A novel capillary gel electrophoresis based fragment analysis method for the rapid detection of important thrips species on alfalfa in Turkey

Year 2022, Volume: 62 Issue: 3, 5 - 11, 30.09.2022

Ali Ferhan Morca , Cenk Yücel , Aydemir Barış , Ekrem Atakan , Ali Çelik

https://doi.org/10.16955/bitkorb.1078737

Abstract

Thrips cause significant yield reduction in several industrial crops. Since these pests are also included in the quarantine organisms of Turkey, the rapid detection of agents is important to prevent their spread to new areas. Mitochondrial cytochrome oxidase I (COI) barcoding gene assay; one of the molecular methods is widely used in thrips identification. However, as the COI gene has a very short fragment length, it is very difficult to distinguish fragment sizes on agarose gel after PCR. In this study, a new identification method was developed by integrating the Capillary Gel Electrophoresis (CGE) system for Thrips tabaci Lideman, Frankliniella occidentalis (Pergande) and Frankliniella intonsa (Trybom) species, using primer pairs previously used by different researchers. The assay produces strong signals obtained by minimizing the margin of error in the separation of fragment lengths close to each other, especially in the short fragment length COI gene. Therefore, by eliminating the gel electrophoresis step, reliable detections could be obtained without exposure to hazardous chemicals. The novel method shortened the detection time and minimized the process mistakes on the detection of a single thrips with a low DNA concentration. Total 83 thrips individual (52 F. intonsa, 31 F. occidentalis) were able to be detected with this capillary gel electrophoresis based fragment analysis. The novel method is evaluated as unique, specific and quick for the detection of three different thrips species. It is also thought to be able to utilize for identification of different thrips species with short fragment sizes in the foreseeable future.

Keywords

Frankliniella intonsa, Frankliniella occidentalis, mitochondrial cytochrome oxidase I, multiplex polymerase chain reaction, Thysanoptera

Supporting Institution

Tarım ve Orman Bakanlığı Tarımsal Araştırmalar ve Politikalar Genel Müdürlüğü Ankara Zirai Mücadele Merkez Araştırma Enstitüsü Müdürlüğü

Project Number

TAGEM-BS-14/09-02/02-08

Thanks

This work was supported by the General Directorate of Agricultural Research and Policies, Directorate of Central Plant Protection Research Institute, Turkey [Project No. TAGEM-BS-14/09-02/02-08].

Türkiye'de yoncada önemli thrips türlerinin hızlı tespiti için yeni bir kapiler jel elektroforez tabanlı fragment analiz yöntemi

Abstract

Thripsler, birçok endüstriyel üründe önemli verim kayıplarına neden olur. Bu zararlılar Türkiye'deki karantina organizmaları arasında yer aldığından, etmenlerin hızlı tespiti yeni alanlara yayılmalarını önlemek için önemlidir. Mitokondriyal sitokrom oksidaz I (COI) barkodlama geninin analizleri; moleküler yöntemlerden biri olarak thrips teşhislerinde yaygın olarak kullanılmaktadır. Ancak COI geninin fragman uzunluğu çok kısa olduğundan, PCR sonrası agaroz jel üzerinde fragman boyutlarını ayırt etmek çok zordur. Bu çalışmada, daha önce farklı araştırmacılar tarafından kullanılan primer çiftleri kullanılarak Thrips tabaci Lideman, Frankliniella occidentalis (Pergande) ve Frankliniella intonsa (Trybom) türleri için Kapiler Jel Elektroforez (CGE) sistemi entegre edilerek yeni bir tanımlama yöntemi geliştirilmiştir. Analiz, özellikle kısa parça uzunluklu COI geninde birbirine yakın parça uzunluklarının ayrılmasında hata payını en aza indirerek, elde edilmiş güçlü sinyaller üretir. Bu sebeple, jel elektroforezi adımı ortadan kaldırılarak, tehlikeli kimyasallara maruz kalmadan güvenilir tespitler elde edilmiştir. Yeni yöntem, tespit süresini kısaltmış ve düşük DNA konsantrasyonuna sahip tek bir thripsin saptanmasındaki işlem hatalarını da en aza indirmiştir. Kapiler jel elektroforezi tabanlı fragman analizi ile toplam 83 thrips bireyi (52 F. intonsa, 31 F. occidentalis) tespit edilebilmiştir. Yeni yöntem, üç farklı thrips türünün tespiti için benzersiz, spesifik ve hızlı olarak değerlendirilmektedir. Ayrıca yakın gelecekte kısa fragman boyutlarına sahip farklı thrips türlerinin tanımlanmasında da kullanılabileceği düşünülmektedir.

Keywords

Frankliniella intonsa, Frankliniella occidentalis, Mitokondriyal sitokrom oksidaz I, Multipleks polimeraz zincir reaksiyonu, Thysanoptera

Project Number

TAGEM-BS-14/09-02/02-08

References

• Balou MM., Tong X.L., Chen X.X., 2012 A new record and description of a new species of the genus thrips with an updated key to species from Iran. J Insect Sci 12:1–15. https://doi.org/10.1673/031.012.9001
• Bravo-Pérez D., Santillán-Galicia M.T., Johansen-Naime R.M., González-Hernández H., Segura-León O.L., Ochoa-Martínez D.L., Guzman-Valencia S., 2018. Species diversity of thrips (Thysanoptera) in selected avocado orchards from Mexico based on morphology and molecular data. Journal of integrative agriculture, 17(11), 2509-2517.
• Brunner P.C., Chatzivassiliou E.K., Katis N.I., Frey J.E. 2004., Host-associated genetic differentiation in Thrips tabaci (Insecta; Thysanoptera), as determined from mtDNA sequence data. Heredity. 93: 364-370. PMID: 15241445
• Danks H.V., 1988. Systematic in support of entomology. Annual Review of Entomology, 33, 217–296.
• EPPO., 2008. https://gd.eppo.int/taxon/THRIPL/distribution/TR Accessed 01 January 2021
• EPPO., 2015. https://gd.eppo.int/taxon/FRANOC/distribution/TR Accessed 01 January 2021
• Fekrat L., Zaki Aghl M., Tahan V., 2015. Application of the LAMP assay as a diagnostic technique for rapid identification of Thrips tabaci (Thysanoptera: Thripidae). Journal of economic entomology, 108(3), 1337-1343.
• Folmer O., Black M., Hoeh W., Lutz R., Vrijenhoek R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3, 294–299.
• Gariepy T.D., Kuhlmann U., Haye T., Gillott C., Erlandson M., 2005. A Single-Step Multiplex PCR Assay for the Detection of European Peristenus spp., Parasitoids of Lygus spp. Biocontrol. Sci. Technol., 15(5): 481- 495.
• Glover R.H., Collins D.W., Walsh K., Boonham N., 2010. Assessment of loci for DNA barcoding in the genus Thrips (Thysanoptera: Thripidae). Molecular Ecology Resources, 10(1), 51-59. doi: 10.1111/j.1755-0998.2009. 02723.x PMID: 21564990
• Haung C.G., Hsu J.C., Haymer D.S., Lin G.C. Wu W.J., 2009. Rapid Identification of the Medditerranean Fruit Fly (Diptera: Tephritidae) by Loop Mediated Isothermal Amplification. J. Econ. Entomol., 102(3): 1239-1246.
• Hebert, P.D.N., Cywinska A., Ball S.L., deWaard, J.R., 2003a. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London, Series B 270, 313–321.
• Hebert P.D.N., Ratnasingham S., deWaard J. R., 2003b. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London, Series B 270, S96–S99.
• Hebert P.D.N., Penton EH., Burns J.M., Janzen D.H., Hallwachs W., 2004b. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences of the United States of America 101, 14 812–14 817.
• Huang K., Lee S., Yeh Y., Shen G., Mei E., Chang C., 2010. Taqman Real-Time Quantitative PCR for Identification of Western Flower Thrips (Frankliniella occidentalis) for Plant Quarantine. Biol. Lett., 6(4): 555–557.
• Iftikhar R., Ashfaq M., Rasool A., Hebert P.D., 2016. DNA barcode analysis of thrips (Thysanoptera) diversity in Pakistan reveals cryptic species complexes. PLoS One, 11(1), e0146014.
• Jinbo U., Kato T., Ito M., 2011. Current progress in DNA barcoding and future implications for entomology. Entomological Science, 14(2), 107-124.
• Kadirvel P., Srinivasan R., Hsu Y.C., Su F.C., De La Pena R., 2013. Application of cytochrome oxidase I sequences for phylogenetic analysis and identification of thrips species occurring on vegetable crops, J Econ Entomol, 106 (1), 408- 418.
• Karakuş M., Yılmaz B., Özbel Y., Töz S., 2017. A new cost and time effective method for multilocus microsatellite typing (MLMT) studies: Application of Leishmania tropica isolates and clinical samples from Turkey. Journal of microbiological methods, 141, 97-100.
• Karimi J., Hassani-Kakhki M., Awal M.M., 2010. Identifying thrips (Insecta: Thysanoptera) using DNA barcodes. J Cell Mol Res. 2: 35–41.
• Kerékgyártó M., Németh N., Kerekes T., Rónai Z., Guttman A., 2013. Ultrafast haplotyping of putative microRNA-binding sites in the WFS1 gene by multiplex polymerase chain reaction and capillary gel electrophoresis. Journal of Chromatography A, 1286, 229-234.
• Leite L.A.R., 2012. Mitochondrial pseudogenes in insect DNA barcoding: differing points of view on the same issue, Biota Neotropica, 12 (3), 301-308.
• Lindeman K., 1889. The most damaging insects of tobacco in Bessarabia, Bulletin de la Société Imperial des Naturalistes de Moscou, 1., 15, 61-75.
• Mehle N., Trdan S., 2012. Traditional and modern methods for the identification of thrips (Thysanoptera) species. Journal of Pest Science, 85(2), 179-190.
• Marullo R., Mercati F., Vono G., 2020. DNA Barcoding: A Reliable Method for the Identification of Thrips Species (Thysanoptera, Thripidae) Collected on Sticky Traps in Onion Fields. Insects, 11(8), 489.
• Mound L.A., 2013. Homologies and host-plant specificity: Recurrent problems in the study of thrips. Florida Entomologist, 96, 318–322. Przybylska A., Fiedler Z., Kucharczyk H., Obrępalska-Stęplowska A., 2015. Detection of the quarantine species Thrips palmi by loop-mediated isothermal amplification. PLoS ONE, 10, e0122033.
• Przybylska A., Fiedler Z., Obrępalska-Stęplowska A., 2016. PCR-RFLP Method to Distinguish Frankliniella occidentalis, Frankliniella intonsa, Frankliniella pallida and Frankliniella tenuicornis. J. Plant Prot. Res., 56: 60-66.
• Rebijith K.B., Asokan R., Krishna V., Ranjitha H.H., Krishna Kumar N.K. Ramamurthy V.V., 2014. DNA Barcoding and Elucidation of Cryptic Diversity in Thrips (Thysanoptera), Florida Entomologist, 97 (4), 1328-1347.
• Rugman-Jones P.F., Hoddle M.S., Mound L.A., Stouthamer. R., 2006. Molecular identification key for pest species of Scirtothrips (Thysanoptera: Thripidae). Journal of Economic Entomology, 99, 1813 –1819.
• Rugman-Jones P.F., Hoddle M.S., Stouthamer R., 2010. Nuclear-mitochondrial barcoding exposes the global pest Western flower thrips (Thysanoptera: Thripidae) as two sympatric cryptic species in its native California. J Econ Entomol. 103: 877–886. PMID: 20568635
• Sabahi S., Fekrat L., Zakiaghl M., 2018. A simple and rapid molecular method for simultaneous identification of four economically important thrips species. J. Agr. Sci. Tech. Vol. 19: 1279-1290
• Saccaggi D.L., Kruger K., Pietersen G., 2008. A Multiplex PCR Assay for the Simultaneous Identification of Three Mealy Bug Species (Hemiptera: Pseudococcidae). Bull. Entomol. Res., 98: 27-33.
• Skarlinsky T., Funderburk J., 2016. A key to some Frankliniella (Thysanoptera: Thripidae) larvae found in Florida with descriptions of the first instar of selected species. Florida Entomologist, 99, 463–470.
• Toda S., Murai T., 2007. Phylogenetic analysis based on mitochondrial COI gene sequences in Thrips tabaci Lindeman (Thysanoptera: Thripidae) in relation to reproductive forms and geographic distribution. Appl Entomol Zool. 42: 309.
• Toda S., Hirose T., Kakiuchi K., Kodama H., Kijima K., Mochizuki M., 2013. Occurrence of a Novel Strain of Scirtothrips dorsalis (Thysanoptera: Thripidae) in Japan and Development of Its Molecular Diagnostics. Appl. Entomol. Zool., 49(2): 231-239.
• Xie Y., Mound L.A., Zhang H., 2019. A new species of Heliothrips (Thysanoptera, Panchaetothripinae), based on morphological and molecular data, Zootaxa, 4638 (1), 143-150.
• Yokoyama E., Kishida K., Uchimura M., Ichinphe S., 2006. Comparison between agarose gel electrophoresis and capillary electrophoresis for variable numbers of tandem repeat typing of Mycobacterium tuberculosis, Journal of Microbiological Methods, 65, 425-431
• Zhang D.X., Hewitt G.M., 1997. Assessment of the universality and utility of a set of conserved mitochondrial primers in insects. Insect Molecular Biology, 6, 143–150.
• Zhang G., Meng X., Min L., Qiao W., Wan F., 2012. Rapid Diagnosis of the Invasive Species, Frankliniella occidentalis (Pergande): A Species–Specific COI Marker. Journal of Applied Entomology, 136(6): 410–420.
• zur Strassen R., 2003. Die Terebranten Thysanopteren Europas. Verlag Goecke and Evers, Kentern, Germany pp 277.