3): 250-263. doi: 10.1080/09670874.2015.1043972
[2] 杨旸. 香蕉枯萎病菌两个生理小种fgb和fmk基因的克隆与序列分析[D]. 广州: 华南农业大学, 2010. [3] 吕顺, 曾莉莎, 刘文清, 等.大蕉枯萎病病原菌鉴定及TEF-1α序列分析[J].植物病理学报, 2014, 44(4): 337-348. http://www.cnki.com.cn/Article/CJFDTOTAL-ZWBL201404001.htm [4] 刘景梅, 王璧生, 陈霞, 等.广东香蕉枯萎病菌生理小种RAPD技术的建立[J].广东农业科学, 2004(4): 43-45. http://www.cnki.com.cn/Article/CJFDTOTAL-GDNY200404017.htm [5] FOULY H M. ITS ribosomal DNA phylogeny of Gaeumannomyces graminis[J]. Arab J Biotech, 2002, 7(1): 45-52. http://www.academia.edu/8410521/ITS_ribosomal_DNA_phylogeny_of_Gaeumannomyces_graminis
[6] 李志芳, 冯自力, 赵丽红, 等.棉花主要真菌病害病原菌ITS-RFLP快速鉴定方法[J].中国棉花, 2013, 40(12): 13-16. doi: 10.3969/j.issn.1000-632X.2013.12.004 [7] 王文华. 香蕉枯萎镰刀菌1、4号小种rDNA-ITS序列分析及4号小种遗传转化体系建立[D]. 海口: 海南大学, 2007. [8] 王洪凯, 张天宇, 张猛.应用5.8S rDNA及ITS区序列分析链格孢种级分类[J].菌物学报, 2001, 20(2): 168-173. http://www.cnki.com.cn/Article/CJFDTOTAL-JWXT200102004.htm [9] BURGERMEISTER W, BRAASCH H, METGE K, et al. ITS-RFLP analysis, an efficient tool for differentiation of Bursaphelenchus species[J]. Nematology, 2009, 11(5): 649-668. doi: 10.1163/156854108/399182
[10] MARTÍNEZ-CULEBRAS P V, RAMÓN D. An ITS-RFLP method to identify black Aspergillus isolates responsible for OTA contamination in grapes and wine[J]. Int J Food Microbiol, 2007, 113(2): 147-153. doi: 10.1007/s00217-009-1109-x
[11] VIAUD M, PASQUIER A, BRYGOO Y. Diversity of soil fungi studied by PCR-RFLP of ITS[J]. Mycol Res, 2000, 104(9): 1027-1032. doi: 10.1017/S0953756200002835
[12] ERLAND S, HENRION B, MARTIN F, et al. Identification of the ectomycorrhizal basidiomycete Tylospora fibrillosa Donk by RFLP analysis of the PCR-amplified ITS and IGS regions of ribosomal DNA[J]. New Phytol, 1994, 126(3): 525-532. doi: 10.1111/nph.1994.126.issue-3
[13] 陈永青, 姜子德, 戚佩坤. RAPD分析与ITS序列分析在拟茎点霉分类鉴定上的应用[J].菌物学报, 2002, 21(1): 39-46. http://www.cnki.com.cn/Article/CJFDTOTAL-JWXT200201011.htm [14] GARDES M, BRUNS T D. ITS-RFLP matching for identification of fungi[J]. Methods Mol Biol, 1996, 50: 177-186. doi: 10.1385/0-89603-323-6:177/fulltext.html
[15] PANDEY A K, REDDY M S, SURYANARAYANAN T S. ITS-RFLP and ITS sequence analysis of a foliar endophytic Phyllosticta from different tropical trees[J]. Mycol Res, 2003, 107(4): 439-444. doi: 10.1017/S0953756203007494
[16] MILLS P R, SREENIVASAPRASAD S, BROWN A E. Detection and differentiation of Colletotrichum gloeosporioides isolates using PCR[J]. FEMS Microbiol Lett, 1992, 98(1/2/3): 137-143. http://uwispace.sta.uwi.edu/dspace/bitstream/handle/2139/38062/1471-2148-13-130.xml;sequence=1
[17] 张瑞萍, 王家军, 魏崃, 等.一株大豆腐霉菌(PSHH1) rDNA的ITS序列鉴定[J].大豆科学, 2015, 34(5): 867-873. http://www.cnki.com.cn/Article/CJFDTOTAL-DDKX201505023.htm [18] 岳万松. 云南牛肝菌的分子鉴定及其多糖抗氧化性研究[D]. 昆明: 云南民族大学, 2015. [19] 刘新锐, 谢宝贵, 熊芳, 等.高温平菇种质资源的ITS-RFLP分析[J].热带作物学报, 2010, 31(9): 1509-1513. http://www.cnki.com.cn/Article/CJFDTOTAL-RDZX201009015.htm [20] 张文娟, 康帅, 魏锋, 等.基于ITS序列分析鉴别冬虫夏草与古尼虫草[J].药物分析杂志, 2015(9): 1551-1555. http://www.cnki.com.cn/Article/CJFDTOTAL-YWFX201509008.htm [21] 曾莉莎, 吕顺, 刘文清, 等.基于多基因序列分析对尖孢镰孢菌古巴专化型(香蕉枯萎病菌)生理小种的鉴定[J].菌物学报, 2014, 33(4): 867-882. http://www.cnki.com.cn/Article/CJFDTOTAL-JWXT201404014.htm [22] 张俊华, 张明, 韩英鹏, 等.大豆灰斑病菌生理小种PCR-RFLP分子检测[J].中国油料作物学报, 2010, 32(1): 128-131. http://www.cnki.com.cn/Article/CJFDTOTAL-ZGYW201001026.htm
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