European Journal of Plant Pathology

, Volume 153, Issue 2, pp 571–582 | Cite as

Non-destructive DNA extraction from aphids: the application in virus - vector studies of Banana bunchy top virus (BBTV)

  • N. Robbertse
  • B. A. Omondi
  • I. M. Millar
  • K. Krüger
  • A. E. C. JoosteEmail author


Banana bunchy top virus (BBTV), one of the most devastating viruses of banana, is transmitted by the banana aphid, Pentalonia nigronervosa. The high degree of morphological similarity between P. nigronervosa and the related experimental vector P. caladii makes it difficult to distinguish the two species. DNA barcoding can be used as an alternative for the rapid identification of these aphid species. However, standard molecular identification techniques for small insects usually require maceration of the sample to obtain sufficient amounts of DNA, resulting in the loss of voucher specimens. In this study, a non-destructive DNA extraction method for aphids was optimised, allowing the specimens to remain intact for use as vouchers for further morphological studies. Sufficient DNA concentrations were obtained from the aphid extractions and were used for species identification through direct sequencing of PCR products. Extracted DNA was further used to detect BBTV in the insect vector, P. nigronervosa. Approximately 78% of the aphids collected from symptomatic plants in infected plantations in KwaZulu-Natal tested positive for BBTV, as well as some aphids collected in other African countries. The BBTV viral strain identified from South Africa grouped with the “South Pacific” clade. This non-destructive DNA extraction method can be used in an early detection management strategy, especially in epidemiology and virus-vector studies of Banana bunchy top disease (BBTD).


Banana bunchy top virus KwaZulu-Natal Pentalonia nigronervosa Voucher specimens 



As authors we would like to thank our partners in the Learning Alliance Network, at the University of Kisangani, for collecting and donating samples from outside Kenya and Uganda. The Professional Development Programme (PDP, Agricultural Research Council) and the University of Pretoria is acknowledged for support of N. Robbertse during her studies. We thank Janine Snyman (University of Pretoria) for identification of aphids collected in traps.

Compliance with ethical standards

Conflict of interest

We hereby state that no conflict of interest are included in this manuscript and that no humans or animals were used in the study. All authors agreed to the publishing of this manuscript.


  1. Böhm, A., Bartel, D., Szucsich, N. U., & Pass, G. (2011). Confocal imaging of the exo-and endoskeleton of Protura after non-destructive DNA extraction. Soil Organisms, 83(3), 335–345.Google Scholar
  2. Castalanelli, M. A., Severtson, D. L., Brumley, C. J., Szito, A., Foottit, R. G., Grimm, M., Munyard, K., & Groth, D. M. (2010). A rapid non-destructive DNA extraction method for insects and other arthropods. Journal of Asia-Pacific Entomology, 13(3), 243–248.CrossRefGoogle Scholar
  3. Coquerel, C. (1859). Observations entomologiques sur divers insectes recueillis à Madagascar. Annales de la Société entomologique de France, 3(7), 239–260.Google Scholar
  4. Dale, J. L., Phillips, D. A., & Parry, J. N. (1986). Double-stranded RNA in banana plants with bunchy top disease. Journal of General Virology, 67(2), 371–375.CrossRefGoogle Scholar
  5. Dheepa, R., & Paranjothi, S. (2010). Transmission of cucumber mosaic virus (CMV) infecting banana by aphid and mechanical methods. Emirates Journal of Food and Agriculture, 22(2), 117.CrossRefGoogle Scholar
  6. 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(5), 294–299.Google Scholar
  7. Foottit, R. G., Maw, H. E. L., Pike, K. S., & Miller, R. H. (2010). The identity of Pentalonia nigronervosa Coquerel and P. Caladii van der Goot (Hemiptera: Aphididae) based on molecular and morphometric analysis. Zootaxa, 2358, 25–38.CrossRefGoogle Scholar
  8. Gondwe, W. T., Mwenebanda, B. M. L., Natha, E., & Mutale, P. (2007). Banana bunchy top disease in Mozambique and Zambia. Infomusa, 16(1/2), 38–39.Google Scholar
  9. Guindon, S., & Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology, 52(5), 696–704.CrossRefGoogle Scholar
  10. Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In Nucleic Acids Symposium Series (Vol. 41, pp. 95–98).Google Scholar
  11. Hu, J. S., Wang, M., Sether, D., Xie, W., & Leonhardt, K. W. (1996). Use of polymerase chain reaction (PCR) to study transmission of banana bunchy top virus by the banana aphid (Pentalonia nigronervosa). Annals of Applied Biology, 128(1), 55–64.CrossRefGoogle Scholar
  12. Hunter, S. J., Goodall, T. I., Walsh, K. A., Owen, R., & Day, J. C. (2008). Nondestructive DNA extraction from blackflies (Diptera: Simuliidae): Retaining voucher specimens for DNA barcoding projects. Molecular Ecology Resources, 8(1), 56–61.CrossRefGoogle Scholar
  13. Iskra-Caruana, M. L., Galzi, S., & Laboureau, N. (2008). A reliable IC one-step RT-PCR method for the detection of BBrMV to ensure safe exchange of Musa germplasm. Journal of Virological Methods, 153(2), 223–231.CrossRefGoogle Scholar
  14. Jinbo, U., Kato, T., & Ito, M. (2011). Current progress in DNA barcoding and future implications for entomology. Entomological Science, 14(2), 107–124.CrossRefGoogle Scholar
  15. Jooste, A. E. C., Wessels, N., & van der Merwe, M. (2016). First report of banana bunchy top virus in banana (Musa spp.) from South Africa. Plant Disease, 100(6), 1251.CrossRefGoogle Scholar
  16. Kumar, P. L., Hanna, R., Alabi, O. J., Soko, M. M., Oben, T. T., Vangu, G. H. P., & Naidu, R. A. (2011). Banana bunchy top virus in sub-Saharan Africa: Investigations on virus distribution and diversity. Virus Research, 159(2), 171–182.CrossRefGoogle Scholar
  17. Lee, W., Kim, H., Lim, J., Choi, H. R., Kim, Y., Kim, Y. S., et al. (2011). Barcoding aphids (Hemiptera: Aphididae) of the Korean peninsula: Updating the global data set. Molecular Ecology Resources, 11(1), 32–37.CrossRefGoogle Scholar
  18. Noordam, D. (2004). Aphids of java. Part V: Aphidini (Homoptera: Aphididae). Zoologische Verhandelingen.Google Scholar
  19. Pillay, M., Blomme, G., Rodrigues, E., & Ferreira, A. (2005). Presence of banana bunchy top virus in Angola. Infomusa, 14, 44–45.Google Scholar
  20. Posada, D. (2008). jModelTest: Phylogenetic model averaging. Molecular Biology and Evolution, 25(7), 1253–1256.CrossRefGoogle Scholar
  21. Ronquist, F., & Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19(12), 1572–1574.CrossRefGoogle Scholar
  22. Rowley, D. L., Coddington, J. A., Gates, M. W., Norrbom, A. L., Ochoa, R. A., Vandenberg, N. J., et al. (2007). Vouchering DNA-barcoded specimens: Test of a nondestructive extraction protocol for terrestrial arthropods. Molecular Ecology Notes, 7(6), 915–924.CrossRefGoogle Scholar
  23. Sambrook, J., & Russell, D. (2001). Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory.Google Scholar
  24. Savory, F., & Ramakrishnan, U. (2015). Cryptic diversity and habitat partitioning in an economically important aphid species complex. Infection, Genetics and Evolution, 30, 230–237.CrossRefGoogle Scholar
  25. Sethusa, M. T., Millar, I. M., Yessoufou, K., Jacobs, A., van der Bank, M., & van der Bank, H. (2014). DNA barcode efficacy for the identification of economically important scale insects (Hemiptera: Coccoidea) in South Africa. African Entomology, 22(2), 257–266.CrossRefGoogle Scholar
  26. Stern, D. L., & Foster, W. A. (1997). The evolution of sociality in aphids: A clone’s-eye view. In The evolution of social behavior in insects and arachnids (pp. 150–165). Cambridge: Cambridge University Press.CrossRefGoogle Scholar
  27. Thomas, J. E., Iskra-Caruana, M. L., & Jones, D. R. (1994). Banana bunchy top disease. Paper presented at the Musa disease fact sheet no. 4 (INIBAP), Montpellier, France.Google Scholar
  28. Thomson, D., & Dietzgen, R. G. (1995). Detection of DNA and RNA plant viruses by PCR and RT-PCR using a rapid virus release protocol without tissue homogenization. Journal of Virological Methods, 54(2), 85–95.CrossRefGoogle Scholar
  29. Van der Goot, P. (1917). A contribution to the knowledge of the aphids of java. Contrib. Faune Indes Neérl., 1(3), 1–301.Google Scholar
  30. Watanabe, S., Greenwell, A. M., & Bressan, A. (2013). Localization, concentration, and transmission efficiency of banana bunchy top virus in four asexual lineages of Pentalonia aphids. Viruses, 5(2), 758–776.CrossRefGoogle Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2019

Authors and Affiliations

  • N. Robbertse
    • 1
    • 2
  • B. A. Omondi
    • 3
  • I. M. Millar
    • 4
  • K. Krüger
    • 2
  • A. E. C. Jooste
    • 1
    • 5
    Email author
  1. 1.Plant Microbiology Division, Agricultural Research Council-Plant Protection Research (ARC-PPR)PretoriaSouth Africa
  2. 2.Department of Zoology and EntomologyUniversity of PretoriaPretoriaSouth Africa
  3. 3.Bioversity InternationalKampalaUganda
  4. 4.Biosystematics Division, Agricultural Research Council-Plant Protection Research (ARC-PPR)PretoriaSouth Africa
  5. 5.Crop Protection Division, Agricultural Research Council - Tropical and Subtropical Crops (ARC-TSC)NelspruitSouth Africa

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