中国农业科学 ›› 2022, Vol. 55 ›› Issue (17): 3334-3342.doi: 10.3864/j.issn.0578-1752.2022.17.006
崔江宽(),任豪豪(
),曹梦园,陈昆圆,周博,蒋士君,汤继华(
)
收稿日期:
2022-03-16
接受日期:
2022-04-03
出版日期:
2022-09-01
发布日期:
2022-09-07
通讯作者:
汤继华
作者简介:
崔江宽,E-mail: 基金资助:
CUI JiangKuan(),REN HaoHao(
),CAO MengYuan,CHEN KunYuan,ZHOU Bo,JIANG ShiJun,TANG JiHua(
)
Received:
2022-03-16
Accepted:
2022-04-03
Online:
2022-09-01
Published:
2022-09-07
Contact:
JiHua TANG
摘要:
【目的】 玉米孢囊线虫(Heterodera zeae)为孢囊线虫属定居型半内寄生线虫,可侵染多种禾本科作物的根部,玉米孢囊线虫的发生和传播扩散将会对我国玉米生产造成严重威胁。本研究旨在建立玉米孢囊线虫的快速分子检测体系,以期从近缘属种可以准确地检测出玉米孢囊线虫,为玉米孢囊线虫的监测预警和防控打下技术基础。【方法】 从我国河南、河北、甘肃、山东、湖南、广西和北京共收集20个玉米孢囊线虫近缘属种群体作为供试线虫;选取13条含有10个碱基的随机引物,采用RPAD技术对供试孢囊线虫进行多态性分析,筛出玉米孢囊线虫特异性RPAD片段,并将其转化为玉米孢囊线虫SCAR-PCR引物;采用PCR方法测试玉米孢囊线虫特异性引物的准确性以及检测技术体系的稳定性、灵敏度和实效性。【结果】 通过RAPD比对分析,发现随机引物OPA03能在玉米孢囊线虫基因组DNA中扩增出一条468 bp的特异性片段;将该片段回收测序,根据片段序列信息,利用Primer 5.0设计一对特异性引物HzF1/HzR1;特异性检测结果表明,HzF1/HzR1仅在玉米孢囊线虫群体中扩增出大小为393 bp的特异性片段,其他5种16个孢囊群体(禾谷孢囊线虫、菲利普孢囊线虫、大豆孢囊线虫、旱稻孢囊线虫和甜菜孢囊线虫)和4个其他种群(水稻干尖线虫、马铃薯腐烂茎线虫、落选短体线虫和咖啡短体线虫)均没有扩增出目的条带;以含有玉米孢囊线虫的混合种群孢囊DNA为模板时,特异性引物HzF1/HzR1也能扩增出来单一明亮的393 bp的特异片段,而不含玉米孢囊线虫的混合种群不能扩增出该片段;该体系实现了玉米孢囊线虫准确、稳定的检测目的;对建立的玉米孢囊线虫快速检测技术体系灵敏度和实用范围测试,表明该检测体系对玉米孢囊线虫单个孢囊和单条幼虫均有灵敏的检测能力,检出值不低于1/2 000个孢囊和1/80条2龄幼虫。【结论】 本研究建立的玉米孢囊线虫SCAR-PCR快速分子检测技术,可用于玉米孢囊线虫单一样本和混合种群的快速检测,对玉米孢囊线虫的孢囊和2龄幼虫均有灵敏的检测能力,检测引物特异性强,检测方法便捷稳定,灵敏度高。
崔江宽,任豪豪,曹梦园,陈昆圆,周博,蒋士君,汤继华. 玉米孢囊线虫SCAR-PCR快速分子检测技术[J]. 中国农业科学, 2022, 55(17): 3334-3342.
CUI JiangKuan,REN HaoHao,CAO MengYuan,CHEN KunYuan,ZHOU Bo,JIANG ShiJun,TANG JiHua. SCAR-PCR Rapid Molecular Detection Technology of Heterodera zeae[J]. Scientia Agricultura Sinica, 2022, 55(17): 3334-3342.
表1
用于玉米孢囊线虫SCAR-PCR特异性检测的线虫种群"
种群编号 Population code | 种类 Species | 来源 Origin | 寄主植物 Host plant |
---|---|---|---|
Hz01 | 玉米孢囊线虫H. zeae | 广西来宾Laibin, Guangxi | 玉米Zea mays |
Hz02 | 玉米孢囊线虫H. zeae | 河南省濮阳市清丰县Qingfeng County, Puyang, Henan | 玉米Zea mays |
Hz03 | 玉米孢囊线虫H. zeae | 河南省许昌市长葛Changge, Xuchang, Henan | 玉米Zea mays |
Hz04 | 玉米孢囊线虫H. zeae | 河南省许昌市禹州Yuzhou, Xuchang, Henan | 玉米Zea mays |
Ha01 | 禾谷孢囊线虫H. avenae | 河南省周口市扶沟县Fugou County, Zhoukou, Henan | 小麦Triticum aestivum |
Ha02 | 禾谷孢囊线虫H. avenae | 河南省洛阳市偃师Yanshi, Luoyang, Henan | 小麦Triticum aestivum |
Ha03 | 禾谷孢囊线虫H. avenae | 河北省沙河市Shahe, Hebei | 小麦Triticum aestivum |
Ha04 | 禾谷孢囊线虫H. avenae | 山东省烟台市莱州Laizhou, Yantai, Shandong | 小麦Triticum aestivum |
Hf01 | 菲利普孢囊线虫H. filipjevi | 河南省商丘市虞城县Yucheng County, Shangqiu, Henan | 小麦Triticum aestivum |
Hf02 | 菲利普孢囊线虫H. filipjevi | 河南省洛阳市栾川县Luanchuan County, Luoyang, Henan | 小麦Triticum aestivum |
Hf03 | 菲利普孢囊线虫H. filipjevi | 河北省邯郸市Handan, Hebei | 小麦Triticum aestivum |
Hf04 | 菲利普孢囊线虫H. filipjevi | 山东省滕州市Tengzhou, Shandong | 小麦Triticum aestivum |
He01 | 旱稻孢囊线虫H. elachista | 河南省信阳市潢川县Huangchuan County, Xinyang, Henan | 水稻Oryza sativa |
He02 | 旱稻孢囊线虫H. elachista | 河南省新乡市获嘉县Huojia County, Xinxiang, Henan | 水稻Oryza sativa |
He03 | 旱稻孢囊线虫H. elachista | 湖南省长沙市Changsha, Hunan | 水稻Oryza sativa |
Hg01 | 大豆孢囊线虫H. glycines | 河南省周口市淮阳县Huaiyang County, Zhoukou, Henan | 大豆Glycine max |
Hg02 | 大豆孢囊线虫H. glycines | 河南省南阳市内乡县Neixiang County, Nanyang, Henan | 大豆Glycine max |
Hg03 | 大豆孢囊线虫H. glycines | 甘肃省庆阳市Qingyang, Gansu | 大豆Glycine max |
Hg04 | 大豆孢囊线虫H. glycines | 河北省廊坊市Langfang, Hebei | 大豆Glycine max |
Hs01 | 甜菜孢囊线虫H. schachtii | 中国农业科学院Chinese Academy of Agricultural Sciences | 甜菜Beta vulgaris |
Ab01 | 水稻干尖线虫A. besseyi | 河南农业大学Henan Agricultural University | 水稻Oryza sativa |
Dd01 | 马铃薯腐烂茎线虫D. destructor | 河南农业大学Henan Agricultural University | 马铃薯Solanum tuberosum |
Pn01 | 落选短体线虫P. neglectus | 河南农业大学Henan Agricultural University | 小麦Triticum aestivum |
Pc01 | 咖啡短体线虫P. coffeae | 河南农业大学Henan Agricultural University | 小麦Triticum aestivum |
图2
OPA03对玉米孢囊线虫及其近缘属种单孢囊DNA的扩增 M:DL 2000 DNA Marker;1—7:玉米孢囊线虫 Hz01-1、Hz01-2、Hz02-1、Hz02-2、Hz03-1、Hz03-2、Hz04 种群 Hz01-1, Hz01-2, Hz02-1, Hz02-2, Hz03-1, Hz03-2, Hz04 population of H. zeae;8—12:禾谷孢囊线虫 Ha01种群、菲利普孢囊线虫 Hf02种群、大豆孢囊线虫 Hg04种群、甜菜孢囊线虫 Hs01种群、旱稻孢囊线虫 He01种群 Ha01 population of H. avenae, Hf02 population of H. filipjevi, Hg04 population of H. glycines, Hs01 population of H. schachtii, He01 population of H. elachista;13:阴性对照Negative control;红框内为玉米孢囊线虫特异性条带The red box indicates the specific bands of H. zeae"
图3
玉米孢囊线虫特异性引物对玉米孢囊线虫及其近缘种群的SCAR-PCR检测 M:DL 2000 DNA Marker;1—8:玉米孢囊线虫Hz01-1、Hz01-2、Hz02-1、Hz02-2、Hz03-1、Hz03-2、Hz04-1、Hz04-2种群 Hz01-1, Hz01-2, Hz02-1, Hz02-2, Hz03-1, Hz03-2, Hz04-1 and Hz04-2 population of H. zeae;9:6种孢囊线虫混合种群孢囊DNA DNA of cyst of H. zeae and other 5 related species populations;10:6种孢囊线虫混合种群J2幼虫DNA DNA of J2 of H. zeae and other 5 related species populations;11:不含玉米孢囊线虫的5种孢囊线虫混合种群孢囊DNA DNA of cyst of 5 related species populations without H. zeae;12:不含玉米孢囊线虫的5种孢囊线虫混合种群2龄幼虫DNA DNA of J2 of 5 related species populations without H. zeae;13—17:禾谷孢囊线虫Ha01种群、菲利普孢囊线虫Hf02种群、大豆孢囊线虫Hg04种群、旱稻孢囊线虫He01种群和甜菜孢囊线虫Hs01种群 Ha01 population of H. avenae, Hf02 population of H. filipjevi, Hg04 population of H. glycines, He01 population of H. elachista, Hs01 population of H. schachtii;18—21:水稻干尖线虫 Ab01种群、马铃薯腐烂茎线虫 Dd01种群、落选短体线虫 Pn01种群和咖啡短体线虫 Pc01种群 Ab01 population of A. besseyi, Dd01 population of D. destructor, Pn01 population of P. neglectus, Pc01 population of P. coffeae;22:阴性对照Negative control"
[1] | SRIVASTAVA A N, CHAWLA G. Maize cyst nematode, Heterodera zeae: A key nematode pest of maize and its management. New Delhi, India: Director, IARl, 2005: 8-9. |
[2] | WU H Y, QIU Z Q, MO A S, LI J Q, PENG D L. First report of Heterodera zeae on maize in China. Plant Disease, 2017, 101(7): 1330. |
[3] | CUI J K, ZHOU B, JIAO Y J, LÜ Y, LU J F, MENG H G, JIANG S J. First report of Heterodera zeae on maize (Zea mays L.) in Henan Province, China. Plant Disease, 2020, 104(7): 2031. |
[4] | LUC M, SIKORA R A, BRIDGE J. Plant Parasitic Nematodes in Subtropical and Tropical Agriculture. UK: CABI Publishing, 2005: 122. |
[5] | BAJAJ H K, KANWAR R S. Parasitization of maize by Heterodera avenae and H. filipjevi. Nematologia Mediterranea, 2005, 33: 203-207. |
[6] | BAHETI B L, DODWADIYA M, RATHORE B S, BHATI S S. Bioagents: An effective and ecofriendly option for the management of maize cyst nematode, Heterodera zeae on sweet corn (Zea mays L. Saccharata). Journal of Biopesticides, 2015, 8(2): 141-146. |
[7] | XIAO D X, FENG Y X, ZHOU Y, XUAN Y H, CHEN L J, WANG Y Y, LIU X Y, DUAN Y X, ZHU X F. First report of Heterodera elachista on Zea mays and Echinochloa crusgalli var. mitis in Liaoning Province, China. Plant Disease, 2019, 103(6): 1433. |
[8] | RINGER C E, SARDANELLI S, KRUSBERG L R. Investigations of the host range of the corn cyst nematode Heterodera zeae from Maryland. Journal of Nematology, 1987, 19(1): 97-106. |
[9] | SHAHZAD S, GHAFFAR A. Tomato, a natural host of Heterodera zeae in Pakistan. International Nematology Network Newsletter, 1986, 3(4): 38. |
[10] | QASIM M, GHAFFAR A. Heterodera zeae in the rhizosphere of declining almond trees. Nematologia Mediterranea, 1986, 14(1): 159. |
[11] | SRIVASTAVA A N, JAISWAL R K. Host and non-host status of plant species for the maize cyst nematode, Heterodera zeae, in India. Nematologia Mediterranea, 2011, 39: 203-206. |
[12] | CORREIA F J S, ABRANTES I M. Characterization of Heterodera zeae populations from Portugal. Journal of Nematology, 2005, 37(3): 328-335. |
[13] | MAQBOOL M A. Occurrence of root-knot and cyst nematodes in Pakistan. Nematologia Mediterranea, 1981, 9(2): 211-212. |
[14] | ABOUL-EID H Z, GHORAB A I. The occurrence of Heterodera zeae in maize fields in Egypt. Egyptian Journal of Phytopathology, 1983, 13(1/2): 51-61. |
[15] | CHINNASRI B, TANGCHITSOMKID N, TOIDA Y. Heterodera zeae on maize in Thailand. Japanese Journal of Nematology, 1994, 24(1): 35-38. |
[16] | SHARMA S B, PANDE S, SAHA M, KAUSHAL K K, LAL M, SINGH M, SINGH K, POKHAREL R, UPRETI R P, SINGH K. Plant parasitic nematodes associated with rice and wheat based cropping systems in Nepal. International Journal of Nematology, 2001, 11(1): 35-38. |
[17] |
SARDANELLI S, KRUSBERG L, GOLDEN A M. Corn cyst nematode, Heterodera zeae, in the United States. Plant Disease, 1981, 65(7): 622.
doi: 10.1094/PD-65-622 |
[18] | SKANTAR A M, HANDOO Z A, ZANAKIS G N, TZORTZAKAKIS E A. Molecular and morphological characterization of the corn cyst nematode, Heterodera zeae, from Greece. Journal of Nematology, 2012, 44(1): 58-66. |
[19] | ASGHARI R, POURJAM E, HEYDAR R, TANHA MAAFI Z, LATIFI A M. First report of corn cyst nematode, Heterodera zeae in Afghanistan. Australasian Plant Disease Notes, 2013, 8(1): 93-96. |
[20] |
SUBBOTIN S A, WAEYENBERGE L, MOENS M. Identification of cyst forming nematodes of the genus Heterodera (Nematoda: Heteroderidae) based on the ribosomal DNA-RFLP. Nematology, 2000, 2(2): 153-164.
doi: 10.1163/156854100509042 |
[21] | 彭德良, SUBBOTIN S, MOENS M. 小麦禾谷胞囊线虫(Heterodera avenae)的核糖体基因(rDNA)限制性片段长度多态性研究. 植物病理学报, 2003, 33(4): 323-329. |
PENG D L, SUBBOTIN S, MOENS M. rDNA restriction fragment length polymorphism of Heterodera avenae in China. Acta Phytopathologica Sinica, 2003, 33(4): 323-329. (in Chinese) | |
[22] |
WILLIAMS J G, KUBELIK A R, LIVAK K J, RAFALSKI J A, TINGEY S V. DNA polymorphism amplified by arbitrary primers are useful as genetic marker. Nucleic Acids Research, 1990, 18(22): 6531-6535.
doi: 10.1093/nar/18.22.6531 |
[23] |
WELSH J, MCCLELLAND M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research, 1990, 18(24): 7213-7218.
doi: 10.1093/nar/18.24.7213 |
[24] | CASWELL-CHEN E P, WILLIAMSON V M, WU F F. Random amplified polymorphic DNA analysis of Heterodera cruciferae and H. schachtii populations. Journal of Nematology, 1992, 24(3): 343-351. |
[25] | 刘佳, 徐娇, 代君丽, 李洪连. 中国黄淮麦区燕麦孢囊线虫荥阳群体致病型相关RAPD标记的建立. 麦类作物学报, 2013, 33(6): 1294-1299. |
LIU J, XU J, DAI J L, LI H L. RAPD markers of Heterodera avenae Xingyang population from Huang-huai floodplain in China. Journal of Triticeae Crops, 2013, 33(6): 1294-1299. (in Chinese) | |
[26] |
PARAN I, MICHELMORE R W. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theoretical and Applied Genetics, 1993, 85(8): 985-993.
doi: 10.1007/BF00215038 |
[27] |
FULLANODO A, BARRENA E, VIRIBAY M, BARRENA I, SALAZAR A, RITTER E. Identification of potato cyst nematode species Globodera rostochiensis and G. pallida by PCR using specific primer combinations. Nematology, 1999, 1(2): 157-163.
doi: 10.1163/156854199508126 |
[28] | AMIRI S, SUBBOTIN S A, MOENS M. Identification of the beet cyst nematode Heterodera schachtii by PCR. European Journal of Plant Pathology, 2002, 108(6): 497-506. |
[29] |
OU S Q, PENG D L, LIU X M, LI Y, MOENS M. Identification of Heterodera glycines using PCR with sequence characterised amplified region (SCAR) primers. Nematology, 2008, 10(3): 397-403.
doi: 10.1163/156854108783900212 |
[30] | 代君丽, 徐姣, 刘佳. Heterodera filipjevi许昌群体SCAR标记的建立. 植物病理学报, 2018, 48(2): 239-247. |
DAI J L, XU J, LIU J. Development of SCAR markers related to Heterodera filipjevi Xuchang population. Acta Phytopathologica Sinica, 2018, 48(2): 239-247. (in Chinese) | |
[31] | 彭德良. 小麦和大豆孢囊线虫毒性、分子诊断及线虫病害生防因子研究[D]. 北京: 中国农业大学, 2001. |
PENG D L. Studies on virulence, molecular diagnosis of cereal cyst nematode (Heterodera avenae) and soybean cyst nematode (Heterodera glycines) and biocontrol agents of nematode diseases[D]. Beijing: China Agricultural University, 2001. (in Chinese) | |
[32] | 亓晓莉, 彭德良, 彭焕, 龙海波, 黄文坤, 贺文婷. 基于SCAR标记的小麦禾谷孢囊线虫快速分子检测技术. 中国农业科学, 2012, 45(21): 4388-4395. |
QI X L, PENG D L, PENG H, LONG H B, HUANG W K, HE W T. Rapid molecular diagnosis based on SCAR marker system for cereal cyst nematode. Scientia Agricultura Sinica, 2012, 45(21): 4388-4395. (in Chinese) | |
[33] | 郑经武, 李德葆. 植物线虫分子分类及鉴定研究新进展. 植物病理学报, 1998, 28(1): 1-4. |
ZHENG J W, LI D B. Recent progress on molecular taxonomy and identification of plant nematodes. Acta Phytopathologica Sinica, 1998, 28(1): 1-4. (in Chinese) | |
[34] | POWERS T O, SANDALL L J. Estimation of genetic divergence in Meloidogyne mitochondrial DNA. Journal of Nematology, 1988, 20(4): 505-511. |
[35] | RADICE A D, POWERS T O, SANDALL L J, RIGGS R D. Comparisons of mitochondrial DNA from the sibling species Heterodera glycines and H. schachtiin. Journal of Nematology, 1988, 20(3): 443-450. |
[36] |
SUBBOTIN S A, WAEYENBERGE L, MOLOKANOVA I, MOENS M. Identification of Heterodera avenae group species by morphometrics and rDNA-RFLPs. Nematology, 1999, 1(2): 195-207.
doi: 10.1163/156854199508018 |
[37] | 卓侃, 宋汉达, 王宏洪, 陶冶, 张洪玲, 陆秀红, 黄金玲, 刘志明, 廖金铃. 旱稻孢囊线虫在广西的发生及其rDNA-ITS异质性分析. 中国水稻科学, 2014, 28(1): 78-84. |
ZHUO K, SONG H D, WANG H H, TAO Y, ZHANG H L, LU X H, HUANG J L, LIU Z M, LIAO J L. Occurence of Heterodera elachista in Guangxi region and its intra-species heterogeneity in rDNA-ITS region. Chinese Journal of Rice Science, 2014, 28(1): 78-84. (in Chinese) | |
[38] | 郑经武, 许建平, 陈卫良, 龚鸿飞, 李德葆. 四种植物线虫的RAPD比较及鉴定研究. 农业生物技术学报, 1998, 6(2): 108, 116. |
ZHENG J W, XU J P, CHEN W L, GONG H F, LI D B. Molecular comparison and identification of four plant nematodes using randomly amplified polymorphic DNA (RAPD) markers. Journal of Agricultural Biotechnology, 1998, 6(2): 108, 116. (in Chinese) | |
[39] | 喻盛甫, 王扬, 胡先奇, 白万明. 4种常见根结线虫基因组DNA的RAPD分析. 植物病理学报, 1998, 28(4): 359-365. |
YU S F, WANG Y, HU X Q, BAI W M. RAPD analysis of four most common Meloidogyne spp.. Acta Phytopathologica Sinica, 1998, 28(4): 359-365. (in Chinese) | |
[40] | MENG Q P, LONG H, XU J H. PCR assays for rapid and sensitive identification of three major root-knot nematodes, Meloidogyne incognita, M. javanica and M. arenaria. Acta Phytopathologica Sinica, 2004, 34(3): 204-210. |
[41] | 葛建军, 刁鹏, 陈洪俊, MARICE M. 马铃薯金线虫的分子检测技术. 植物检疫, 2008, 22(1): 21-23. |
GE J J, DIAO P, CHEN H J, MARICE M. Research on the molecular diagnosis for the Globodera rostochiensiss. Plant Quarantine, 2008, 22(1): 21-23. (in Chinese) | |
[42] |
PENG H, QI X L, PENG D L, LONG H B, HE X F, HUANG W K, HE W T. Sensitive and direct detection of Heterodera filipjevi in soil and wheat roots by species-specific SCAR-PCR assays. Plant Disease, 2013, 97(10): 1288-1294.
doi: 10.1094/PDIS-02-13-0132-RE |
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