芋疫霉菌的巢式PCR检测

兰成忠; 卢学松; 姚锦爱; 丁雪玲; 蒋军喜

doi:10.19303/j.issn.1008-0384.2019.01.012

芋疫霉菌的巢式PCR检测

doi: 10.19303/j.issn.1008-0384.2019.01.012

兰成忠^{1, 2,},
卢学松¹,
姚锦爱¹,
丁雪玲¹,
蒋军喜^2, ,

1.
福建省作物有害生物监测与治理重点实验室/福建省农业科学院植物保护研究所, 福建福州 350013
2.
江西农业大学农学院, 江西南昌 330045

基金项目:

国家自然科学基金项目 31400025

福建省农业科学院青年科技英才百人计划项目 YC2015-4

福建省农业科学院植物保护创新团队建设项目 STIT2017-1-8

详细信息

作者简介:
兰成忠(1979-), 男, 硕士, 副研究员, 主要从事植物病害防治技术研究(E-mail:lczhong7911@126.com)

通讯作者:
蒋军喜(1964-), 男, 博士, 教授, 博士生导师, 主要从事植物病害综合治理研究(E-mail:jxau2011@126.com)

中图分类号: S435.72
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出版历程
- 收稿日期: 2017-12-05
- 修回日期: 2018-11-25
- 刊出日期: 2019-01-28

Nested-PCR Detection of Taro Leaf Blight Pathogen Phytophthora colocasiae

1.
Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pest, Institute of Plant Protection/Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, China
2.
College of Agronomy, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China

摘要

摘要: 目的建立芋疫霉菌快速、准确的PCR检测技术，为芋疫病流行规律监测和综合防控提供科学依据。方法根据芋疫霉菌与其他疫霉菌种类Ypt1基因序列差异，设计了1对芋疫霉菌PCR检测特异引物PCOF/PCOR，并对该引物的特异性、灵敏性和应用性进行了验证。结果在优化的反应体系与扩增条件下，PCOF/PCOR引物能特异性地从芋疫霉菌基因组DNA中扩增出1条172 bp的条带，而其他供试病原菌均无扩增条带。在25 μL PCR反应体系中，PCOF/PCOR引物对芋疫霉菌基因组DNA的检测灵敏度为100 pg，而以疫霉菌Ypt1基因通用引物ph1F/Yph2R为第一轮引物，PCOF/PCOR为第二轮引物，进行巢式PCR扩增，能检测到10 fg芋疫霉菌基因组DNA，检测灵敏度提高了10 000倍。采用巢式PCR，可从芋疫病发病的叶片和未显症叶片组织中检测到芋疫霉菌，检出率分别为100%和57.5%。结论所建立的巢式PCR可应用于芋疫霉菌的快速、特异和高灵敏度检测。
- 芋疫霉菌 /
- 特异引物 /
- 巢式PCR /
- 分子检测
Abstract: Objective To develop a PCR assay for rapid and accurate detection, epidemiology information, and integrated disease management on Phytophthora colocasiae, the pathogen of taro phytophthora blight. Method A pair of species-specific primers, PCOF/PCOR, for P. colocasiae was designed based on the differences in Ras-related protein (Ypt1) gene sequence between P. colocasiae and other species in the same genus. The specificity, sensitivity and applicability of the primers were evaluated. Result With the optimized reaction conditions and amplification, PCOF/PCOR amplified only a single band of 172 bp with genomic DNA extracted from all P.colocasiae strains, while the other tested pathogens had no corresponding band. The sensitivity of conventional PCR method using PCOF/PCOR as primers was 100 pg of genomic DNA in a 25 μL reaction solution. Whereas, the newly developed nested-PCR performed using Ypt1 gene universal primers ph1F/Yph2R for the first-round and PCOF/PCOR for the second-round increased 10 000-fold on the sensitivity to 10 fg. The nested-PCR methodology could positively detected P. colocasiae 100% in diseased leaves or 57.5% in symptom-free infected tissues. Conclusion The newly established nested-PCR assay could be used for rapid, specific and sensitive detection of P. colocasiae.
- Phytophthora colocasiae /
- specific primer /
- nested-PCR /
- molecular detection

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图 1 特异引物PCOF/PCOR PCR扩增产物电泳

注：泳道M为2 000 bp DNA Marker，泳道1~4、12为芋疫霉菌，泳道5~10分别为番茄晚疫病菌、辣椒疫霉菌、大豆疫霉菌、豇豆疫霉菌、樟疫霉菌和恶疫霉菌，泳道11为阴性对照。

Figure 1. Agarose electrophoresis of PCR amplified products with specific primers PCOF/PCOR

Note:M: 2 000 bp DNA Marker, 1-4, 12: Phytophthora colocasiae, 5-10: P. infestans, P. capsici, P. sojae, P. vignae, P. cinnamomi, P.cactorum, 11: Negative control.

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图 2 巢式PCR特异性检测电泳

注：①A为巢式PCR第一轮扩增检测结果，B为巢式PCR第二轮扩增检测结果。②泳道M为DL 2 000 bp DNA Marker，泳道1~2为芋疫霉菌，泳道3~8分别为辣椒疫霉菌、豇豆疫霉菌、番茄晚疫病菌、大豆疫霉菌、香蕉枯萎病菌和番茄灰霉病菌，泳道9为阴性(无菌水)对照。

Figure 2. Specificity of Nest-PCR for detection of Phytophthora colocasiae

Note:①A: First round of nested-PCR; B: Second round of nested-PCR.② M: 2 000 bp DNA Marker, 1-2: Phytophthora colocasiae, 3-8: P. capsici, P. vignae, P. infestans, P. sojae, Fusarium oxysoporum f. sp. cubense, Botrytis cinerea, 9: Negative control.

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图 3 常规PCR和巢式PCR对芋疫霉菌基因组DNA系列浓度的检测灵敏度

注：①A为常规PCR灵敏性检测结果，B为巢式PCR灵敏性检测结果。②泳道M为DL 2 000 bp DNA Marker，泳道1为100 ng·μL^-1，泳道2为10 ng·μL^-1，泳道3为1 ng·μL^-1，泳道4为100 pg·μL^-1，泳道5为10 pg·μL^-1，泳道6为1 pg·μL^-1，泳道7为100 fg·μL^-1，泳道8为10 fg·μL^-1，泳道9为1 fg·μL^-1，泳道10为100 ag·μL^-1，泳道11为阴性对照，泳道12为阳性对照。

Figure 3. Sensitivity of conventional PCR and nested PCR

Note: ①A: Sensitivity of conventional PCR, B: Sensitivity of nested-PCR.②M: 2 000 bp DNA Marker, 1-10:100 ng·μL^-1, 10 ng·μL^-1, 1 ng·μL^-1, 100 pg·μL^-1, 10 pg·μL^-1, 1 pg·μL^-1, 100 fg·μL^-1, 10 fg·μL^-1, 1 fg·μL^-1, 100 ag L^-1, 11: Negative control, 12: Positive control.

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图 4 巢式PCR对植物叶片组织芋疫霉菌的PCR扩增检测

注：M为2 000 bp DNA Marker，泳道1为阳性对照，泳道2、4、6、8为健康叶片，泳道3为人工接种发病叶片，泳道5、7为田间自然发病叶片，泳道9为阴性对照。

Figure 4. Detection of Phytophthora colocasiaea from leave samples by Nest-PCR

Note:M: DL 2000bp DNA marker, 1: Positive control, 2, 4, 6, 8: Healthy leaves, 3: Artificial inoculation leaves, 5, 7: Naturally diseased leaves in the field, 9: Negative control.

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表 1 供试菌株

Table 1. Tested strains

序号 No.	种名Species	寄主host	来源 Locality	数量Number of isolate
1	芋疫霉菌Phytophthora colocasiae	芋Colocasia esculenta	福建Fujian	15
2	芋疫霉菌P.colocasiae	芋C.esculenta	广西Guangxi	5
3	芋疫霉菌P.colocasiae	芋C.esculenta	广东Guangdong	5
4	芋疫霉菌P.colocasiae	芋C.esculenta	海南Hainan	6
5	恶疫霉菌P.cactorum	草莓Fragaria ananassa	福建Fujian	3
6	辣椒疫霉P.capsici	辣椒Capsicum annuum	福建Fujian	10
7	瓜疫霉菌P.melonis	黄瓜Cucumis sativus	福建Fujian	11
8	大豆疫霉菌P.sojae	大豆Glycine max	福建Fujian	3
9	烟草疫霉菌P.nicotianae	烟草Nicotiana tabacum	福建Fujian	2
10	樟疫霉菌	蓝莓Vaccinium corymbosum	福建Fujian	3
11	棕榈疫霉菌P.Palmivora	木瓜Chaenomeles sinensis	福建Fujian	6
12	苜蓿疫霉菌P.medicaginis	苜蓿Medicago Sativa	未知Unkown	1
13	致病疫霉菌P.infestans	番茄Solanum lycopersicum	福建Fujian	8
14	苎麻疫霉菌P.boehmeriae	棉花Gossypium spp	新疆Xinjiang	1
15	隐地疫霉菌P.cryptogea	非洲菊Gerbera jamesonii	未知Unkown	1
16	豇豆疫霉菌p.vignae	豇豆Vigna unguiculata	福建Fujian	6
17	荔枝霜疫霉菌Peronophythora litchi	荔枝Litchi chinensis	福建Fujian	1
18	瓜果腐霉Pythium aphanidermatum	黄瓜Cucumis sativus	福建Fujian	1
19	茄病镰孢菌Fusarium solani	番茄S.lycopersicum	福建Fujian	4
20	黄瓜枯萎病菌F.oxysporum.sp.cucumebrium	黄瓜Cucumis sativus	福建Fujian	6
21	香蕉枯萎病菌F.oxysporum f.sp.cubense	香蕉Musa nana	福建Fujian	6
22	甘薯蔓割病菌F.oxysporum f.sp.batatas	甘薯Dioscorea esculenta	福建Fujian	1
23	西瓜枯萎病菌F.oxysporium f.sp.niveum	西瓜Citrullus lanatus	福建Fujian	6
24	豇豆枯萎病菌F.oxysporum f.sp.tracheiphlium	豇豆V.unguiculata	福建Fujian	3
25	黄瓜炭疽病菌C.orbiculare	黄瓜C.sativus	福建Fujian	3
26	大豆炭疽病菌C.truncatum	大豆G.max	福建Fujian	3
27	胶孢炭疽菌C.gloeosporioides	柑橘Citrus reticulata	福建Fujian	1
28	香蕉炭疽病菌C.musae	香蕉Musa nana	福建Fujian	1
29	辣椒炭疽C.capsici	辣椒C.annuum	福建Fujian	1
30	立枯丝核菌Rhizoctonia solani	番茄S.lycopersicum	福建Fujian	1
31	番茄早疫病菌Alternaria solani	番茄S.lycopersicum	福建Fujian	1
32	烟草赤星病菌A.alternata	烟草N.tabacum	福建Fujian	1
33	番茄灰霉病菌Botrytis cinerea	番茄S.lycopersicum	福建Fujian	1

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