Abstract
Twelve Heterodera species are considered of major economic significance in cereals, of which Heterodera avenae, H. latipons and H. filipjevi are the most important. Precise identification and quantification of these nematodes are necessary to develop effective integrated pest control. This study reports on the use of the mitochondrial cytochrome oxidase subunit 1 (COI) gene to develop qPCR assays that could be used for the identification and quantification of H. avenae and H. latipons. Two qPCR primer sets, each comprising two primers and a probe, were designed for each of both species. After optimization, the qPCR assays using a single second-stage juvenile (J2) were able to identify and quantify H. avenae and H. latipons. Their specificity was confirmed by the lack of amplification of J2 of 14 other Heterodera species. A qPCR using DNA extracted from 120 J2 + eggs of H. avenae and H. latipons resulted in steady Ct-values (Ct = 22.33 ± 0.1 and Ct = 21.83 ± 0.12, respectively). Dilution series of DNA extracted from 120 J2 + eggs of the two species were made. The assays for both species resulted in a standard curve showing a highly significant linearity between the Ct-values and the dilution rates (R2 = 0.99; slope = −3.03 and R2 = 0.99; slope = −3.28 for H. avenae and H. latipons, respectively). The two qPCR assays provide a sensitive and valid tool for rapid detection and quantification of the two species whether they occur alone or in mixtures with other species.
Similar content being viewed by others
References
Abidou, H., El-Ahmed, A., Nicol, J. M., Bolat, N., Rivoal, R., & Yahyaoui, A. (2005). Occurrence and distribution of species of the Heterodera avenae group in Syria and Turkey. Nematologia Mediterranea, 33, 195–201.
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.
Brown, R. H. (1985). The selection of management strategies for controlling nematodes in cereals. Agriculture, Ecosystems and Environment, 12, 371–388.
Bustin, A. S., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J., & Wittwer, C. T. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. The American Association for Clinical Chemistry, 55, 611–612.
Christoforou, M., Pantelides, I. S., Kanetis, L., Ioannou, N., & Tsaltas, D. (2014). Rapid detection and quantification of viable potato cyst nematodes using qPCR in combination with propidium monoazide. Plant Pathology, 63, 1185–1192.
Cunha, A., Azevedo, R. B. R., Emmons, S. W., & Leroi, A. M. (1999). Developmental biology-Variable cell number in nematodes. Nature, 402, 253–253.
Dababat, A. A., Imren, M., Orakci, G. E., Ashrafi, S., Yavuzaslanoglu, E., Toktay, H., Pariyar, S. R., Elekcioglu, H. I., Morgounov, A., & Mekete, T. (2014). The importance and management strategies of cereal cyst nematodes, Heterodera spp., in Turkey. Euphytica, 202, 173–188.
Ferris, V. R., Subbotin, S. A., Ireholm, A., Spiegel, Y., Faghhihi, J., & Ferris, J. (1999). Ribosomal DNA sequence analysis of Heterodera filipjevi and H. latipons isolates from Russia and comparisons with other nematode isolates. Russian Journal of Nematology, 7, 121–125.
Fusco, V., Quero, G. M., Morea, M., Blaiotta, G., & Visconti, A. (2011). Rapid and reliable identification of Staphylococcus aureus harbouring the enterotoxin gene cluster (egc) and quantitative detection in raw milk by real time PCR. International Journal of Food Microbiology, 144, 528–537.
Goto, K., Sato, E., & Toyota, K. (2009). A novel detection method for the soybean cyst nematode Heterodera glycines Ichinohe using soil compaction and real-time PCR. Nematological Research, 39, 1–7.
Hillis, D. M., & Dixon, M. T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. The Quarterly Review of Biology, 66, 411–453.
Holterman, M., van der Wurff, A., van den Elsen, S., van Megen, H., Bongers, T., Holovachov, O., Bakker, J., & Helder, J. (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.
Imren, M., Waeyenberge, L., Viaene, N., Toktay, H., Dababat, A., & Elekcioğlu, İ. H. (2012). Molecular characterization of cereal cyst nematodes from South Anatolian region in Turkey using ITS-rDNA sequences. Turkey Journal of Entomology, 36, 491–499.
Kingsnorth, C. S., Kingsnorth, A. J., Lyons, P. A., Chwaesczynska, D. M., & Asher, M. J. C. (2003). Real-time analysis of Polymyxa beta GST expression in infected sugar beet. Molecular Plant Pathology, 4, 171–176.
Madani, M., Vovlas, N., Castillo, P., Subbotin, S. A., & Moens, M. (2004). Molecular characterization of cyst nematode species (Heterodera spp.) from the Mediterranean Basin using RFLPs and sequences of ITS-rDNA. Journal of Phytopathology, 152, 229–234.
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.
McDonald, A. H., & Nicol, J. M. (2005). Nematode parasites of cereals. In M. Luc, R. A. Sikora, & J. Bridge (Eds.), Plant-parasitic nematodes in subtropical and tropical agriculture (pp. 131–191). Wallingford: CAB International.
Meagher, J. W. (1977). World dissemination of the cereal-cyst nematode (Heterodera avenae) and its potential as a pathogen of wheat. Journal of Nematology, 9, 9–15.
Mokrini, F., Waeyenberge, L., Viaene, N., Andaloussi, F. A., & Moens, M. (2014). The β-1,4-endoglucanase gene is suitable for the molecular quantification of the root-lesion nematode, Pratylenchus thornei. Nematology, 16, 789–796.
Nakhla, M. K., Owens, K. J., Li, W., Wei, G., Skantar, A. M., & Levy, L. (2010). Multiplex real-time PCR assays for the identification of the potato cyst and tobacco cyst nematodes. Plant Disease, 94, 959–965.
Nicol, J.M. (2002). Important nematode pests. In: Curtis, B.C., Rajaram, S. & Macpherson, H.G. (Eds). Bread wheat improvement and production. FAO, Rome, Italy, Plant Production and Protection Series 30, pp. 345–366.
Nicol, J. M., & Rivoal, R. (2008). Global knowledge and its application for the integrated control and management of nematodes on wheat. In A. Ciancio & K. G. Mukerji (Eds.), Integrated management and biocontrol of vegetable and grain crops nematodes (Vol. 2, pp. 243–287). Dordrecht: Springer.
Oberhänsli, T., Altenbach, D., & Bitterlin, W. (2011). Development of a duplex TaqMan real-time PCR for the general detection of phytoplasmas and 18S rRNA host genes in fruit trees and other plants. Bulletin of Insectology, 64, 37–38.
Peng, H., Qi, X., Peng, D., Long, H., He, X., Huang, W., & He, W. (2013). Sensitive and direct detection of Heterodera filipjevi in soil and wheat roots by species-specific SCAR-PCR assays. Plant Disease, 97, 1288–1294.
QI, X.l., Peng, D.L., Peng, H., Long, H.B., Huang, W.K. & He, W.T. (2012). Rapid, Molecular Diagnosis Based on SCAR Marker System for Cereal Cyst Nematode. Science Agriculture Sinica 45, 4388–4395.
Rasmussen, R., Morrison, T., Herrmann, M. & Wittwe, C. (1998). Quantitative PCR by continuous fluorescence monitoring of a double strand DNA specific binding dye. Biochemica, 2, 8–11.
Rivoal, R., & Cook, R. (1993). Nematode pests of cereals. In K. Evans, D. L. Trudgill, & J. M. Webster (Eds.), Plant-parasitic nematodes in temperate agriculture (pp. 259–303). Wallingford: CAB International.
Rivoal, R., Valette, S., Bekal, S., Gauthier, J. B., & Yahyaoui, A. (2003). Genetic and phenotypic diversity in the graminaceous cyst nematode complex, inferred from PCR-RFLP of ribosomal DNA and morphometric analysis. European Journal of Plant Pathology, 109, 227–241.
Sahin, E., Nicol, J.M., Elekçioğlu, I.H., Yorgancilar, O., Yildirim, A.F., Tülek, A., Hekimhan, H., Yorgancilar, A., Kilinç, A.T., Bolat, N. & Erginbaş-Orakci, G. (2009). Frequency and diversity of cereal Nematodes on the central Anatolian Plateau of Turkey. Proceedings of the First Workshop of the International Cereal Cyst Nematode Initiative, 21–23 October 2009 (pp. 100–105). Antalya, Turkey: CIMMYT.
Sato, E., Min, Y. Y., Shirakashi, T., Wada, S., & Toyota, K. (2007). Detection of the root-lesion nematode, Pratylenchus penetrans (Cobb), in a nematode community using real-time PCR. Japanese Journal of Nematology, 37, 87–92.
Scholz, U. (2001). Biology, pathogenity and control of cereal cyst nematode Heterodera latipons Franklin on wheat and barley under semiarid conditions and interactions with common root rot Bipolaris sorokiniana (Sacc.) Shoemaker [teleomorph: Cochliobolus sativus (Ito et Kurib.). PhD, University of Bonn, Bonn, Germany.
Smiley, R. W., & Nicol, J. M. (2009). Nematodes which challenge global wheat production. In B. F. Carver (Ed.), Wheat science and trade (pp. 171–187). Wiley-Blackwell: Ames.
Subbotin, S. A., Mundo-Ocampo, M., & Baldwin, J. G. (2010). Description and diagnosis of Heterodera species. In D. J. Hunt & R. N. Perry (Eds.), Systematics of Cyst Nematodes (Nematoda: Heteroderinae) (pp. 35–449). Leiden: Brill.
Toumi, F., Hassan, G., Waeyenberge, L., Viaene, N., Dababat, A., Nicol, M. J., Ogbonnaya, C.F., Al-Assas, K., Abou Al-Fadil, T. & Moens, M. (2012). Distribution of cereal cyst nematodes (Heterodera spp.) in wheat and barley fields in northeastern regions of Syria. Proceedings of the third workshop of the international cereal cyst nematode initiative, 22–23 October 2012 (pp. 310), Adana, Turkey: CIMMYT.
Toumi, F., Waeyenberge, L., Viaene, N., Dababat, A., Nicol, J. M., Ogbonnaya, F., & Moens, M. (2013a). Development of a species-specific PCR to detect the cereal cyst nematode Heterodera latipons. Nematology, 15, 709–717.
Toumi, F., Waeyenberge, L., Viaene, N., Dababat, A., Nicol, J. M., Ogbonnaya, F., & Moens, M. (2013b). Development of two species-specific primer sets to detect the cereal cyst nematodes Heterodera avenae and Heterodera filipjevi. European Journal of Plant Pathology, 136, 613–624.
Toyota, K., Shirakashia, T., Satoa, E., Wadaa, S., & Mina, Y. Y. (2010). 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.
Waeyenberge, L., Viaene, N., Subbotin, S.A. & Moens, M. (2009). Molecular identification of Heterodera spp., an overview of fifteen years of research. Proceedings of the first workshop of the international cereal cyst nematode initiative, 21–23 October 2009 (pp. 109–114), Antalya, Turkey: CIMMYT.
Wang, A. L., Hou, S. Y., Peng, D. L., Zhang, G., Huang, W. K., & Yao, Q. (2010). Studies on occurrence and distribution of cereal cyst nematodes of wheat in Qinghai province. Journal of Anhui Agricultural Science, 38, 13231–13233.
Yan, G. P., & Smiley, R. W. (2009). Distinguishing Heterodera filipjevi and H. avenae using polymerase chain reaction-restriction fragment length polymorphism and cyst morphology. Phytopathology, 100, 216–2.
Yan, L., Gary, W. L., Shien, L., Clarissa, B., & Vincent, P. K. (2014). Quantitative field testing Heterodera glycines from metagenomic DNA samples isolated directly from soil under agronomic production. PLoS ONE, 9, e89887.
Yavuzaslanoglu, E., Elekçioğlu, H., Nicol, J. M., Yorgancilar, O., Hodson, D., Yildirim, F. A., Yorgancilar, A., & Bolat, B. (2012). Distribution, frequency and occurrence of cereal nematodes on the Central Anatolian Plateau in Turkey and their relationship with soil physicochemical properties. Nematology, 14, 839–854.
Acknowledgments
The first author appreciates Monsanto’s Beachell-Borlaug International Scholars Program – MBBISP for the financial support for his PhD research. The authors thank the suppliers of the Heterodera samples, i.e., Drs L. Al-Banna, M. Christoforou, A. Dawabah, S. Hajjar, J. Hallmann, G. Hassan, M. Imren, N. Kachouri, G. Karssen, S. Kornobis, F. Mokrini, D. Peng, R. Rivoal, D. Saglam, R. Smiley, N. Grujić and Z. Tanha Maafi.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Toumi, F., Waeyenberge, L., Viaene, N. et al. Development of qPCR assays for quantitative detection of Heterodera avenae and H. latipons . Eur J Plant Pathol 143, 305–316 (2015). https://doi.org/10.1007/s10658-015-0681-0
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10658-015-0681-0