福建新记录入侵害虫木瓜秀粉蚧的分子检测鉴定
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摘要
【目的】基于核糖体28S rDNA基因序列的种特异性PCR方法,快速鉴定福建新纪录入侵性害虫——木瓜秀粉蚧(Paracoccus marginatus Williams and Granara de Willink)。【方法】通过1对扩增粉蚧28S rDNA的通用引物(S3660/A335)扩增以木瓜秀粉蚧为靶标,以野外采集的另外10种粉蚧为对照的11种粉蚧的基因片段序列。根据所得基因序列结果并参照GeneBank数据库中已知粉蚧的28S rDNA基因序列,设计出木瓜秀粉蚧28S rDNA的种特异性引物(28S-ParF/28S-MarR),对该种特异性引物的效果进行检验。【结果】该种特异性引物对木瓜秀粉蚧的28S rDNA基因具有扩增能力,得到产物大小为446 bp的扩增片段,对其他10种粉蚧均无扩增效果。该引物不仅对木瓜秀粉蚧雌成虫具有稳定的扩增效果,对不同虫态、不同寄主来源以及不同地区来源的木瓜秀粉蚧均有稳定的扩增效果。【结论】对福建发现的木瓜秀粉蚧进行分子鉴定并建立了木瓜秀粉蚧快速分子检测技术,可用于识别和防控木瓜秀粉蚧,有助于遏制木瓜秀粉蚧的入侵危害。
【Objective】Paracoccus marginatus Williams and Granara de Willink is an invasive pest with strong diffusion and fecundity. It has caused serious damage to the papaya industry in Central America,Florida(USA), Guam(USA) and India. Pa. marginatus was first discovered in Fujian Province(Fuzhou and Zhangzhou) in 2017, showing great potential risks to papaya and other fruit crops, as well as flower industry in Fujian province. Because of the small body size and similar morphological characteristics, the morphological identification of mealybugs was inefficient. Rapid molecular identification of different species could be achieved through the use of DNA barcoding technology. Therefore, a technology for rapid molecular identification of Pa. marginatus was established based on the species-specific PCR method.【Methods】A species-specific PCR method based on ribosomal DNA-28 S gene fragment(28 S rDNA) was exploited to establish one technology for rapid detection and identification of Pa.marginatus. The additional 10 species of mealybugs(Phenacoccus solenopsis, Dysmicoccus boninsis,Nipaecoccus viridis, Phenacoccus solani, Pseudococcus comstocki, Pseudococcus cryptus, Planococcus lilacinus, Pseudococcus odermatti, Planococcus minor and Phenacoccus madeirensi) were collected in the fields as the contrast. In order to ensure the uniqueness of the source of DNA, the DNA templates were all extracted from one single female adult of these 11 species of mealybugs, respectively.28 S rDNA of the 11 species was amplified by a pair of universal primers(S3660/A335). The obtained partial fragments of 28 S rDNA were sequenced. And the phylogenetic tree was established by using a Neighbor-joining(NJ) method. According to the obtained 28 S rDNA gene partial sequence of the 11 species and 28 S rDNA gene sequences of Paracoccus galzerae in GeneBank database, the sequence alignment and analysis were performed on DNAMAN. 28 S rDNA species-specific primers(28 S-ParF/28 S-MarR) for Pa. marginatus were designed by selecting the sites with large differences in the sequence. And then, the specific effects, versatility and sensitivity of the specific primers were examined.【Results】The comparative results showed that the similarity between Pa. marginatus and Paracoccus marginatus isolate S3-668, KP692333 in the GenBank database was 100%. It was also indicated that the mealybugs were identified as Pa. marginatus by molecular identification. Phylogenetic analysis indicated that Pa. marginatus from Fuzhou and Zhangzhou was clustered in a clade. And that combined with Paracoccus galzerae(inter-species genetic distance is 0.058) to form a clade of the genus Paracoccus. The results of specificity tests showed that all Pa. marginatus specimens could be detected positively and a 446 bp fragment of the 28 S rDNA of Pa. marginatus was obtained by the species-specific primers, while there was no cross reactions with other 10 species of mealybugs. The species-specific primers not only had a stable amplification effect on female adults, but also were proved to be applicable for the2 ndinstar nymphs and the 3 rdinstar nymphs. Pa. marginatus from three different regions(Fuzhou and Zhangzhou in Fujian province, Jinghong in Yunnan province) and six different host plants(Carica papaya, Solanum melongena, Plumeria rubra, Solanum tuberosum, Tithonia diversifolia and Duranta erecta) was also successfully detected by the species-specific primers.【Conclusion】Molecular identification of Pa. marginatus first reported in Fujian province was carried out based on 28 S rDNA molecular markers. It was proved by experiments that the 28 S rDNA species-specific primers had ideal and stable specificity for Pa. marginatus and could be used to identify Pa. marginatus accurately. A rapid molecular detection technique for Pa. marginatus was established based on the species-specific PCR method.The technology has the characteristics of accuracy, rapidity, sensitivity and simplicity. Our present results indicated that the rapid detection technique should be useful in quarantine at ports, in pest detection and in monitoring during transportation of papaya and other fruit tree seedlings, as well as flowers.However, in view of the fact that no other mealybugs of the genus Paracoccus has been reported in China, this study can provide a reference for the molecular identification for the closely related species of Pa. marginatus.
【Objective】Paracoccus marginatus Williams and Granara de Willink is an invasive pest with strong diffusion and fecundity. It has caused serious damage to the papaya industry in Central America,Florida(USA), Guam(USA) and India. Pa. marginatus was first discovered in Fujian Province(Fuzhou and Zhangzhou) in 2017, showing great potential risks to papaya and other fruit crops, as well as flower industry in Fujian province. Because of the small body size and similar morphological characteristics, the morphological identification of mealybugs was inefficient. Rapid molecular identification of different species could be achieved through the use of DNA barcoding technology. Therefore, a technology for rapid molecular identification of Pa. marginatus was established based on the species-specific PCR method.【Methods】A species-specific PCR method based on ribosomal DNA-28 S gene fragment(28 S rDNA) was exploited to establish one technology for rapid detection and identification of Pa.marginatus. The additional 10 species of mealybugs(Phenacoccus solenopsis, Dysmicoccus boninsis,Nipaecoccus viridis, Phenacoccus solani, Pseudococcus comstocki, Pseudococcus cryptus, Planococcus lilacinus, Pseudococcus odermatti, Planococcus minor and Phenacoccus madeirensi) were collected in the fields as the contrast. In order to ensure the uniqueness of the source of DNA, the DNA templates were all extracted from one single female adult of these 11 species of mealybugs, respectively.28 S rDNA of the 11 species was amplified by a pair of universal primers(S3660/A335). The obtained partial fragments of 28 S rDNA were sequenced. And the phylogenetic tree was established by using a Neighbor-joining(NJ) method. According to the obtained 28 S rDNA gene partial sequence of the 11 species and 28 S rDNA gene sequences of Paracoccus galzerae in GeneBank database, the sequence alignment and analysis were performed on DNAMAN. 28 S rDNA species-specific primers(28 S-ParF/28 S-MarR) for Pa. marginatus were designed by selecting the sites with large differences in the sequence. And then, the specific effects, versatility and sensitivity of the specific primers were examined.【Results】The comparative results showed that the similarity between Pa. marginatus and Paracoccus marginatus isolate S3-668, KP692333 in the GenBank database was 100%. It was also indicated that the mealybugs were identified as Pa. marginatus by molecular identification. Phylogenetic analysis indicated that Pa. marginatus from Fuzhou and Zhangzhou was clustered in a clade. And that combined with Paracoccus galzerae(inter-species genetic distance is 0.058) to form a clade of the genus Paracoccus. The results of specificity tests showed that all Pa. marginatus specimens could be detected positively and a 446 bp fragment of the 28 S rDNA of Pa. marginatus was obtained by the species-specific primers, while there was no cross reactions with other 10 species of mealybugs. The species-specific primers not only had a stable amplification effect on female adults, but also were proved to be applicable for the2 ndinstar nymphs and the 3 rdinstar nymphs. Pa. marginatus from three different regions(Fuzhou and Zhangzhou in Fujian province, Jinghong in Yunnan province) and six different host plants(Carica papaya, Solanum melongena, Plumeria rubra, Solanum tuberosum, Tithonia diversifolia and Duranta erecta) was also successfully detected by the species-specific primers.【Conclusion】Molecular identification of Pa. marginatus first reported in Fujian province was carried out based on 28 S rDNA molecular markers. It was proved by experiments that the 28 S rDNA species-specific primers had ideal and stable specificity for Pa. marginatus and could be used to identify Pa. marginatus accurately. A rapid molecular detection technique for Pa. marginatus was established based on the species-specific PCR method.The technology has the characteristics of accuracy, rapidity, sensitivity and simplicity. Our present results indicated that the rapid detection technique should be useful in quarantine at ports, in pest detection and in monitoring during transportation of papaya and other fruit tree seedlings, as well as flowers.However, in view of the fact that no other mealybugs of the genus Paracoccus has been reported in China, this study can provide a reference for the molecular identification for the closely related species of Pa. marginatus.
引文
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[2] MILLER D R,WILLIAMS D J,HAMON A B. Notes on a new mealybug(Hemiptera:Coccoidea:Pseudococcidae)pest in Florida and the Caribbean:the papaya mealybug,Paracoccus marginatus Williams and Granara de Willink[J]. Insecta Mundi,1999,13(3-4):179-181.
[3] WILLIAMS D J,GRANARA DE WILLINK D C. Mealybugs of Central and South America[M]. Wallingford,UK:CAB International,1992:635.
[4] AMARASEKARE K G,CHONG J H,EPSKY N D,MANNION C M. Effect of temperature on the life history of the mealybug Paracoccus marginatus(Hemiptera:Pseudococcidae)[J]. Journal of Economic Entomology,2008,101(6):1798-1804.
[5] WAKER A,HOY M,MEYERDIRK D. Papaya mealybug,Paracoccus marginatus Williams and Granara de Willink(Insecta:Hemiptera:Pseudococcidae)[M]. EENY-302. Featured Creatures. Entomology and Nematology Department,Florida Cooperative Extension Service,Institute of Food and Agricultural Sciences,University of Florida,Gainesville,FL.2003.
[6] SENI A,CHONGTHAM S. Papaya mealybug Paracoccus marginatus Williams&Granara de Willink(Hemiptera:Pseudococcidae),a current threat to agriculture-a review[J]. Agricultural Reviews,2013,34(3):216-222.
[7] MUNIAPPAN R,SHEPARD B M,WATSON G W,CARNER G R,SARTIAMI D,RAUF A,HAMMING M D. First report of the papaya mealybug,Paracoccus marginatus(Hemiptera:Pseudococcidae),in Indonesia and India[J]. Journal of Agricultural&Urban Entomology,2014,25(1):37-40.
[8] GALANIHE L D,JAYASUNDERA M U P,VITHANA A,ASSELAARACHCHI N,WATSON G W. Occurrence,distribution and control of papaya mealybug,Paracoccus marginatus(Hemiptera:Pseudococcidae),an invasive alien pest in Sri Lanka[J].Tropical Agricultural Research&Extension,2011,13(3):81-86.
[9] SAENGYOT S,BURIKAM I. Host plants and natural enemies of papaya mealybug,Paracoccus marginatus(Hemiptera:Pseudococcidae)in Thailand[J]. Thai Journal of Agricultural Science,2011,44(3):197-205.
[10] CHEN S P,WONG J Y,WU W J. Preliminary report on the occurrence of papaya mealybug,Paracoccus marginatus Williams and Granara de Willink,in Taiwan[J]. Journal of Taiwan Agricultural Research,2011,60(1):72-76.
[11] WU F Z,LIU Z H,SHEN H,YU F,MA J,HU X N,ZENG L.Morphological and molecular identification of Paracoccus marginatus(Hemiptera:Pseudococcidae)in Yunnan,China[J]. Florida Entomologist,2014,97(4):1469-1473.
[12]顾渝娟,刘海军,梁帆,马骏,李海林.一种重要的有害生物—木瓜粉蚧[J].植物检疫,2015,29(2):57-60.GU Yujuan,LIU Haijun,LIANG Fan,MA Jun,LI Hailin. An important pest:papaya mealybug(Paracoccus marginatus)[J].Plant Quarantine,2015,29(2):57-60.
[13]卢辉,卢芙萍,梁晓,伍春玲,陈青.木瓜秀粉蚧在海南的适生性及空间分布型研究[J].热带作物学报,2016,37(10):1962-1968.LU Hui,LU Fuping,LIANG Xiao,WU Chunling,CHEN Qing.The potential geographic distribution and spatial pattern of Paracoccus marginatus in Hainan province[J]. Chinese Journal of Tropical Crops,2016,37(10):1962-1968.
[14]何衍彪,万宣伍,詹儒林,孙光明,刘映红,许再福,赵艳龙.基于DNA序列的12种粉蚧亲缘关系分析[J].热带作物学报,2011,32(12):2324-2330.HE Yanbiao,WUAN Xuanwu,ZHAN Rulin,SUN Guangming,LIU Yinghong,XU Zaifu,ZHAO Yanlong. Genetic relationship of 12 species of mealybugs(Hemiptera:Pseudococcidae)based on DNA sequences[J]. Chinese Journal of Tropical Crops,2011,32(12):2324-2330.
[15] GULLAN P J,KAYDAN M B,HARDY N B. Molecular phylogeny and species recognition in the mealybug genus Ferrisia Fullaway(Hemiptera:Pseudococcidae)[J]. Systematic Entomology,2010,35(2):329-339.
[16]王芙蓉,吴薇,荣德福.分子生物学技术在检疫性昆虫鉴定中的应用前景[J].安徽农业科学,2008,36(8):3149-3151,3162.WANG Furong,WU Wei,RONG Defu. Application prospect of molecular biological techniques in identification of quarantine insect[J]. Journal of Anhui Agriculture Science,2008,36(8):3149-3151,3162.
[17]王玉生.我国常见粉蚧类害虫双基因条形码鉴定技术研究[D].北京:中国农业科学院,2016.WANG Yusheng. Identification of common mealybug species based on double genes barcoding techniques in China[D]. Beijing:Chinese Academy of Agricultural Sciences,2016.
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