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Monitoring and Rapid Identification Techniques for Stored Product Insects in Imported Rice
|關鍵字:||imported rice;進口米;stored-product insects;quarantine;light trap;SCAR;multiplex PCR;積穀昆蟲;檢疫;燈光誘引器;序列特徵化增幅區域;複合式PCR||出版社:||昆蟲學系所||引用:||Agustí N, de Vicente MC, Gabarra R. 1999. Development of sequence amplified characterized region (SCAR) markers of Helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis. Mol Ecol 8: 1467-1474. Agustí N, de Vicente MC, Gabarra R. 2000. Developing SCAR markers to study predation on Trialeurodes vaporariorum. Insect Mol Biol 9: 263-268. Athanassiou CG, Kavallieratos NG, Trematerra P. 2006. Responses of Sitophilus oryzae (Coleoptera: Curculionidae) and Tribolium confusum (Coleoptera: Tenebrionidae) to traps baited with pheromones and food volatiles. European J Entomol 103: 371-378. Ball SL, Hebert PDN, Burian SK, Webb JM. 2005. Biological identifications of mayflies (Ephemeroptera) using DNA barcodes. J North Am Benthol Soc 24: 508-524. Bashir TL, Birkinshaw A, Farman D, Hall DR, Hodges RJ. 2003. 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Molecular characterization of RAPD and SCAR marker linked to frog-eye leaf spot resistance gene in soybean. Chinese Sci Bull 45: 460-466.||摘要:||
台灣自2002年加入世界貿易組織（World Trade Organization, WTO），開始進口稻米長期貯藏，害蟲監測也因此成為後續管理之重要一環。本研究主要在探討進口米貯藏期間之害蟲發生情形，並建立害蟲族群變化之監測方法與分子快速鑑定技術。首先，自2003至2006年間，針對進口米害蟲進行危害調查及進倉後長期監測之結果顯示，進口米檢疫採樣之樣品，含活蟲之比例為12％，但並未發現檢疫害蟲；後續貯藏階段以袋內取樣監測之全部樣品，共檢測出11種害蟲及1種害蟎。在常溫倉中，害蟲於貯藏三個月後發生，平均密度每公斤低於10隻；六個月後，害蟲則普遍大量發生，密度每公斤高於35隻；低溫倉（15-18°C）貯藏，害蟲延至貯藏六個月後才開始發生，且自2004年之後害蟲發生密度每公斤低於1隻，遠較常溫倉為低。
其次，利用部分積穀害蟲善飛及具趨光性，評估應用燈光誘引器監測害蟲族群變化之結果顯示，在3處（林內、莿桐、六甲）常溫倉捕獲之初級及次級害蟲種類相極為相似，其中對初級害蟲穀蠹（Rhyzopertha dominica (Fabricius)）、麥蛾（Sitotroga cerealella (Olivier)）、粉斑螟蛾（Cadra cautella (Walker)）及次級害蟲角胸粉扁蟲（Cryptolestes pusillus Schonherr）、背圓粉扁蟲（Ahasverus advena (Waltl)）、茶蛀蟲（Liposcelis divinatorius Müller）等有較佳誘引效果。貯米袋內直接取樣則顯示，此3處常溫倉初級害蟲均以米象（Sitophilus oryzae (L.)）為主，與燈光誘引器所誘得之蟲相差異極大，而次級害蟲則與燈光誘引器所誘得之蟲相較一致。此等結果顯示，燈光誘引器在進口糙米倉之害蟲監測應用上，適合做為監測米象以外大部分積穀害蟲消長之工具。
最後，為解決檢疫樣品中常發現害蟲幼體，卻難以從形態鑑定其種類的問題，而開發分子快速鑑定技術。應用序列特徵化增幅區域（sequence characterized amplified region, SCAR）技術，建立3組複合式PCR（Multiplex PCR）鑑定試劑組，分別命名為SCAR-I、-II與-III。每一試劑組內含可同時鑑別外米綴蛾（Corcyra cephalonica (Stainton)）、粉斑螟蛾、麥蛾及印度穀蛾（Plodia interpunctella (Hübner)）等四種主要鱗翅目積穀害蟲之專一性引子對；其中SCAR-I可分別擴增得205、550、324及382 bp之專一片段，SCAR-II可分別擴增出341、565、261及170 bp片段，SACAR-III可分別擴增得514、555、445及299 bp片段。利用此3組複合式PCR於進口穀物之害蟲樣品，可快速、簡易及精準地鑑定出此四種鱗翅目害蟲之種類，顯示該技術足以開發於進口米檢疫害蟲檢查快速鑑定之應用。
Since Taiwan became a member of the World Trade Organization (WTO) in 2002, the government began to import rice for long-term storage. As a result, monitoring foreign species of insect pests in the imported rice has become one of important matters. The aims of this study were to understand the occurrence of key pests in the imported rice and to set up the methods for monitoring and rapid identification of species of stored product insects. First, a survey conducted during 2003-2006 showed that live insects and mites were intercepted in 12% of the inspected samples of imported brown rice, but none of them were listed as quarantine pests in Taiwan. Eleven species of insects and one species of mite were found after their storage in warehouses. For storage at room temperature for three months, the average number of insects in the brown rice was below 10 insects per kilogram, while the number increased to 35 insects per kilogram after stored for six months. The occurrence of pest insects in low temperature warehouses (15-18°C) was delayed until six months of storage, while the number was below 1 insect per kilogram since 2004.
Some stored-product pests can be trapped by light due to their positive phototactic behavior; we therefore further evaluated light trap efficacy to develop methods for monitoring stored product insects. Light traps were used for long-term monitoring in the same batch of USA-imported brown rice stored separately in Linnei, Cihtong (Yunlin County), Liujia (Tainan City), and Renwu (Kaohsiung City). The results of light-trapping in the common storehouses in Linnei, Cihtong, and Liujia showed similar compositions of primary and secondary pests, and have better trapping efficacy on the primary pests of Sitotroga cerealella (Olivier), Rhyzopertha dominica (Fabricius), and Cadra cautella (Walker) and the secondary pests of Cryptolestes pusillus Schonherr, Ahasverus advena (Waltl), and Liposcelis divinatorius Müller. However, the direct survey in stored rice bags showed that Sitophilus oryzae was the key primary pest in these common storehouses, which was different from the light-trapped results. However, the minor species of the secondary pests and their ratio of amount when surveyed by two different methods were similar. These results indicate that light traps used to monitor the population fluctuation of most stored-product pests in imported brown rice are applicable.
Finally, insect pests intercepted from imported brown rice are frequently as immature forms, and their morphological identification is difficult. To solve this problem, a DNA identification method based on a sequence-characterized amplified region-polymerase chain reaction (SCAR-PCR) was developed. Here, three sets of multiplex SCAR-PCR mixtures, namely SCAR-I, -II, and -III, were finally developed with each set composed of four species-specific primer pairs derived from the genomic DNA of four major lepidopterous stored-product pests: Corcyra cephalonica (Stainton), C. cautella, S. cerealella, and Plodia interpunctella (Hübner). The SCAR-I amplicons of C. cephalonica, C. cautella, S. cerealella, and P. interpunctella were 205, 550, 324, and 382 bp, respectively, while those of SCAR-II were 341, 565, 261, and 170 bp, and those of SCAR-III were 514, 555, 445, and 299 bp. These multiplex PCR mixtures could sensitively and unambiguously detect and identify individuals among the four lepidopterous pests intercepted in imported stored-products.
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