Pratylenchus penetrans is one of the most economically damaging plant-parasitic nematodes and is found on a wide variety of crops. Correct identification and quantification of this nematode are necessary for providing advice to farmers, but are not easily obtained with the traditional way of microscopic observation. We developed a qPCR assay to detect and quantify P. penetrans in a short but accurate manner. A qPCR primer set, including two primers and a TaqMan probe, was designed based on the sequence of the β-1,4-endoglucanase gene. The assay was optimized by using the primers in a qPCR assay with SYBR green I dye and setting the qPCR program to different annealing temperatures ranging from 60 °C to 64 °C. Based on the Ct-values, we retained the program with an annealing temperature of 63 °C. The assay with the probe was very sensitive as it was able to detect a single individual of P. penetrans, even when mixed with up to 80 individuals of P. thornei. The specificity of the reaction was confirmed by the lack of amplification of DNA from 28 populations of 18 other Pratylenchus species and from plant-parasitic nematodes from nine other genera. DNA from 21 different isolates from P. penetrans was amplified. DNA extraction from 80 individuals and quantification by qPCR was repeated four times; Ct-values showed consistent results (Ct = 24.4 ± 0.4). A dilution series from DNA of P. penetrans resulted in a standard curve showing a highly significant linearity between the Ct-values and the dilution rates (R2 = 0.99; slope = −3.23; E = 104 %). The tests showed a high correlation between the real numbers of nematodes and the numbers detected by the qPCR. The developed qPCR assay provides a sensitive means for the rapid detection and reliable quantification of individuals of this pest. This method does not require expertise in nematode taxonomy and morphology, and can be used as a rapid diagnostic tool in research, as well as in diagnostic labs and extension services advising farmers for pest management.
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Al-Banna, L., Ploeg, A. T., Williamson, V. M., & Kaloshian, I. (2004). Discrimination of six Pratylenchus species using PCR and species-specific primers. Journal of Nematology, 36, 142–146.
Ball-Coelho, B., Bruin, A. J., Roy, R. C., & Riga, E. (2003). Forage pearl millet and marigold as rotation crops for biological control of root-lesion nematodes in potato. Agronomy Journal, 95, 282–292.
Belair, G., Dauphinais, N., Fournier, Y., Dangi, O. P., & Clement, M. F. (2005). Effect of forage and grain pearl millet on Pratylenchus penetrans and potato yields in Quebec. Journal of Nematology, 37, 78–82.
Bernard, E. C., & Laughlin, C. W. (1976). Relative susceptibility of selected cultivars of potato to Pratylenchus penetrans. Journal of Nematology, 8, 839–842.
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.
Castillo, P., & Vovlas, N. (2007). Pratylenchus (Nematoda: Pratylenchidae): diagnosis, biology, pathogenicity and management. Nematology Monographs and Perspectives 6, Brill, 530 pp.
Cunha, A., Azevedo, R. B. R., Emmons, S. W., & Leroi, A. M. (1999). Developmental biology - Variable cell number in nematodes. Nature, 402, 253.
De Luca, F., Fanelli, E., Di Vito, M., Reyes, A., & De Giorgi, C. (2004). Comparison of the sequences of the D3 expansion of the 26S ribosomal genes reveals different degrees of heterogeneity in different populations and species of Pratylenchus from the Mediterranean region. European Journal of Plant Pathology, 111, 949–957.
Green, C. D., & Verdejo, S. (1985). Colonisation of Pisum sativum and Phaeseolus vulgaris by Pratylenchus penetrans in sterile media. Nematologica, 31, 451–457.
Holgado, R., Skau, K. A. O., & Magnusson, C. (2009). Field damage in potato by lesion nematode Pratylenchus penetrans, its association with tuber symptoms and its survival in storage. Nematologia Mediterranea, 37, 25–29.
Holterman, M., van der Wurff, A., van den Elsen, S., van Megen, H., Bongers, T., Holovachov, O., et al. (2006). Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Molecular Biology and Evolution, 23, 1792–1800.
Kingsnorth, C. S., Kingsnorth, A. J., Lyons, P. A., Chwaesczynska, D. M., & Asher, M. J. C. (2003). Real-time analysis pf Polymyxa beta GST expression in infected sugar beet. Molecular Plant Pathology, 4, 171–176.
Loof, P. A. A. (1991). The family Pratylenchidae Thorne, 1949. In W. R. Nickle (Ed.), Manuel of agricultural nematology (pp. 363–421). NY, USA, Marcel Dekker: New York.
Luc, M. (1987). A reapparaisal of Tylenchina (Nemata). 7. The family Pratylenchidae Thorne, 1949. Revue de Nématologie, 10, 203–218.
Madani, M., Subbotin, S. A., & Moens, M. (2005). Quantitative detection of the potato cyst nematode, Globodera pallida, and the beet cyst nematode, Heterodera schachtii, using Real-Time PCR with SYBR green I dye. Molecular and Cellular Probes, 19, 81–86.
Meskine, M., & Abbad Andaloussi, F. (1992). Importance et répartition des nématodes phytoparasites associés aux cultures de blé et orge au Maroc. Al Awamia, 80, 46–51.
Mokrini, F., Abbad Andaloussi, F., Alaoui, Y., & Troccoli, A. (2009). Importance and distribution of the main cereal nematodes in Morocco. In: Cereal Cyst Nematodes: Status, Research and Outlook. The first of the International Cereal Cyst Nematode Initiative. 21–23 October, Antalya, Turkey, 45–50.
Mokrini, F., Waeyenberge, L., Viaene, N., & Moens, M. (2012). Occurence of nematodes of the Heterodera avenae group and Pratylenchus spp. on wheat and barley in Morocco. (Abstr.) Proceedings of the 31st International Symposium of the European Society of Nematologists Adana, Turkey.
Olthof, T. H. A. (1986). Reaction of six Solanum tuberosum cultivars to Pratylenchus penetrans. Journal of Nematology, 18, 54–58.
Olthof, T. H. A. (1987). Effects of fumigants and systemic pesticides on Pratylenchus penetrans and potato yield. Journal of Nematology, 19, 424–430.
Olthof, T. H. A. (1989). Effects of fumigants and non-fumigants nematicides on Pratylenchus penetrans and yield of potato. Supplement to the Journal of Nematology, 21, 645–649.
Olthof, T. H. A., & Potter, J. W. (1973). The relationship between populations densities of Pratylenchus penetrans and crop losses in summer-maturing vegetables in Ontario. Phytopathology, 63, 577–582.
Orui, Y. (1996). Discrimination of the main Pratylenchus species in Japan by PCR-RFLP analysis. Applied Entomology and Zoology, 31, 505–514.
Pudasaini, M. P., Viaene, N., & Moens, M. (2007). The influence of host and temperature on the vertical migration of Pratylenchus penetrans. Nematology, 9, 439–449.
Sato, E., Min, Y. Y., Toyota, K., & Takada, A. (2009). Relationships between the damage to radish caused by the root-lesion nematode Pratylenchus penetrans, its density prior to cultivation and the soil nematode community structure evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis. Soil Science and Plant Nutrition, 55, 478–484.
Seinhorst, J. W. (1998). The common relation between population density and plant weight in pot and microplot experiments with various nematode plant combinations. Fundamental and Applied Nematology, 21, 459–468.
Subbotin, S. A., & Moens, M. (2006). Molecular taxonomy and phylogeny. In R. N. Perry & M. Moens (Eds.), Plant nematology (pp. 33–58). Wallingford: CABI Publishing.
Subbotin, S. A., Sturhan, D., Chizhov, V. N., Vovlas, N., & Baldwin, J. G. (2006). Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3 expansion fragments of the 28S rRNA gene sequences. Nematology, 8, 455–474.
Subbotin, S. A., Ragsdale, E. J., Mullens, T., Roberts, P. A., Mundo-Ocampo, M., & Baldwin, J. G. A. (2008). Phylogenetic framework for root lesion nematodes of the genus Pratylenchus (Nematoda): evidence from 18S and D2-D3 expansion segments of 28S ribosomal RNA genes and morphological characters. Molecular Phylogenetics and Evolution, 48, 491–505.
Talavera, M., Itou, K., & Mizukubo, T. (2001). Reduction of nematode damage by root colonization with arbuscular (Glomus spp.) in tomato Meloidogyne incognita (Tylenchida: Meloidogynidae) and carrot Pratylenchus penetrans pathosystems. Applied Entomology and Zoology, 36, 387–392.
Toyota, J. B., Shirakashi, T., Sato, E., & Wada, S. (2008). Development of a real-time PCR method for the potato-cyst nematode Globodera rostochiensis and the root-knot nematode Meloidogyne incognita. Journal of Plant Nutrition and Soil Science, 54, 72–76.
Troccoli, A., Lamberti, F., & Greco, N. (1992). Pratylenchus species occurring in Algeria (Nematoda, Pratylenchidae). Nematologica Mediterranea, 20, 97–103.
Uehara, T., Mizukubo, T., Kushida, A., & Momota, Y. (1998). Identification of Pratylenchus penetrans (Cobb) by PCR using ITS-based species-specific primers. Japanese Journal of Nematology, 28, 1–7.
Uehara, T., Kushida, A., & Momota, Y. (1999). Rapid and sensitive identification of Pratylenchus spp. using reverse dot blot hybridization. Nematology, 1, 549–555.
Waeyenberge, L., Moens, M., Pinochet, J., & Vrain, T. C. (2000). Molecular characterization of Pratylenchus species using r DNA restriction fragment length polymorphisms. Nematology, 2, 135–142.
Waeyenberge, L., Viaene, N., & Moens, M. (2009). Species-specific duplex PCR for the detection of Pratylenchus penetrans. Nematology, 11, 847–857.
Yan, G., Smiley, R. W., & Okubara, P. A. (2012). Detection and quantification of Pratylenchus thornei in DNA extracted from soil Using Real-Time PCR. Phytopathology, 102, 14–22.
The first author thanks the awarding of a PhD scholarship from the Islamic Development Bank (IDB). The authors wish to warmly thank Ms Nancy de Sutter for maintaining the Pratylenchus populations. We also express our gratitude to Mr. Rachid Tahzima for his valuable comments on this manuscript. We finally thank Dr. Benaouda Hassan, Director of INRA Kenitra (Morocco), and Mr. Tahiri sidi Mohamed, for the logistic assistance during the field sampling.
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Mokrini, F., Waeyenberge, L., Viaene, N. et al. Quantitative detection of the root-lesion nematode, Pratylenchus penetrans, using qPCR. Eur J Plant Pathol 137, 403–413 (2013). https://doi.org/10.1007/s10658-013-0252-1
- β-1,4-endoglucanase gene