CN102747137B - 向日葵黑茎病菌的鉴定方法 - Google Patents

向日葵黑茎病菌的鉴定方法 Download PDF

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CN102747137B
CN102747137B CN201110397732.5A CN201110397732A CN102747137B CN 102747137 B CN102747137 B CN 102747137B CN 201110397732 A CN201110397732 A CN 201110397732A CN 102747137 B CN102747137 B CN 102747137B
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sunflower
black stem
phoma
identifying method
primer
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CN102747137A (zh
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赵君
周洪友
景岚
娜仁
张之为
侯亚光
石必显
雷中华
王玉杰
曹雄
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Inner Mongolia Agricultural University
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Abstract

本发明公开了一种向日葵黑茎病菌的鉴定方法，通过采用特定引物对向日葵DNA进行扩增，该方法能够特异性的鉴定黑茎病。采用该方法能对向日葵种子进行带菌检测能够控制此病害的远距离的传播，对田间发病植株进行早期的快速鉴定也是控制病害扩展蔓延的重要的环节。

Description

向日葵黑茎病菌的鉴定方法
技术领域
本发明涉及病菌鉴定方法，具体而言涉及一种向日葵黑茎病菌的鉴定方法。
背景技术
向日葵黑茎病(Sunflower Phoma black stem)是向日葵上由种子携带传播的一种重要的、毁灭性的真菌病害，也是我国的一种对外检疫性病害。引起该病的病原菌的有性态为Leptosphaeria lindquistii属于子囊菌亚门小球腔菌属，无性态为Phoma macdonaldii属于半知菌亚门茎点霉属。向日葵黑茎病菌在向日葵的整个生长期都可进行侵染。该病最典型的症状是病斑初发生于叶柄基部，迅速向茎杆上扩展，形成黑色椭圆形病斑，引起叶片萎蔫干枯。病斑黑色有清晰边缘，严重时可环绕茎杆，导致植株倒伏枯死，茎杆表面产生黑色小粒的分生孢子器，在花盘背面，产生褐色病斑，花盘干枯且小，子粒无法正常灌浆，造成严重减产。
在我国向日葵黑茎病是一种新病害，2008年第一次报道在新疆地区有此病害发生。2011年的调查的结果表明除新疆外，内蒙古西部巴彦淖尔市、东部的阿荣旗地区、宁夏的石嘴山地区、甘肃兰州安宁区、山西的汾阳地区、河北的张家口地区、黑龙江甘南地区等均有不同程度的发生，重病田的发病株率高达100％。重病田对产量的损失超过60％。
发明内容
然而，在向日葵黑茎病株中可能存在其它病原菌混合侵染的现象，如果不能及时确定病原菌类型，就无法做到有针对性的进行防治。本发明的目的是提供一种向日葵黑茎病菌的快速检测方法。为了实现本发明的目的，采用如下技术方案：
本发明一方面涉及向日葵黑茎病菌的鉴定方法，包括如下步骤：
从向日葵种子或发病植株中提取DNA；
采用引物：
F-引物CAAACTGACCATTTCTAGC(正向)；
R-引物AGGGATCCACTCGACGAAGT(反向)；
对提取的向日葵DNA进行PCR扩增，对扩增产物进行凝胶电泳，检测凝胶电泳图在441bp处是否有产物。
在本发明的一个优选实施方式中，在扩增产物凝胶电泳的同时还包括标记电泳条带大小的标记物。
在本发明的一个优选实施方式中，所述的标记物是DL2000。
该方法能够特异性的鉴定黑茎病菌。采用该方法能对向日葵种子进行带菌检测能够控制此病害的远距离的传播；同时，对田间发病植株进行早期的快速鉴定也是控制病害扩展蔓延的重要的环节。
附图说明：
图1：凝胶电泳结果：M：DL2000 Marker；1：向日葵菌核病核盘菌DNA；2：向日葵黄萎病大丽轮枝菌DNA；3：向日葵黑茎病菌DNA；4：向日葵黑斑病菌链格孢菌DNA；CK负对照。
具体实施方式
实施例1：
首先，由于向日葵黑茎病只有新疆有报道，因此我们首先对在其他向日葵种植地区采集的病样通过传统的Koch’s法则结合PCR进行鉴定，确定所采集的病样是由Phoma macdonaldi引起的黑茎病。
合成引物：
F-引物CAAACTGACCATTTCTAGC(正向)；
R-引物AGGGATCCACTCGACGAAGT(反向)；
从向日葵种子或发病植株中提取DNA，采用合成的引物进行PCR扩增，所述的PCR采用本领域公知的步骤进行，然后对扩增产物进行凝胶电泳，如果在凝胶电泳的441bp上有显示，如附图1所示，则表示该向日葵种子或发病植株感染了黑茎病，该方法能够特异性的鉴定黑茎病。采用该方法能对向日葵种子进行带菌检测能够控制此病害的远距离的传播，对田间发病植株进行早期的快速鉴定也是控制病害扩展蔓延的重要的环节。
当理解的是，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，而所有这些改进和变换都应属于本发明所附权利要求的保护范围。

Claims (3)

1.一种向日葵黑茎病菌的鉴定方法，包括如下步骤：
从向日葵种子或发病植株中提取DNA；
采用引物：
F-引物：CAAACTGACCATTTCTAGC；
R-引物：AGGGATCCACTCGACGAAGT；
对提取的向日葵DNA进行PCR扩增，对扩增产物进行凝胶电泳，检测凝胶电泳图在441bp处是否有产物。
2.根据权利要求1所述的鉴定方法，在扩增产物凝胶电泳的同时还包括标记物。
3.根据权利要求2所述的鉴定方法，所述的标记物是DL2000。
CN201110397732.5A 2011-12-05 2011-12-05 向日葵黑茎病菌的鉴定方法 Active CN102747137B (zh)

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CN103122375A (zh) * 2012-11-06 2013-05-29 新疆出入境检验检疫局检验检疫技术中心 向日葵黑茎病菌实时荧光pcr检测试剂盒
CN102943118B (zh) * 2012-12-06 2014-03-19 天津出入境检验检疫局动植物与食品检测中心 同步检测向日葵黑茎病菌和向日葵茎溃疡病菌的三重pcr引物及其扩增方法和用途
CN111662996A (zh) * 2020-04-30 2020-09-15 宁波检验检疫科学技术研究院 一种引物组及其在基于微流控芯片用于快速检测向日葵黑茎病菌的应用

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