玉米大斑病菌和小斑病菌交配型多重PCR 检测方法的建立与应用

doi:10.3864/j.issn.0578-1752.2020.03.006

中国农业科学 ›› 2020, Vol. 53 ›› Issue (3): 527-538.doi: 10.3864/j.issn.0578-1752.2020.03.006

玉米大斑病菌和小斑病菌交配型多重PCR 检测方法的建立与应用

代玉立¹,甘林¹,滕振勇²,杨静民³,祁月月⁴,石妞妞¹,陈福如¹,杨秀娟¹()

¹ 福建省农业科学院植物保护研究所/福建省作物有害生物监测与治理重点实验室,福州 350013
² 福建省种子管理总站,福州 350001
³ 建瓯市农业农村局,福建建瓯 353100
⁴ 浙江天丰生物科学有限公司,浙江金华 321000

收稿日期:2019-08-13 接受日期:2019-09-18 出版日期:2020-02-01 发布日期:2020-02-13
通讯作者: 杨秀娟
作者简介:代玉立,E-mail：dai841225@126.com。
基金资助:
福建省属公益类科研院所专项(2017R1025-3);福建省属公益类科研院所专项(2018R1025-1);国家重点研发计划(2018YFD0200706);福建省农业科学院青年自由探索项目(AA2018-14);福建省农业科学院青年科技英才百人计划(YC2016-4);福建省农业科学院植物保护创新团队(STIT2017-1-8)

Establishment and Application of a Multiple PCR Method to Detect Mating Types of Exserohilum turcicum and Bipolaris maydis

DAI YuLi¹,GAN Lin¹,TENG ZhenYong²,YANG JingMin³,QI YueYue⁴,SHI NiuNiu¹,CHEN FuRu¹,YANG XiuJuan¹()

¹ Institute of Plant Protection, Fujian Academy of Agricultural Sciences/Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Fuzhou 350013
² Fujian Seed Management Station, Fuzhou 350001
³ Jianou Municipal Bureau of Agriculture and Rural Affairs, Jianou 353100, Fujian
⁴ Zhejiang Tianfeng Biological Science Co. Ltd, Jinhua 321000, Zhejiang

Received:2019-08-13 Accepted:2019-09-18 Online:2020-02-01 Published:2020-02-13
Contact: XiuJuan YANG

摘要/Abstract

摘要：

【目的】由大斑突脐蠕孢（Exserohilum turcicum）和玉蜀黍平脐蠕孢（Bipolaris maydis）引起的玉米大斑病和小斑病是玉米生产上重要的叶部真菌病害,本研究旨在建立玉米大斑病菌和小斑病菌交配型多重PCR检测方法,为玉米大斑病菌和小斑病菌的交配型田间分布和有性生殖研究提供技术方法。【方法】根据GenBank中已登录的玉米大斑病菌（登录号MAT1-1：GU997138和MAT1-2：GU997137）和小斑病菌（登录号MAT1-1：X68399和MAT1-2：X68398）交配型基因序列,利用Primer Premier 5.0软件设计2种病原菌交配型多重PCR检测特异性引物,采用单因素法对引物的退火温度以及扩增程序中延伸时间和循环数等重要参数进行优化,建立玉米大斑病菌和小斑病菌交配型多重PCR检测方法,并对2种病原菌的交配型多重PCR检测灵敏度和特异性进行检验。同时,对田间采集的129株玉米大斑病菌和194株玉米小斑病菌单孢菌株的交配型进行多重PCR检测,以明确建立的交配型多重PCR检测方法的适用性。【结果】建立的多重PCR方法和设计的交配型特异引物StMAT01-2F/R、StMAT02-3F/R、ChMAT01-3F/R和ChMAT02-2F/R可分别扩增出MAT1-1、MAT1-2型菌株大小为816、132 bp（大病斑菌）与490、136 bp（小病斑菌）的特异性目的条带。25 μL多重PCR扩增体系：2×Multiplex PCR Mix 12.5 μL,引物各10 pmol,DNA模板100 ng,退火温度为57.2℃（大病斑菌）和55.0℃（小斑病菌）,35个循环。该多重PCR对玉米大斑病菌MAT1-1、MAT1-2型单孢菌株的检测灵敏度分别为0.1、0.01 ng基因组DNA,而对玉米小斑病菌MAT1-1、MAT1-2交配型的检测灵敏度均为0.1 ng基因组DNA。该交配型多重PCR检测方法对玉米大斑病菌和小斑病菌特异性很强,能够很好地区分玉米大斑病菌和小斑病菌相应的近缘种和14株其他真菌菌株。不同地理来源的玉米大斑病菌和小斑病菌交配型检测结果表明,该多重PCR能够准确地检出129株玉米大斑病菌和194株玉米小斑病菌的交配型,且检测结果与随机抽取的菌株杂交验证结果完全吻合。【结论】构建的玉米大斑病菌和小斑病菌交配型多重PCR检测方法灵敏度高、特异性好、操作简便,能够准确、快速地检测出玉米大斑病菌和小斑病菌的交配型,为玉米大斑病菌和小斑病菌的交配型田间分布和监测及有性生殖研究提供了可靠的技术方法。

关键词: 玉米大斑病菌, 玉米小斑病菌, 交配型, 多重PCR, 灵敏度, 特异性

Abstract:

【Objective】 Northern corn leaf blight (NCLB) and southern corn leaf blight (SCLB), caused by Exserohilum turcicum and Bipolaris maydis, respectively, are the most important foliar fungal diseases affecting maize production. The objective of this study is to establish a multiple PCR method to detect mating types of E. turcicum and B. maydis, and to provide a technical method for the study of mating type distribution in the field and sexual reproduction of E. turcicum and B. maydis. 【Method】 Mating type-specific primers for the two pathogens were designed on the basis of mating type gene sequences of E. turcicum (accession numbers: GU997138 for MAT1-1; GU997137 for MAT1-2) and B. maydis (accession numbers: X68399 for MAT1-1; X68398 for MAT1-2) obtained from GenBank, and the important parameters of primer annealing temperatures, extension times and amplification cycles in the amplification program were optimized using the single factor method. A multiple PCR method was established to detect mating types of E. turcicum and B. maydis, and the sensitivity and specificity of the multiple PCR were also assessed. Meanwhile, the mating types of 129 strains of E. turcicum and 194 strains of B. maydis from field-collections were detected by the multiple PCR to determine the adaptability of the established method.【Result】The expected 816, 132 bp (E. turcicum), and 490, 136 bp (B. maydis) target fragments were amplified specifically using the multiple PCR with mating type-specific primers of StMAT01-2F/R, StMAT02-3F/R, and ChMAT01-3F/R, ChMAT02-2F/R from MAT1-1 and MAT1-2 strains, respectively. A 25 μL PCR reaction system consisted of 12.5 μL 2×Multiplex PCR Mix, 10 pmol each primer, and 100 ng DNA template. The annealing temperatures for E. turcicum and B. maydis were 57.2℃ and 55.0℃, respectively, and the number of amplification cycles was 35. The multiple PCR method could reliably detect mating types of E. turcicum at 0.1 ng genomic DNA for MAT1-1 or 0.01 ng DNA for MAT1-2 from single-spore strains, while the sensitivity of the multiple PCR to detect mating types of B. maydis was 0.1 ng genomic DNA for both MAT1-1 and MAT1-2 from pure culture strains. The method exhibited specificity in differentiating mating types of E. turcicum and B. maydis from their closely-related species, as well as 14 other fungal genera. The results of mating types detection of E. turcicum and B. maydis strains from different geographical origins indicated that the multiple PCR could reliably detect mating types of 129 and 194 strains of E. turcicum and B. maydis, respectively. These results were consistent with the verification results of laboratorial cross assays with random selected strains from different locations.【Conclusion】The established multiple PCR method for mating types detection of E. turcicum and B. maydis in this study was characterized as high sensitivity, specificity and user-friendly control, it could accurately and rapidly detect mating types of E. turcicum and B. maydis. This study provides a reliable technique and approach for the study of distribution and monitoring of mating types in the field and sexual reproduction of E. turcicum and B. maydis.

Key words: Exserohilum turcicum, Bipolaris maydis, mating type, multiple PCR, sensitivity, specificity

代玉立,甘林,滕振勇,杨静民,祁月月,石妞妞,陈福如,杨秀娟. 玉米大斑病菌和小斑病菌交配型多重PCR 检测方法的建立与应用[J]. 中国农业科学, 2020, 53(3): 527-538.

DAI YuLi,GAN Lin,TENG ZhenYong,YANG JingMin,QI YueYue,SHI NiuNiu,CHEN FuRu,YANG XiuJuan. Establishment and Application of a Multiple PCR Method to Detect Mating Types of Exserohilum turcicum and Bipolaris maydis[J]. Scientia Agricultura Sinica, 2020, 53(3): 527-538.

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图/表 7

表1

供试菌株信息"

菌株编号 Code of strains	学名 Scientific name	菌株来源 Source of strains	采集年份 Year of collection	交配型 Mating type
1	玉米大斑病菌E. turcicum	福建建瓯 Jianou, Fujian	2018	MAT1-2
2	玉米大斑病菌E. turcicum	福建建瓯 Jianou, Fujian	2018	MAT1-2
3	玉米大斑病菌E. turcicum	福建南靖 Nanjing, Fujian	2018	MAT1-1
4	玉米大斑病菌E. turcicum	福建南靖 Nanjing, Fujian	2018	MAT1-1
5	玉米大斑病菌E. turcicum	福建松溪 Songxi, Fujian	2018	MAT1-1
6	玉米大斑病菌E. turcicum	福建松溪 Songxi, Fujian	2018	MAT1-2
7	玉米大斑病菌E. turcicum	福建南靖 Nanjing, Fujian	2019	MAT1-2
8	玉米大斑病菌E. turcicum	福建建瓯 Jianou, Fujian	2019	MAT1-1
9	玉米大斑病菌E. turcicum	福建屏南 Pingnan, Fujian	2016	MAT1-2
10	玉米大斑病菌E. turcicum	福建屏南 Pingnan, Fujian	2016	MAT1-2
11	玉米大斑病菌E. turcicum	福建建瓯 Jianou, Fujian	2019	MAT1-2
12	玉米大斑病菌E. turcicum	福建建瓯 Jianou, Fujian	2019	MAT1-1
13	玉米小斑病菌B. maydis	福建武夷山 Wuyishan, Fujian	2015	MAT1-2
14	玉米小斑病菌B. maydis	福建建阳 Jianyang, Fujian	2014	MAT1-2
15	玉米小斑病菌B. maydis	福建建阳 Jianyang, Fujian	2014	MAT1-2
16	玉米小斑病菌B. maydis	福建建瓯 Jianou, Fujian	2016	MAT1-2
17	玉米小斑病菌B. maydis	福建屏南 Pingnan, Fujian	2016	MAT1-2
18	玉米小斑病菌B. maydis	福建建瓯 Jianou, Fujian	2016	MAT1-1
19	玉米小斑病菌B. maydis	安徽霍邱 Huoqiu, Anhui	2017	MAT1-2
20	玉米小斑病菌B. maydis	海南三亚 Sanya, Hainan	2018	MAT1-2
21	玉米小斑病菌B. maydis	浙江泰顺 Taishun, Zhejiang	2017	MAT1-2
22	玉米小斑病菌B. maydis	福建屏南 Pingnan, Fujian	2016	MAT1-1
23	玉米小斑病菌B. maydis	福建屏南 Pingnan, Fujian	2016	MAT1-1
24	玉米小斑病菌B. maydis	广东惠州 Huizhou, Guangdong	2018	MAT1-2
近缘种及其他真菌Closely related and other fungal species
25	參子凸脐蠕孢Exserohilum frumentacei	CGMCC	2009	ND
26	小柄凸脐蠕孢Exserohilum pedicellatum	CGMCC	2009	ND
27	芦苇凸脐蠕孢Exserohilum phragmitis	CGMCC	2009	ND
28	突脐蠕孢属Exserohilum sp.	广东广州 Guangzhou, Guangdong	2016	ND
29	水稻胡麻斑病菌Bipolaris oryzae	福建建瓯 Jianou, Fujian	2017	ND
30	双色平脐蠕孢Bipolaris bicolor	福建建瓯 Jianou, Fujian	2017	ND
31	玉米圆斑病菌Bipolaris zeicola	四川 Sichuan	ND	ND
32	麦根腐平脐蠕孢Bipolaris sorokiniana	安徽 Anhui	ND	ND
33	玉米弯孢叶斑病菌Curvularia lunata	福建霞浦 Xiapu, Fujian	2016	ND
34	玉米画眉草弯孢叶斑病菌Curvularia eragrostidis	福建莆田 Putian, Fujian	2016	ND
35	番茄早疫病菌Alternaria solani	福建 Fujian	ND	ND
36	稻曲病菌Ustilaginoidea virens	福建 Fujian	2016	ND
37	胶孢炭疽菌Colletotrichum gloeosporioides	福建福州 Fuzhou, Fujian	2017	ND
38	尖镰孢Fusarium oxysporum	福建漳州 Zhangzhou, Fujian	2014	ND
39	桃褐腐病菌Monilinia laxa	福建Fujian	ND	ND
40	柑橘酸腐病菌Oospora citriaurantii	福建 Fujian	ND	ND
41	玉米纹枯病菌Rhizoctonia solani	福建 Fujian	2017	ND
42	油菜菌核病菌Sclerotinia sclerotiorum	安徽宁国 Ningguo, Anhui	2004	ND
43	柑橘疮痂病菌Sphaceloma fawcetti	福建 Fujian	ND	ND
44	哈茨木霉Trichoderma harzianum	福建 Fujian	ND	ND
45	稻瘟病菌Magnaporthe oryzae	福建 Fujian	ND	ND
46	苹果红粉病菌Trichothecium roseum	福建 Fujian	ND	ND

表1

图1

表2

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图4

表3

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菌株来源 Source of strains	病原菌 Pathogen	菌株数量 Strain numbers ^a	交配型的多重PCR和杂交检测结果 Results of mating type detection using multiple PCR and cross assay ^b
菌株来源 Source of strains	病原菌 Pathogen	菌株数量 Strain numbers ^a	MAT1-1	MAT1-2
福建建瓯Jianou, Fujian	玉米大斑病菌E. turcicum	64 (10)	+ (2)	+ (8)
福建建瓯Jianou, Fujian	玉米小斑病菌B. maydis	12 (5)	+	+ (5)
福建松溪Songxi, Fujian	玉米大斑病菌E. turcicum	20 (8)	+ (1)	+ (7)
福建屏南Pingnan, Fujian	玉米大斑病菌E. turcicum	15 (5)	+	+ (5)
福建屏南Pingnan, Fujian	玉米小斑病菌B. maydis	24 (10)	+ (3)	+ (7)
福建福州Fuzhou, Fujian	玉米小斑病菌B. maydis	20 (8)	+ (2)	+ (6)
福建莆田Putian, Fujian	玉米大斑病菌E. turcicum	1	ND	+
福建莆田Putian, Fujian	玉米小斑病菌B. maydis	8 (5)	+	+ (5)
福建南靖Nanjing, Fujian	玉米大斑病菌E. turcicum	20 (10)	+ (1)	+ (9)
福建南靖Nanjing, Fujian	玉米小斑病菌B. maydis	11 (5)	+	+ (5)
福建龙岩Longyan, Fujian	玉米大斑病菌E. turcicum	8 (5)	+	+ (5)
福建龙岩Longyan, Fujian	玉米小斑病菌B. maydis	9 (5)	+ (1)	+ (4)
安徽霍邱Huoqiu, Anhui	玉米小斑病菌B. maydis	26 (8)	+ (1)	+ (7)
海南三亚Sanya, Hainan	玉米大斑病菌E. turcicum	1	ND	+
海南三亚Sanya, Hainan	玉米小斑病菌B. maydis	24 (6)	+ (2)	+ (4)
浙江泰顺Taishun, Zhejiang	玉米小斑病菌B. maydis	27 (8)	+ (3)	+ (5)
广东Guangdong	玉米小斑病菌B. maydis	33 (10)	+ (4)	+ (6)
总计 Total	玉米大斑病菌E. turcicum	129 (38)	+ (4)	+ (34)
总计 Total	玉米小斑病菌B. maydis	194 (70)	+ (16)	+ (54)

玉米大斑病菌和小斑病菌交配型多重PCR 检测方法的建立与应用

Establishment and Application of a Multiple PCR Method to Detect Mating Types of Exserohilum turcicum and Bipolaris maydis

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引物名称 Primer name	引物序列 Primer sequence (5′→3′)	退火温度 Tm (℃)	产物大小 Size (bp)	目标基因 Target gene
StMAT01-2	F: TGCCTTTGTTGGATTTCG	57.2	816	E. turcicum MAT1-1
StMAT01-2	R: CATCGTTCTGGCTGTGGG	57.2	816	E. turcicum MAT1-1
StMAT02-3	F: TACACCAAACAACATCGCTCCT	57.2	132	E. turcicum MAT1-2
StMAT02-3	R: TCGGCGTCGTAGAACAAG	57.2	132	E. turcicum MAT1-2
ChMAT01-3	F: ACCACGAGACAACATACGC	55.0	490	B. maydis MAT1-1
ChMAT01-3	R: GTTTGAGGTGAGGGCAGA	55.0	490	B. maydis MAT1-1
ChMAT02-2	F: GGCGAAGTTTGTGGGTTT	55.0	136	B. maydis MAT1-2
ChMAT02-2	R: GCGATGTCTGGGCTGATT	55.0	136	B. maydis MAT1-2

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