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Variable ITS-copy number at different developmental stages of Meloidogyne hapla and M. chitwoodi

  • Everaldo Antônio LopesEmail author
  • David Mark Roberts
  • Vivian Carol Blok
Article
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Abstract

Quantitative polymerase chain reaction (qPCR) can be used for the accurate quantification of plant-parasitic nematodes from soil samples. Unlike the traditional Baermann funnel and centrifugal flotation techniques followed by visual enumeration, all developmental stages of plant-parasitic nematodes can be detected and quantified by using qPCR. However, little is known about the amount of DNA between different stages of plant-parasitic nematodes. Here, we show that ITS-copy number varies with developmental stage of Meloidogyne chitwoodi and M. hapla. The number of copies of ITS was lower in cell-stage eggs (51 ± 7.4 for M. hapla and 31 ± 6.0 for M. chitwoodi) than in second-stage juveniles (1345 ± 130.6 for M. hapla and 1036 ± 140.3 for M. chitwoodi) and females (19,508 ± 3367.3 for M. hapla and 9049 ± 316.7 for M. chitwoodi).

Keywords

Number of copies qPCR Quantification Real-time PCR Root-knot nematode 

Notes

Acknowledgments

The authors are grateful for FERA (The Food and Environment Research Agency, United Kingdom) for providing nematode inoculum. E.A. Lopes thanks CNPq (Proc. 304663/2014-0) and The James Hutton Institute for Post-Doctoral Training.

Funding

This study was funded by CNPq (PDE 233650/2014–8) and the Rural and Environment Science and Analytical Services (RESAS) Division of the Scottish Government.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. Berry, S. D., Fargette, M., Spaull, V. W., Morand, S., & Cadet, P. (2008). Detection and quantification of root-knot nematode (Meloidogyne javanica), lesion nematode (Pratylenchus zeae) and dagger nematode (Xiphinema elongatum) parasites of sugarcane using real-time PCR. Molecular and Cellular Probes, 22, 168–176.CrossRefGoogle Scholar
  2. CABI (2018). Invasive species. Retrieved March 1, 2018, from http://www.cabi.org/isc. Accessed 1 March 2018.
  3. Chan, Y. L., Cai, D., Taylor, P. W. J., Chan, M. T., & Yeh, K. W. (2010). Adverse effect of the chitinolytic enzyme PjCHI-1 in transgenic tomato on egg mass production and embryonic development of Meloidogyne incognita. Plant Pathology, 59, 922–930.CrossRefGoogle Scholar
  4. Cunha, A., Azevedo, R. B. R., Emmons, S. W., & Leroi, A. M. (1999). Variable cell number in nematodes. Nature, 402, 253.CrossRefGoogle Scholar
  5. De Haan, E. G., Dekker, C. C. E. M., Tameling, W. I. L., Den Nijs, L. J. M. F., Van Den Bovenkamp, G. W., & Kooman-Gersmann, M. (2014). The MeloTuber test: A real-time TaqMan PCR-based assay to detect the root-knot nematodes Meloidogyne chitwoodi and M. fallax directly in potato tubers. Bulletin OEPP/EPPO, 44, 166–175.CrossRefGoogle Scholar
  6. De Weerdt, M., Kox, L., Waeyenberge, L., Viaene, N., & Zijlstra, C. (2011). A real-time PCR assay to identify Meloidogyne minor. Journal of Phytopathology, 159, 80–84.CrossRefGoogle Scholar
  7. Evans, A. A. F., & Perry, R. (2009). Survival mechanisms. In R. N. Perry, M. Moens, & J. L. Starr (Eds.), Root-knot nematodes (pp. 201–222). Wallingford: CABI Publishing.CrossRefGoogle Scholar
  8. Ferris, V. R., Ferris, J. M., & Faghihi, J. (1993). Variation in spacer ribosomal DNA in some cyst-forming species of plant parasitic nematodes. Fundamental and Applied Nematology, 16, 177–184.Google Scholar
  9. Goto, K., Sato, E., Gang, L. F., Toyota, K., & Sugito, T. (2010). Comparison of calibration curves prepared by soil compaction and ball milling methods for direct quantification of the potato cyst nematode Globodera rostochiensis in soil. Nematological Research, 40, 41–45.CrossRefGoogle Scholar
  10. Greco, N., & Di Vito, M. (2009). Population dynamics and damage levels. In R. N. Perry, M. Moens, & J. L. Starr (Eds.), Root-knot nematodes (pp. 246–274). Wallingford: CABI Publishing.CrossRefGoogle Scholar
  11. Gugino, B. K., Abawi, G. S., & Ludwig, J. W. (2006). Damage and management of Meloidogyne hapla using oxamyl on carrot in New York. Journal of Nematology, 38, 483–490.Google Scholar
  12. Holeva, R., Phillips, M. S., Neilson, R., Brown, D. J. F., Young, V., Boutsika, K., & Blok, V. C. (2006). Real-time PCR detection and quantification of vector trichodorid nematodes and Tobacco rattle virus. Molecular and Cellular Probes, 20, 203–211.CrossRefGoogle Scholar
  13. Hugall, A., Stanton, J., & Moritz, C. (1999). Reticulate evaluation and the origins of ribosomal internal transcribed spacer diversity in apomictic Meloidogyne. Molecular Biology and Evolution, 16, 157–164.CrossRefGoogle Scholar
  14. Jesus, D. S., Oliveira, C. M. G., Roberts, D., Blok, V., Neilson, R., Prior, T., Balbino, H. M., MacKenzie, K. M., & Oliveira, R. D. L. (2016). Morphological and molecular chracterisation of Aphelenchoides besseyi and A. fujianensis (Nematoda: Aphelenchoididae) from rice and forage grass seeds in Brazil. Nematology, 18, 337–356.CrossRefGoogle Scholar
  15. Jones, J. T., Haegeman, A., Danchin, E. G., Gaur, H. S., Helder, J., Jones, M. G., Kikuchi, T., Manzanilla-López, R., Palomares-Rius, J. E., Wesemael, W. M., & Perry, R. N. (2013). Top 10 plant-parasitic nematodes in molecular plant pathology. Molecular Plant Pathology, 14, 946–961.CrossRefGoogle Scholar
  16. Karssen, G., Wesemael, W., & Moens, M. (2013). Root-knot nematodes. In R. N. Perry, M. Moens, & J. L. Starr (Eds.), Root-knot nematodes (pp. 73–108). Wallingford: CABI Publishing.Google Scholar
  17. Manzanilla-López, R. H. (2012). Methodology and symptomatology. In R. H. Manzanilla-López & N. Marban-Mendoza (Eds.), Practical plant nematology (pp. 89–129). Montecillo: Biblioteca Basica de Agricultura.Google Scholar
  18. Min, Y. Y., Toyota, K., Goto, K., Sato, E., Mizuguchi, S., Abe, N., Nakano, A., & Sawada, E. (2011). Development of a direct quantitative detection method for Meloidogyne incognita in sandy soils and its application to sweet potato cultivated fields in Tokushima prefecture, Japan. Nematology, 13, 95–102.CrossRefGoogle Scholar
  19. Oliveira, C. M. G., Blok, V., Neilson, R., Mróz, T., & Roberts, D. (2017). Hydrolysis probe-based PCR for detection of Pratylenchus crenatus, P. neglectus and P. penetrans. Nematology, 19, 81–91.CrossRefGoogle Scholar
  20. Onkendi, E. M., Kariuki, G. M., Marais, M., & Moleleki, L. N. (2014). The threat of root-knot nematodes (Meloidogyne spp.) in Africa: A review. Plant Pathology, 63, 727–737.CrossRefGoogle Scholar
  21. Raymaekers, M., Smets, R., Maes, B., & Cartuyvels, R. (2009). Checklist for optimization and validation of real-time PCR assays. Journal of Clinical Laboratory Analysis, 23, 145–151.CrossRefGoogle Scholar
  22. Rusin, L. Y., & Malakhov, V. V. (1998). Free-living marine nematodes possess no eutely. Doklady Biological Sciences, 361, 331–333.Google Scholar
  23. Sapkota, R., Skantar, A. M., & Nicolaisen, M. (2016). A TaqMan real-time PCR assay for detection of Meloidogyne hapla in root galls and in soil. Nematology, 18, 147–154.CrossRefGoogle Scholar
  24. Schena, L., Nicosia, M. G. L. D., Sanzani, S. M., Faedda, R., Ippolito, A., & Cacciola, S. O. (2013). Development of quantitative PCR detection methods for phytopathogenic fungi and oomycetes. Journal of Plant Pathology, 95, 7–24.Google Scholar
  25. Toyota, K., Shirakashi, T., Sato, E., Wada, S., & Min, Y. Y. (2008). Development of a real-time PCR method for the potato-cyst nematode Globodera rostochiensis and the root-knot nematode Meloidogyne incognita. Soil Science & Plant Nutrition, 54, 72–76.CrossRefGoogle Scholar
  26. Van Ghelder, C., Reid, A., Kenyon, D., & Esmenjaud, D. (2015). Development of a real time PCR method for the detection of the dagger nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi and X. italiae, and for the quantification of X. index numbers. Plant Pathology, 64, 489–500.CrossRefGoogle Scholar
  27. Viaene, N. M., & Abawi, G. S. (1996). Damage threshold of Meloidogyne hapla to lettuce in organic soil. Journal of Nematology, 28, 537–545.Google Scholar
  28. Wesemael, W. M. L., & Moens, M. (2008). Quality damage on carrots (Daucus carota L.) caused by the root-knot nematode Meloidogyne chitwoodi. Nematology, 10, 261–270.CrossRefGoogle Scholar
  29. Wesemael, W. M. L., Viaene, N., & Moens, M. (2011). Root-knot nematodes (Meloidogyne spp.) in Europe. Nematology, 13, 3–16.CrossRefGoogle Scholar
  30. Yan, G., Smiley, R. W., Okubara, P. A., Skantar, A. M., & Reardon, C. L. (2013). Developing a real-time PCR assay for detection and quantification of Pratylenchus neglectus in soil. Plant Disease, 97, 757–764.CrossRefGoogle Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2019

Authors and Affiliations

  1. 1.Universidade Federal de ViçosaRio ParanaíbaBrazil
  2. 2.The James Hutton InstituteDundeeUK

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