重大检疫性害虫玉米根萤叶甲的种特异性SS-CO Ⅰ快速检测技术研究
|
摘要
玉米根萤叶甲在欧洲和美国是一种严重为害玉米的入侵性害虫,传播速度快,侵入我国的可能性极高。本文针对其难以快速准确进行形态鉴别的问题,以玉米根萤叶甲为研究对象,以其他9种/生物型叶甲总科常见害虫为参照,采用基于COⅠ基因的种特异性PCR方法(species-specific COⅠ, SS-COⅠ),研究其快速分子检测鉴定技术。通过提取10种/生物型叶甲DNA和通用型引物扩增测序,获得其基因片段的碱基序列,并比对分析设计1对玉米根萤叶甲特异性引物(DvvZCE1/DvvZCF1),其扩增片段为462 bp。种特异性检验结果显示,该对引物只对玉米根萤叶甲的COⅠ基因具有扩增能力,对其他常见叶甲类害虫,包括玉米双斑长跗萤叶甲、榆黄毛萤叶甲、黄曲条跳甲、油菜蚤跳甲、黄点直缘跳甲、莲草直胸跳甲、枸杞负泥虫以及褐足角胸叶甲的棕黄型和蓝绿型没有扩增能力,该对引物不仅对成虫有很好的扩增效果,对单粒卵、2龄幼虫以及成虫残体(包括触角、头部、胸部、腹部、前足、后足)也具有同样的扩增效果,其最低检出阈值为102.73 pg/μL(相当于1/122 880头雌性成虫)。玉米根萤叶甲特异性SS-COⅠ检测技术在其口岸检疫,以及有效阻截中具有重要意义。
The western corn rootworm Diabrotica virgifera virgifera LeConte, one of the most devastating and invasive pest insect of field maize in Europe and USA, has most likely invaded China due to its rapid propagation. Identification based on the external morphology of D. virgifera virgifera is blocked by small body size, high degree of similarity and pleomorphism. In this study, a molecular method based on species-specific cytochrome c oxidase subunit I(SS-COⅠ) PCR technique was developed for rapidly identifying the western corn rootworm by using nine other common leaf beetle species as reference. The mitochondrial DNA COⅠ(mtDNA COⅠ) gene from D. virgifera virgifera and nine other leaf beetle species common in China, including Monolepta hieroglyphica(Motschulsky), Pyrrhalta maculicollis(Motschulsky), Phyllotreta striolata(Fabricius), Psylliodes punctifrons Baly, Ophrida xanthospilota Baly, Agasicles hygrophila Selman & Vogt, Lema decempunctata Gebler, and Basilepta fulvipes(Motschulsky) biotype brown-yellow and biotype blue-green, were amplified by using the universal primer pair LCO1490/HCO2198 and sequenced. Moreover, one pair of SS-COⅠ primers(DvvZCE1/DvvZCF1) amplifying a single band of 462 bp from D. virgifera virgifera was designed. The specificity test of the primer pair against the other nine leaf beetle species indicated that it was specific to D. virgifera virgifera and no cross reactions were observed. The method was tested with egg, 2 nd-instar larva, male and female adults as well as adult debris(including antenna, head, thorax, abdomen, foreleg and posterior leg), showing that it was applicable. In addition, the 462 bp DNA fragment could be clearly detected even at the concentration of 102.73 pg/μL, equal to 1/122 880 of a whole female adult of D. virgifera virgifera. The SS-COⅠ marker developed here for rapid identification of D. virgifera virgifera could be used in port quarantine and interception in international transportation of agricultural products.
The western corn rootworm Diabrotica virgifera virgifera LeConte, one of the most devastating and invasive pest insect of field maize in Europe and USA, has most likely invaded China due to its rapid propagation. Identification based on the external morphology of D. virgifera virgifera is blocked by small body size, high degree of similarity and pleomorphism. In this study, a molecular method based on species-specific cytochrome c oxidase subunit I(SS-COⅠ) PCR technique was developed for rapidly identifying the western corn rootworm by using nine other common leaf beetle species as reference. The mitochondrial DNA COⅠ(mtDNA COⅠ) gene from D. virgifera virgifera and nine other leaf beetle species common in China, including Monolepta hieroglyphica(Motschulsky), Pyrrhalta maculicollis(Motschulsky), Phyllotreta striolata(Fabricius), Psylliodes punctifrons Baly, Ophrida xanthospilota Baly, Agasicles hygrophila Selman & Vogt, Lema decempunctata Gebler, and Basilepta fulvipes(Motschulsky) biotype brown-yellow and biotype blue-green, were amplified by using the universal primer pair LCO1490/HCO2198 and sequenced. Moreover, one pair of SS-COⅠ primers(DvvZCE1/DvvZCF1) amplifying a single band of 462 bp from D. virgifera virgifera was designed. The specificity test of the primer pair against the other nine leaf beetle species indicated that it was specific to D. virgifera virgifera and no cross reactions were observed. The method was tested with egg, 2 nd-instar larva, male and female adults as well as adult debris(including antenna, head, thorax, abdomen, foreleg and posterior leg), showing that it was applicable. In addition, the 462 bp DNA fragment could be clearly detected even at the concentration of 102.73 pg/μL, equal to 1/122 880 of a whole female adult of D. virgifera virgifera. The SS-COⅠ marker developed here for rapid identification of D. virgifera virgifera could be used in port quarantine and interception in international transportation of agricultural products.
引文
[1] KRYSAN J L, SMITH R F. Systematics of the Virgifera species group of Diabrotica (Coleoptera: Chrysomelidae: Galerucinae)[J]. Entomography, 1987, 5: 375-484.
[2] MEINKE L J, SAPPINGTON T W, ONSTAD D W, et al. Western corn rootworm (Diabrotica virgifera virgifera LeConte) population dynamics [J]. Agricultural and Forest Entomology, 2009, 11: 29-46.
[3] 中华人民共和国农业部和国家质量监督检验检疫总局. 中华人民共和国进境植物检疫性有害生物名录(共435种),农业部公告第862号[EB/OL]. 2007-05-28.http://www.moa.gov.cn/govpublic/ZZYQLS/201006/t201.
[4] CHIANG H C. Bionomics of the northern and western corn rootworms [J].Annual Review of Entomology,1973,18:47-72.
[5] KRYSAN J L, MILLAR T A. Methods for the study of pest Diabrotica [M]. Springer-Verlag New York Inc, 1986: 260.
[6] LEVINE E, OLOUMI-SADEGHI H. Management of Diabroticite rootworms in corn [J].Annual Review of Entomology, 1991, 36: 229-255.
[7] BRANSON T F, ORTMAN E E. Host range of larvae of western corn rootworm [J]. Journal of Economic Entomology, 1967, 60: 201-203.
[8] BREITENBACH S, HEIMBACH U, GLOYNA K, et al. Possible host plants for larvae of western corn rootworm (Diabrotica virgifera virgifera)[J]. BCPC Symposium Proceedings, 2005, 81: 217-218.
[9] CABI. Diabrotica virgifera virgifera (western corn rootworm)[EB/OL]. (2017-06-22)[2018-01-18]. http://www.cabi.org/isc/datasheet/18637.
[10] GRAY M E, SAPPINGTON T W, MILLER N J, et al. Adaptation and invasiveness of western corn rootworm: Intensifying research on a worsening pest [J]. Annual Review of Entomology, 2009, 54: 303-321.
[11] SPENCER J L, HIBBARD B E, MOESER J, et al. Behaviour and ecology of the western corn rootworm (Diabrotica virgifera virgifera LeConte) (Coleoptera: Chrysomelidae)[J]. Agricultural and Forest Entomology, 2009, 11: 9-27.
[12] MCBRIDE D K. Corn rootworm control trials-1971 [J]. North Dakota Farm Research, 1972, 29: 9-13.
[13] SPIKE B P, TOLLEFSON J J. Yield response of corn subjected to western corn rootworm (Coleoptera: Chrysomelidae) infestation and lodging [J].Journal of Entomology,1991,84(5):1585-1590.
[14] WESSELER J, FALL E H. Potential damage costs of Diabrotica virgifera virgifera infestation in Europe-the ‘no control’ scenario [J]. Journal of Applied Entomology, 2010, 134(5): 385-394.
[15] JENSEN S G. Laboratory transmission of maize chlorotic mottle virus by three species of corn rootworms [J].Plant Disease, 1985, 69(10): 864-868.
[16] ONSTAD D W, CRWODER D W, ISARD S A, et al. Does landscape diversity slow the spread of rotation-resistant western corn rootworm (Coleoptera: Chrysomelidae) [J]. Environmental Entomology, 2003, 32(5): 992-1001.
[17] BACA F. New member of the harmful entomofauna of Yugoslavia Diabrotica virgifera virgifera LeConte (Coleoptera, Chrysomelidae)[J]. Zastita Bilja, 1994, 45(2): 125-131.
[18] GERGINOV L, TOMOV N. The western corn rootworm-Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae)-a new quarantine pest of maize [J].Rasteniev’dni Nauki, 1995, 32(9/10): 159-160.
[19] KISS J, EDWARDS C R, BERGER H K, et al. Monitoring of western corn rootworm (Diabrotica virgifera virgifera LeConte) in Europe 1992-2003 [M]//VIDAL S, KUHLMANN U, EDWARDS C R. Western Corn Rootworm: Ecology and Management, 2005: 29-39.
[20] BAUFELD P, ENZIAN S.Maize growing, maize high-risk areas and potential yield losses due to western corn rootworm (Diabrotica virgifera virgifera) damage in selected European countries [M]//VIDAL S, KUHLMANN U, EDWARDS C R. Western Corn Rootworm: Ecology and Management. Wallingford, UK: CAB International, 2005: 285-302.
[21] 江幸福,罗礼智.玉米切根叶甲入侵我国的定性与定量风险分析[C]//成卓敏.植物保护与现代农业——中国植物保护学会2007年学术年会论文集.北京:中国农业科学技术出版社,2007: 41-47.
[22] 张俊华, 马菲, 曹逸霞, 等. 玉米根萤叶甲在中国的潜在适生区域与检疫措施研究[J]. 植物检疫, 2009, 23(6): 9-12.
[23] KRYSAN J L, SMITH R F, BRANSON T F,et al. A new subspecies of Diabrotica virgifera (Coleoptera: Chrysomelidae): description, distribution, and sexual compatibility [J]. Annals of the Entomological Society of America, 1980, 73(2): 123-130.
[24] SUNNUCKS P, HALES D F. Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in Aphids of the genus Sitobion (Hemiptera: Aphididae)[J]. Molecular Biology and Evolution, 1996, 13(3): 510-524.
[25] FOLMER O, BLACK M, HOEH W, et al. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates [J]. Molecular Marine Biology and Biotechnology, 1994, 3(5): 294-299.
[26] SZALANSKI A L, POWERS T O. Molecular diagnostics of three Diabrotica (Coleoptera: Chrysomelidae) pest species [J].Journal of the Kansas Entomological Society,1996,69(3):260-266.
[27] CLARK T L, MEINKE L J, FOSTER J E. PCR-RFLP of the mitochondrial cytochrome oxidase (subunit I) gene provides diagnostic markers for selected Diabrotica species (Coleoptera: Chrysomelidae)[J]. Bulletin of Entomological Research, 2001, 91(6): 419-427.
[28] 席欧彦, 李晓, 郑飞,等. 杨树萤叶甲DNA条形码研究[J]. 新疆农业科学, 2016, 53(2): 309-316.
[29] CLARK T L, MEINKE L J, FOSTER J E. Molecular phylogeny of Diabrotica beetles (Coleoptera: Chrysomelidae) inferred from analysis of combined mitochondrial and nuclear DNA sequences [J].Insect Molecular Biology,2001,10(4):303-314.
[30] 雍容婧,李志红,陈宏,等.3种根萤叶甲实时荧光PCR检测技术研究[J].植物检疫,2017,31(5):38-43.
[31] 张桂芬,刘万学,郭建英,等.美洲斑潜蝇SS-PCR检测技术研究[J].生物安全学报,2012,21(1):74-78.
[2] MEINKE L J, SAPPINGTON T W, ONSTAD D W, et al. Western corn rootworm (Diabrotica virgifera virgifera LeConte) population dynamics [J]. Agricultural and Forest Entomology, 2009, 11: 29-46.
[3] 中华人民共和国农业部和国家质量监督检验检疫总局. 中华人民共和国进境植物检疫性有害生物名录(共435种),农业部公告第862号[EB/OL]. 2007-05-28.http://www.moa.gov.cn/govpublic/ZZYQLS/201006/t201.
[4] CHIANG H C. Bionomics of the northern and western corn rootworms [J].Annual Review of Entomology,1973,18:47-72.
[5] KRYSAN J L, MILLAR T A. Methods for the study of pest Diabrotica [M]. Springer-Verlag New York Inc, 1986: 260.
[6] LEVINE E, OLOUMI-SADEGHI H. Management of Diabroticite rootworms in corn [J].Annual Review of Entomology, 1991, 36: 229-255.
[7] BRANSON T F, ORTMAN E E. Host range of larvae of western corn rootworm [J]. Journal of Economic Entomology, 1967, 60: 201-203.
[8] BREITENBACH S, HEIMBACH U, GLOYNA K, et al. Possible host plants for larvae of western corn rootworm (Diabrotica virgifera virgifera)[J]. BCPC Symposium Proceedings, 2005, 81: 217-218.
[9] CABI. Diabrotica virgifera virgifera (western corn rootworm)[EB/OL]. (2017-06-22)[2018-01-18]. http://www.cabi.org/isc/datasheet/18637.
[10] GRAY M E, SAPPINGTON T W, MILLER N J, et al. Adaptation and invasiveness of western corn rootworm: Intensifying research on a worsening pest [J]. Annual Review of Entomology, 2009, 54: 303-321.
[11] SPENCER J L, HIBBARD B E, MOESER J, et al. Behaviour and ecology of the western corn rootworm (Diabrotica virgifera virgifera LeConte) (Coleoptera: Chrysomelidae)[J]. Agricultural and Forest Entomology, 2009, 11: 9-27.
[12] MCBRIDE D K. Corn rootworm control trials-1971 [J]. North Dakota Farm Research, 1972, 29: 9-13.
[13] SPIKE B P, TOLLEFSON J J. Yield response of corn subjected to western corn rootworm (Coleoptera: Chrysomelidae) infestation and lodging [J].Journal of Entomology,1991,84(5):1585-1590.
[14] WESSELER J, FALL E H. Potential damage costs of Diabrotica virgifera virgifera infestation in Europe-the ‘no control’ scenario [J]. Journal of Applied Entomology, 2010, 134(5): 385-394.
[15] JENSEN S G. Laboratory transmission of maize chlorotic mottle virus by three species of corn rootworms [J].Plant Disease, 1985, 69(10): 864-868.
[16] ONSTAD D W, CRWODER D W, ISARD S A, et al. Does landscape diversity slow the spread of rotation-resistant western corn rootworm (Coleoptera: Chrysomelidae) [J]. Environmental Entomology, 2003, 32(5): 992-1001.
[17] BACA F. New member of the harmful entomofauna of Yugoslavia Diabrotica virgifera virgifera LeConte (Coleoptera, Chrysomelidae)[J]. Zastita Bilja, 1994, 45(2): 125-131.
[18] GERGINOV L, TOMOV N. The western corn rootworm-Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae)-a new quarantine pest of maize [J].Rasteniev’dni Nauki, 1995, 32(9/10): 159-160.
[19] KISS J, EDWARDS C R, BERGER H K, et al. Monitoring of western corn rootworm (Diabrotica virgifera virgifera LeConte) in Europe 1992-2003 [M]//VIDAL S, KUHLMANN U, EDWARDS C R. Western Corn Rootworm: Ecology and Management, 2005: 29-39.
[20] BAUFELD P, ENZIAN S.Maize growing, maize high-risk areas and potential yield losses due to western corn rootworm (Diabrotica virgifera virgifera) damage in selected European countries [M]//VIDAL S, KUHLMANN U, EDWARDS C R. Western Corn Rootworm: Ecology and Management. Wallingford, UK: CAB International, 2005: 285-302.
[21] 江幸福,罗礼智.玉米切根叶甲入侵我国的定性与定量风险分析[C]//成卓敏.植物保护与现代农业——中国植物保护学会2007年学术年会论文集.北京:中国农业科学技术出版社,2007: 41-47.
[22] 张俊华, 马菲, 曹逸霞, 等. 玉米根萤叶甲在中国的潜在适生区域与检疫措施研究[J]. 植物检疫, 2009, 23(6): 9-12.
[23] KRYSAN J L, SMITH R F, BRANSON T F,et al. A new subspecies of Diabrotica virgifera (Coleoptera: Chrysomelidae): description, distribution, and sexual compatibility [J]. Annals of the Entomological Society of America, 1980, 73(2): 123-130.
[24] SUNNUCKS P, HALES D F. Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in Aphids of the genus Sitobion (Hemiptera: Aphididae)[J]. Molecular Biology and Evolution, 1996, 13(3): 510-524.
[25] FOLMER O, BLACK M, HOEH W, et al. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates [J]. Molecular Marine Biology and Biotechnology, 1994, 3(5): 294-299.
[26] SZALANSKI A L, POWERS T O. Molecular diagnostics of three Diabrotica (Coleoptera: Chrysomelidae) pest species [J].Journal of the Kansas Entomological Society,1996,69(3):260-266.
[27] CLARK T L, MEINKE L J, FOSTER J E. PCR-RFLP of the mitochondrial cytochrome oxidase (subunit I) gene provides diagnostic markers for selected Diabrotica species (Coleoptera: Chrysomelidae)[J]. Bulletin of Entomological Research, 2001, 91(6): 419-427.
[28] 席欧彦, 李晓, 郑飞,等. 杨树萤叶甲DNA条形码研究[J]. 新疆农业科学, 2016, 53(2): 309-316.
[29] CLARK T L, MEINKE L J, FOSTER J E. Molecular phylogeny of Diabrotica beetles (Coleoptera: Chrysomelidae) inferred from analysis of combined mitochondrial and nuclear DNA sequences [J].Insect Molecular Biology,2001,10(4):303-314.
[30] 雍容婧,李志红,陈宏,等.3种根萤叶甲实时荧光PCR检测技术研究[J].植物检疫,2017,31(5):38-43.
[31] 张桂芬,刘万学,郭建英,等.美洲斑潜蝇SS-PCR检测技术研究[J].生物安全学报,2012,21(1):74-78.