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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参考文献(25)

[1]	SHRESTHA S, HU J, FRYXELLl R T, et al.SNP marker identify widely distributed clonal lineages of Phytophthora colocasiae in Vietnam, Hawaii and Hainan island, China[J]. Mycologia, 2014, 106(4): 676-685. doi: 10.3852/13-165
[2]	王汉荣, 方丽, 茹水江, 等.槟榔芋疫病的识别与防治[J].中国蔬菜, 2009(21): 21-22. http://d.old.wanfangdata.com.cn/Periodical/zgsc200921009 WANG H R, SU L, RU S J, et al.Identification and control of taro phytophthora blight[J]. China Vegetables, 2009(21): 21-22.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zgsc200921009
[3]	王向社, 李锐, 胡茂松, 等.海南岛芋疫霉菌生物学特性、致病力、对甲霜灵的敏感性研究[J].热带作物学报, 2001, 22(1): 83-90. doi: 10.3969/j.issn.1000-2561.2001.01.014 WANG X S, LI R, HU M S, et al.Study on the biology, virulence of Phytophthora colocasiae Racib and its sensitivity to metalaxyl[J]. Chinese Journal of Tropical Crops, 2001, 22(1): 83-90.(in Chinese) doi: 10.3969/j.issn.1000-2561.2001.01.014
[4]	QUITUGUA R J, TRUJILLO E E.Survival of Phytophthora colocasiae in field soil at various temperatures and water matric potentials[J]. Plant Disease, 1998, 82(2): 203-207. doi: 10.1094/PDIS.1998.82.2.203
[5]	刘独臣.四川芋疫病发生规律及防治技术[J].长江蔬菜, 2013, 18: 118-119. doi: 10.3865/j.issn.1001-3547.2013.18.038 LIU D C.Epidemiology and control of taro phytophthora blight in Sichuan[J]. Journal of Changjiang Vegetables, 2013, 18: 118-119.(in Chinese) doi: 10.3865/j.issn.1001-3547.2013.18.038
[6]	VASGUEZ E A.Yield loss in taro due to Phytophthora leaf blight[J]. Journal of Root Crops, 1990, 16: 48-50. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=8af3982881021ea62339fcdf98cf4996
[7]	方辉, 张惠琴, 陈孝赏, 等.双炔酰菌胺防治红香芋疫病的效果及应用技术[J].浙江农业科学, 2016, 57(6): 899 -900, 911. http://d.old.wanfangdata.com.cn/Periodical/zjnykx201606033 FANG H, ZHANG H Q, CHEN X S, et al.Effect and application technology of mandipropamid in control taro phytophthora blight[J]. Journal of Zhejiang Agricultural Sciences, 2016, 57(6):899-900, 911.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zjnykx201606033
[8]	莫俊杰, 胡汉桥, 梁钾贤, 等.芋疫病抗病性鉴定及不同品系遗传多样性分析[J].广东海洋大学学报, 2012, 32(4): 67-72. http://d.old.wanfangdata.com.cn/Periodical/zjhydxxb201204014 MO J J, HU H Q, LIANG J X, et al.Resistance identification of taro to Phytophthora colocasiae and genetic diversity analysis of Colocasia esculenta[J]. Journal of Guangdong Ocean University, 2012, 32(4): 67-72.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zjhydxxb201204014
[9]	NATH V S, SENTHIL M, HEGDE V M, et al.Evaluation of fungicides on Indian isolates of Phytophthora colocasiae causing leaf blight of taro[J]. Archives of Phytopathology and Plant Protection, 2013, 46(5): 548-555. doi: 10.1080/03235408.2012.749688
[10]	LIN M J, KO W H.Occurrence of isolates of Phytophthora coloscsiae in Taiwan with homothallic behavior and its significance[J]. Mycologia, 2008, 100(5): 727-734. doi: 10.3852/08-070
[11]	叶泉清, 钟佳铃, 陈媚, 等.槟榔芋疫霉菌生物学特性、致病力测定及田间防治药剂筛选[J].南方农业学报, 2016, 47(4): 588-593. doi: 10.3969/j:issn.2095-1191.2016.04.588 YE Q Q, ZHONG J L, CHEN M, et al.Biological characteristics, virulence of Phytophthora colocasiae Racib.From Colacasia esculenta L.var. cormosus Chang and fungicides screening for field control[J]. Journal of Southern Agriculture, 2016, 47(4): 588-593.(in Chinese) doi: 10.3969/j:issn.2095-1191.2016.04.588
[12]	NATH V S, HEGDE V M, JEEVA M L, et al.Rapid and sensitive detection of Phytophthora colocasiae responsible for the taro blight using conventional and real-time PCR assay[J]. FEMS Micobiology Letters, 2014, 352: 174-183. doi: 10.1111/fml.2014.352.issue-2
[13]	MARTIN F N, ABAD Z G, BALCI Y, et al.Identification and detection of Phytophthora: reviewing our progress, identifying our needs[J]. Plant Disease, 2012, 96(8): 1080-1103. doi: 10.1094/PDIS-12-11-1036-FE
[14]	ROLLINS L, COSTS K, ELLIOTT M, et al.Comparison of five detection and quantification methods for Phytophthora ramorum in stream and irrigation water[J]. Plant Disease, 2016, 100(6): 1202-1211. doi: 10.1094/PDIS-11-15-1380-RE
[15]	王晓杰, 康振生, 黄丽丽.PCR技术在植物病害检测中的应用[J].云南农业大学学报, 2005, 20(2): 179-182. doi: 10.3969/j.issn.1004-390X.2005.02.006 WANG X J, KANG Z S, HUANG L L.Application of PCR technology on the detection of plant disease[J]. Journal of Yunnan Agricultural University, 2005, 20(2): 179-182.(in Chinese) doi: 10.3969/j.issn.1004-390X.2005.02.006
[16]	DRENTH A, WAGELS G, SMITH B, et al.Development of a DNA-based method for detection and identification of Phytophthora species[J]. Australasian Plant Pathology, 2005, 35: 147-159. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=f29b87735298dd378bc7471b766c4cc1
[17]	李依韦, 银玲.rDNA-ITS序列分析在植物病原真菌分类鉴定中的应用[J].内蒙古民族大学学报(自然科学版), 2012, 27(1): 66-67. doi: 10.3969/j.issn.1671-0185.2012.01.021 LI Y W, YIN L.Application of rDNA-ITS sequences in plant disease fungi classification and identification[J]. Journal of Inner Mongolia University for Nationalities (Natural Sciences), 2012, 27(1): 66-67.(in Chinese) doi: 10.3969/j.issn.1671-0185.2012.01.021
[18]	傅华英, 葛丹凤, 李晓燕, 等.甘蔗赤条病菌巢式PCR检测[J].植物保护学报, 2017, 44(2): 276-282. http://d.old.wanfangdata.com.cn/Periodical/zwbhxb201702014 FU H Y, GE D F, LI X Y, et al.Nested-PCR detection of Acidovorax avenae subsp.avenae, the pathogen of red stripe on sugarcane[J]. Journal of Plant Protection, 2017, 44(2): 276-282.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zwbhxb201702014
[19]	KONIG S, SCHWENKBIER L, POLLOK S, et al.Potential of Ypt1 and ITS gene regions for the detection of Phytophthora species in a lab-on-a-chip DNA hybridization array[J]. Plant Pathology, 2015, 64: 1176-1189. doi: 10.1111/ppa.2015.64.issue-5
[20]	LAN C Z, LIU P Q, LI B J, et al.Development of a specific PCR assay for the rapid and sensitive detection of Phytophthora capsici[J]. Australasian Plant Pathology, 2013, 42: 379-384. doi: 10.1007/s13313-012-0185-8
[21]	杨万风, 刘艳, 刘翔, 等.巢式PCR检测菜豆细菌性萎蔫病菌[J].浙江农业学报, 2015, 27(7): 1202-1207. doi: 10.3969/j.issn.1004-1524.2015.07.15 YANG W F, LIU Y, LIU X, et al.Detection of Curtobacterium flaccumfaciens pv.flaccumfaciens using nested PCR[J]. Acta Agriculturae Zhejiangensis, 2015, 27(7): 1202-1207.(in Chinese) doi: 10.3969/j.issn.1004-1524.2015.07.15
[22]	ZHANG Z G, LI Y Q, FAN H, et al.Molecular detection of Phytophthora capsici in infected plant tissues, soil and water[J]. Plant Pathology, 2006, 55(6): 770-775. doi: 10.1111/ppa.2006.55.issue-6
[23]	赵杰.ITS序列分析及其在植物真菌病害分子检测中的应用[J].陕西农业科学, 2004(4): 35-37. doi: 10.3969/j.issn.0488-5368.2004.04.016 ZHAO J.ITS sequence analysis and its application in molecular detection of plant fungal diseases[J]. Shanxi Journal of Agricultural Sciences, 2004(4): 35-37.(in Chinese) doi: 10.3969/j.issn.0488-5368.2004.04.016
[24]	SCHENA L, COOKE D E L.Assessing the potential of regions of the nuclear and mitochondrial genome to develop a "molecular tool box" for the detection and characterization of Phytophthora species[J]. Journal of Microbiological Methods, 2006, 67: 70-85. doi: 10.1016/j.mimet.2006.03.003
[25]	VOLOSSIOUK T, ROBB E J, NAZAR R N.Direct DNA extraction for PCR-mediated assays of soil organisms[J]. Applied and Enviromental Microbiology, 1995, 61(11): 3972-3976. http://d.old.wanfangdata.com.cn/OAPaper/oai_pubmedcentral.nih.gov_167704