琯溪蜜柚亚洲柑橘叶点霉与首都叶点霉ITS-RFLP快速鉴定

罗金水; 郑域茹; 卢松茂; 林智明; 林艺华

doi:10.19303/j.issn.1008-0384.2018.02.016

琯溪蜜柚亚洲柑橘叶点霉与首都叶点霉ITS-RFLP快速鉴定

doi: 10.19303/j.issn.1008-0384.2018.02.016

福建省热带作物科学研究所, 福建漳州 363001

基金项目:

福建省科技计划项目——省属公益类科研院所基本科研专项 2012GY01

福建省自然科学基金 2010J01113

详细信息

作者简介:
罗金水(1978-), 男, 硕士, 副研究员, 研究方向:植物保护(E-mail:13709383906@126.com)

中图分类号: S436
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- 被引次数: 0
出版历程
- 收稿日期: 2017-12-12
- 修回日期: 2018-01-07
- 刊出日期: 2018-02-01

A Rapid Identification Method for Phyllosticta citriasiana and Phyllosticta capitalensis in Citrus grandis by ITS-RFLP

Fujian Institute of Tropical Crops, Zhangzhou, Fujian 363001, China

摘要

摘要: 亚洲柑橘叶点霉Phyllosticta citriasiana是引起琯溪蜜柚黑斑病的病原菌。首都叶点霉Phyllosticta capitalensis是分离自琯溪蜜柚的一种腐生真菌。在本研究建立2种叶点霉属真菌的ITS-RFLP快速鉴定方法。首先采集平和县琯溪蜜柚样品，分离纯化获得目的菌P. citriasiana和P. capitalensis，提取这2种病菌的菌丝体总DNA，使用ITS1和ITS4引物对扩增rDNA-ITS基因片段，再用限制性内切酶Acc Ⅰ酶切扩增产物。结果表明，P. citriasiana和P. capitalensis这2种菌的rDNA-ITS基因酶切分别产生了2个和3个特异条带。通过条带的数量和大小使这两种真菌得以区分，该方法快速、简便。
- 琯溪蜜柚 /
- 黑斑病 /
- 限制性内切酶片段长度多态性分析技术
Abstract: Phyllosticta citriasiana is a new pathogen of Citrus grandis which causes black spot on the fruits, while Phyllosticta capitalensis is another fungus which is saprophytic and seprated from Citrus grandis. For distinguishing from each other, a rapid identification method named ITS-RFLP was established in this research. First of all, the target pathogens were seperated from the desease samples which were collected from the Citrus grandis Osbeck orchard at Pinghe city in Fujian. Then the mycelium total DNA were extracted for amplifying the rDNA-ITS gene fragments by using ITS1 and ITS4 primers. And then the amplification products were digested by the restriction enzyme of Acc Ⅰ. The results showed that the rDNA-ITS of P. citriasiana was digested into two fragments, and that of P. capitalensis into three fragments. Thus, the two kinds of fungi can be distinguished from each other by the fragment number and size. This technology is quite rapid and simple.
- Citrus grandis (L.) Osbeck /
- Citrus black spot /
- ITS-RFLP

HTML全文

图 1 P.citriasiana和P.capitalensis的PDA培养菌落

注：A为P.citriasiana，B为P.capitalensis。

Figure 1. Culture on the PDA of P.citriasiana and P. capitalensis

下载: 全尺寸图片幻灯片

图 2 病菌DNA PCR检测

注：M为DL 2000 DNA Marker，1~4为P.citriasiana的rDNA-ITS扩增产物；5为空泳道；6~8为P.capitalensis的rDNA-ITS扩增产物。

Figure 2. DNA PCR detection of pathogens

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图 3 分离自蜜柚的31个叶点霉属真菌菌株的系统进化树分析

Figure 3. Phylogenetic tree analysis of the 31 Phyllosticta strains seperated from Citrus grandis

下载: 全尺寸图片幻灯片

图 4 病菌rDNA-ITS使用AccⅠ酶切产物电泳结果

注：M为DL 500 DNA Marker，1、5为P.citriasiana菌株的rDNA-ITS扩增产物，3、7为P.capitalensis菌株的rDNA-ITS扩增产物，2为P.citriasiana菌株酶切过夜产物，4为P.capitalensis菌株酶切过夜产物，6为P.citriasiana菌株酶切1.5 h产物，8为P.capitalensis菌株酶切1.5 h产物。

Figure 4. rDNA-ITS of the pathogens degested by AccⅠ

下载: 全尺寸图片幻灯片

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