DNA条形码技术对重大潜在入侵害虫大洋臀纹粉蚧的鉴定有效性研究
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摘要
【目的】粉蚧是一类重要的世界性检疫性害虫,对果蔬产业以及水果的进出口贸易造成了巨大威胁。通常,口岸截获的粉蚧多为若虫或残体,加之隐存种的存在和较小的近缘种间差异,严重影响了基于形态学特征的粉蚧类害虫识别鉴定的准确性和及时性。本研究旨在明确DNA条形码技术对重大潜在入侵害虫大洋臀纹粉蚧Planococcus minor(Maskell)的鉴定有效性。【方法】以旅检截获的36头大洋臀纹粉蚧为对象、其近缘种柑橘臀纹粉蚧Pl.citri(Risso)为参照,以线粒体COI基因5'端和3'端序列以及核糖体28S r DNA D2-D3区段序列为分子标记进行比对分析,以K-2-P模型计算种内种间遗传距离,以最大似然法(maximum likelihood,ML)构建进化树并进行系统发育分析,同时利用Species Identifier物种识别软件评价3种基因片段对大洋臀纹粉蚧的鉴定效果。【结果】当分别以COI基因5'端和3'端序列为分子标记时,截获大洋臀纹粉蚧的碱基序列与NCBI中大洋臀纹粉蚧的序列一致性分别为100%和99%~100%,而与近缘种柑橘臀纹粉蚧COI基因5'端和3'端的核苷酸序列一致性分别为97%~98%和96%~98%;且大洋臀纹粉蚧和柑橘臀纹粉蚧分别存在5个和11个稳定的物种特异性识别位点;系统发育分析显示,截获的大洋臀纹粉蚧均与数据库中的大洋臀纹粉蚧聚为一支。当以28S r DNA D2-D3区段序列为分子标记时,臀纹粉蚧属各物种间高度保守,无法区分大洋臀纹粉蚧与其近缘种柑橘臀纹粉蚧;种间遗传距离仅为0.004。此外,物种识别软件评价结果显示,基于COI基因5'端和3'端序列的鉴定结果完全正确,而基于28S r DNA D2-D3区段序列的鉴定结果却存在45.2%~61.9%的模糊鉴定。【结论】基于COI基因5'端和3'端的DNA条形码技术完全可用于大洋臀纹粉蚧的快速准确鉴定及检测,对有效阻截其入侵和进一步扩散蔓延意义重大。
【Aim 】Mealybugs are one of the most important quarantine pests that injury a variety of tropical fruits and vegetables worldwide. Usually,mealybugs intercepted in the quarantine inspection include eggs,nymphs or debris,and there exist several cryptic species and small difference in related species,which make them difficult to be identified by traditional morphological identification methods correctly and timely. In this study,the validity of DNA barcoding in identification of Planococcus minor( Maskell),a potential major alien invasive species to China,was evaluated. 【Methods】The 5'-and 3'-end of the mitochondrial cytochrome c oxidase subunit I( COI) gene and 28 S ribosomal DNA( r DNA) of36 individuals of Pl. minor intercepted during passenger inspection were amplified using universal primers,and Pl. citri( Risso),a related species of Pl. minor,from Langfang,Hebei used as a reference. The obtained partial fragment of 5'- and 3'-end of COI gene and D2-D3 fragment of 28 S r DNA were sequenced. The phylogenetic tree was established by using a maximum likelihood( ML) method.The intra- and inter-species genetic distances were calculated using the Kimura-2-Parameter model.Meanwhile,the validity of Pl. minor identification based on the above three gene fragments was tested with Species Identifier software. 【Results 】When the 5'- and 3'-end of COI gene of Pl. minor were sequenced and BLAST, respectively, the nucleotide sequence identity between the fragments from Gen Bank and our study is 100% and 99%- 100%,respectively. The nucleotide sequence identity of the 5'- and 3'-end of COI gene between Pl. minor and Pl. citri is 97%- 98% and 96%- 98%,respectively. There are 5 and 11 stable species-specific identification sites in the fragments of the 5'- and3'-end of COI gene for Pl. minor and Pl. citri,respectively. Phylogenetic analysis indicated that Pl.minor intercepted during passenger inspection and those from the NCBI clustered in a clade. The sequencing results showed that the D2-D3 fragment of 28 S r DNA among species belonging to the genus Planococcus has high conservation and that it could not be used to distinguish between Pl. minor and Pl.citri. The genetic distance between the two species is 0. 004. The results based on the software of Species Identifier showed that the species identification based on 5'- and 3'-end of COI gene was completely correct,while that based on D2-D3 fragment of 28 S r DNA resulted in 45. 2%- 61. 9% of fuzzy identification. 【Conclusion】Our results suggest that DNA barcoding technique based on 5'-and 3'-end of COI gene can be used to identify and detect Pl. minor rapidly and accurately. The technique will be significant in intercepting and blocking the further spreading of Pl. minor.
【Aim 】Mealybugs are one of the most important quarantine pests that injury a variety of tropical fruits and vegetables worldwide. Usually,mealybugs intercepted in the quarantine inspection include eggs,nymphs or debris,and there exist several cryptic species and small difference in related species,which make them difficult to be identified by traditional morphological identification methods correctly and timely. In this study,the validity of DNA barcoding in identification of Planococcus minor( Maskell),a potential major alien invasive species to China,was evaluated. 【Methods】The 5'-and 3'-end of the mitochondrial cytochrome c oxidase subunit I( COI) gene and 28 S ribosomal DNA( r DNA) of36 individuals of Pl. minor intercepted during passenger inspection were amplified using universal primers,and Pl. citri( Risso),a related species of Pl. minor,from Langfang,Hebei used as a reference. The obtained partial fragment of 5'- and 3'-end of COI gene and D2-D3 fragment of 28 S r DNA were sequenced. The phylogenetic tree was established by using a maximum likelihood( ML) method.The intra- and inter-species genetic distances were calculated using the Kimura-2-Parameter model.Meanwhile,the validity of Pl. minor identification based on the above three gene fragments was tested with Species Identifier software. 【Results 】When the 5'- and 3'-end of COI gene of Pl. minor were sequenced and BLAST, respectively, the nucleotide sequence identity between the fragments from Gen Bank and our study is 100% and 99%- 100%,respectively. The nucleotide sequence identity of the 5'- and 3'-end of COI gene between Pl. minor and Pl. citri is 97%- 98% and 96%- 98%,respectively. There are 5 and 11 stable species-specific identification sites in the fragments of the 5'- and3'-end of COI gene for Pl. minor and Pl. citri,respectively. Phylogenetic analysis indicated that Pl.minor intercepted during passenger inspection and those from the NCBI clustered in a clade. The sequencing results showed that the D2-D3 fragment of 28 S r DNA among species belonging to the genus Planococcus has high conservation and that it could not be used to distinguish between Pl. minor and Pl.citri. The genetic distance between the two species is 0. 004. The results based on the software of Species Identifier showed that the species identification based on 5'- and 3'-end of COI gene was completely correct,while that based on D2-D3 fragment of 28 S r DNA resulted in 45. 2%- 61. 9% of fuzzy identification. 【Conclusion】Our results suggest that DNA barcoding technique based on 5'-and 3'-end of COI gene can be used to identify and detect Pl. minor rapidly and accurately. The technique will be significant in intercepting and blocking the further spreading of Pl. minor.
引文
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Ashfaq M,Noor AR,Mansoor S,2010.DNA-based characterization of an invasive mealybug(Hemiptera:Pseudococcidae)species damaging cotton in Pakistan.Appl.Entomol.Zool.,45(3):395-404.
Bastos CS,De Almeida RP,Vidal Neto FDC,De Araújo GP,2007.Ocorrência de Planococcus minor Maskell(Hemiptera:Pseudococcidae)em algodoeiro no Nordeste do Brasil.Neotrop.Entomol.,36(4):625-628.
Chu D,Liu GX,Fu HB,Xu W,2009.Phylogenetic analysis of mt COIreveals the cryptic lineages in Phenacoccus solenopsis complex(Hemiptera:Pseudococcidae).Acta Entomol.Sin.,52(11):1261-1265.
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Aliabadian M,Kaboli M,Nijman V,Vences M,2009.Molecular identification of birds:performance of distance-based DNAbarcoding in three genes to delimit parapatric species.PLoS ONE,4(1):e4119.
Ashfaq M,Noor AR,Mansoor S,2010.DNA-based characterization of an invasive mealybug(Hemiptera:Pseudococcidae)species damaging cotton in Pakistan.Appl.Entomol.Zool.,45(3):395-404.
Bastos CS,De Almeida RP,Vidal Neto FDC,De Araújo GP,2007.Ocorrência de Planococcus minor Maskell(Hemiptera:Pseudococcidae)em algodoeiro no Nordeste do Brasil.Neotrop.Entomol.,36(4):625-628.
Chu D,Liu GX,Fu HB,Xu W,2009.Phylogenetic analysis of mt COIreveals the cryptic lineages in Phenacoccus solenopsis complex(Hemiptera:Pseudococcidae).Acta Entomol.Sin.,52(11):1261-1265.
Cox JM,1989.The mealybug genus Planococcus(Homoptera:Pseudococcidae).Bull.Brit.Mus.(Nat.Hist.),Entomol.,58(1):1-78.
Dietrich CH,Rakitov RA,Holmes JL,Black WC IV,2001.Phylogeny of the major lineages of Membracoidea(Insecta:Hemiptera:Cicadomorpha)based on 28S rD NA sequences.Mol.Phylogenet.Evol.,18(2):293-305.
Francis AW,Kairo MTK,Roda AL,2012a.Developmental and reproductive biology of Planococcus minor(Hemiptera:Pseudococcidae)under constant temperatures.Fla.Entomol.,95(2):297-303.
Francis AW,Kairo MTK,Roda AL,2012b.Passionvine mealybug,Planococcus minor(Maskell)(Hemiptera:Pseudococcidae).University of Florida Extension.http://edis.ifas.ufl.edu/pdffiles/IN/IN92000.pdf.
Francis AW,Kairo MTK,Roda AL,Liburd OE,Polar P,2012c.The passionvine mealybug,Planococcus minor(Maskell)(Hemiptera:Pseudococcidae),and its natural enemies in the cocoa agroecosystem in Trinidad.Biol.Control,60(3):290-296.
Fu HB,Wang F,Lin Y,Zhao XJ,2008.Planococcus minor,a quarantine pest insect,was firstly interrupted in mainland China.Plant Quarantine,22(6):382-384.[付海滨,王芳,林颖,赵晓军,2008.我国首次截获检疫性有害生物---大洋臀纹粉蚧.植物检疫,22(6):382-384]
García Morales M,Denno BD,Miller DR,Miller GL,Ben-Dov Y,Hardy NB,2016.ScaleN et:a literature-based model of scale insect biology and systematics.http://scalenet.info.
Gu YJ,Chen K,Liu HJ,Hu XN,2013a.Epidemic analysis and control strategy of pests carried by fruits from Thailand.Plant Quarantine,27(1):81-85.[顾渝娟,陈克,刘海军,胡学难,2013a.进境泰国水果携带有害生物疫情分析及防控对策.植物检疫,27(1):81-85]
Gu YJ,Liang F,Ma J,2015.Information analysis of scales on imported plant and plant products.Journal of Biosafety,24(3):208-214.[顾渝娟,梁帆,马骏,2015.中国进境植物及植物产品携带蚧虫疫情分析.生物安全学报,24(3):208-214]
Gu YJ,Liu HJ,He RR,Wu MT,Chen K,Hu XN,2013b.Information analysis of scales on imported fruits from association of Southeast Asian nations.Plant Quarantine,27(5):95-99.[顾渝娟,刘海军,何日荣,武目涛,陈克,胡学难,2013b.东盟输华水果携带检疫性蚧类害虫疫情分析.植物检疫,27(5):95-99]
Hardy NB,Gullan PJ,Hodgson CJ,2008.A subfamily level classification of mealybugs(Hemiptera:Pseudococcidae)based on integrated molecular and morphological data.Syst.Entomol.,33(1):51-71.
He YB,Wan XW,Liu YH,Sun GM,Zhan RL,2012.Mitochondrial COI from Dysmicoccus brevipes(Hemiptera:Pseudococcidae)suggests cryptic lineage and pinpoints the source of the introduction to China.Fla.Entomol.,95(1):183-191.
He YB,Wan XW,Zhan RL,Sun GM,Liu YH,Xu ZF,Zhao YL,2011.Genetic relationship of 12 species of mealybugs(Hemiptera:Pseudococcidae)based on DNA sequences.Chinese Journal of Tropical Crops,32(12):2324-2330.[何衍彪,万宣伍,詹儒林,孙光明,刘映红,许再福,赵艳龙,2011.基于DNA序列的12种粉蚧亲缘关系分析.热带作物学报,32(12):2324-2330]
Hebert PDN,Cywinska A,Ball L,DeW aard JR,2003.Biological identifications through DNA barcodes.Proc.R.Soc.Lond.B Biol.Sci.,270(1512):313-321.
Hebert PDN,Penton EH,Burns JM,Janzen DH,Hallwachs W,2004.Ten species in one:DNA barcoding reveals cryptic species in the neotroical skipper butterfly Astraptes fulgerator.Proc.Natl.Acad.Sci.USA,101(41):14812-14817.
Ho HY,Hung CC,Chuang TH,Wang WL,2007.Identification and synthesis of the sex pheromone of the passionvine mealybug,Planococcus minor(Maskell).J.Chem.Ecol.,33(10):1986-1996.
Hodgson C,Abbas G,Arif MJ,Saeed S,Karar H,2008.Phenacoccus solenopsis Tinsley(Sternorrhyncha:Coccoidea:Pseudococcidae),an invasive mealybug damaging cotton in Pakistan and India,with a discussion on seasonal morphological variation.Zootaxa,1913:1-35.
Huang PY,Lin ZJ,Fang ZP,Cong YH,Lin LL,2015.Survey of scale insect on imported fruits in Xiamen port.Plant Quarantine,29(3):85-89.[黄蓬英,林振基,方志鹏,丛尧华,林玲玲,2015.厦门口岸进口水果截获蚧虫概况.植物检疫,29(3):85-89]
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