Abstract
Plant-parasitic nematodes can cause significant damage to agricultural crops and forests worldwide, resulting in major economic losses. Some nematode species do not occur in all areas and are regulated as quarantine organisms. To avoid introduction and spread of these organisms, fast, simple and reliable detection and identification methods are needed, that help plant diagnostic services such as reference centres or national plant protection organizations (NPPOs) to rapidly identify suspicious nematodes. Real-time PCR is one of the fastest, most sensitive and reliable methods to fulfil this task. It is a DNA-based method that is easy to learn with the only requirement of having a specific thermocycler (Real-time Platform) and the appropriate chemistry. Real-time PCR provides very sensitive detection and species-specific identification with the potential to quantify target organisms if required. Following DNA extraction, results can be seen in 1–3 h and management decisions applied. Real-time PCR can be used for high-throughput analysis of many samples and in some cases for multiplexing, allowing for identification of more than one species in a single reaction. Over the past 15 years, real-time PCR methods have been developed for the main plant-parasitic nematodes, in particular the regulated species. This paper reviews the achievements in plant nematology diagnostics using real-time PCR as the method of choice for fast and reliable detection, identification and even quantification of plant parasitic nematodes.
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References
Adam, M. A. M., Phillips, M. S., & Blok, V. C. (2007). Molecular diagnostic key for identification of single juveniles of seven common and economically important species of root-knot nematode (Meloidogyne spp.). Plant Pathology, 56, 190–197.
Agudelo, P., Lewis, S. A., & Fortnum, B. A. (2011). Validation of a real-time polymerase chain reaction assay for the identification of Meloidogyne arenaria. Plant Disease, 95, 835–838.
Ahmed, M., Sapp, M., Prior, T., Karssen, G., & Back, M. A. (2016). Technological advancements and their importance for nematode identification. The Soil, 2, 257–270.
Anonymous. (2013). EPPO standard PM 7/119 (1). Nematode extraction. EPPO Bulletin, 43, 471–495.
Anonymous. (2016a). EPPO standard PM 7/41 (3). Meloidogyne chitwoodi and Meloidogyne fallax. EPPO Bulletin, 46, 171–189.
Anonymous. (2016b). EPPO standard PM 7/103 (2). Meloidogyne enterolobii. EPPO Bulletin, 46, 190–201.
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.
Blok, V. C. & Powers, T.O. (2009). Biochemical and Molecular Identification. CAB International. Root-knot Nematodes (eds. R.N. Perry, M. Moens and J. L. Starr), 4, 98–118.
Bonants, P., Edema, M., & Robert, V. (2013). Q-bank, a database with information for identification of plant quarantine plant pest and diseases. EPPO Bulletin, 43, 211–215.
Boonham, N., Glover, R., Tomlinson, J., & Mumford, R. (2008). Exploiting generic platform technologies for the detection and identification of plant pathogens. European Journal of Plant Pathology, 121, 355–363.
Braun-Kiewnick, A., Viaene, N., Folcher, L., Ollivier, F., Anthoine, G., Niere, B., Sapp, M., van de Vossenberg, B., Toktay, H., & Kiewnick, S. (2016). Assessment of a new qPCR tool for the detection and identification of the root-knot nematode Meloidogyne enterolobii by an international test performance study. European Journal of Plant Pathology, 144, 97–108.
Cao, A. X., Liu, X. Z., Zhu, S. F., & Lu, B. S. (2005). Detection of the pinewood nematode, Bursaphelenchus xylophilus, using a real time polymerase chain reaction assay. Phytopathology, 95, 566–571.
Castagnone-Sereno, P. (2012). Meloidogyne enterolobii (= M. mayaguensis): Profile of an emerging, highly pathogenic, root-knot nematode species. Nematology, 14, 133–138.
Castagnone-Sereno, P., Vanlerberghe-Masutti, F., & Leroy, F. (1994). Genetic polymorphism between and within Meloidogyne species detected with RAPD markers. Genome, 37, 904–909.
Castagnone-Sereno, P., Danchin, E. G. J., Perfus-Barbeoch, L., & Abad, P. (2013). Diversity and evolution of root-knot nematodes, genus Meloidogyne: New insights from the genomic era. Annual Review of Phytopathology, 51, 203–220.
Castillo, P. & Vovlas, N. (2007). Pratylenchus (Nematoda: Pratylenchidae): Diagnosis, biology, pathogenicity and management. Nematology Monographs and Perspectives 6 (Series Eds: Hunt, D. J. and Perry, R. N.). Leiden, The Netherlands, Brill.
Davies, K. G., Curtis, R. H., & Evans, K. (1996). Serologically based diagnostic and quantification tests for nematodes. Pesticide Science, 47, 81–87.
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.
Den Nijs, L., & Van den Berg, W. (2012). The added value of proficiency tests: Choosing the proper method for extracting Meloidogyne second-stage juveniles from soil. Nematology, 15, 143–151.
Elling, A. (2013). Major emerging problems with minor Meloidogyne species. Phytopathology, 103, 1092–1102.
Esbenshade, P., & Triantaphyllou, A. (1990). Isozyme phenotypes for the identification of Meloidogyne species. Journal of Nematology, 22, 10–15.
Ferris, V., Ferris, J., Faghihi, J., & Ireholm, A. (1994). Comparisons of isolates of Heterodera avenae using 2-D PAGE protein patterns and ribosomal DNA. Journal of Nematology, 26, 144–151.
François, C., Castagnone, C., Boonham, N., Tomlinson, J., Lawson, R., Hockland, S., Quill, J., Vieira, P., Mota, M., & Castagnone-Sereno, P. (2007). Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus. Molecular Plant Pathology, 8, 803–809.
Gamel, S., Letort, A., Fouville, D., Folcher, L., & Grenier, E. (2017). Development and validation of real-time PCR assays based on novel molecular markers for the simultaneous detection and identification of Globodera pallida, G. rostochiensis and Heterodera schachtii. Nematology, 19, 789–804.
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.
Holterman, M., Karssen, G., van den Elsen, S., van Megen, H., Bakker, J., & Helder, H. (2009). Small subunit rDNA-based phylogeny of the Tylenchida sheds light on relationships among some high-impact plant-parasitic nematodes and the evolution of plant feeding. Phytopathology, 99, 227–235.
Holterman, M. H. M., Frey, J.-E., Helder, H., Mooyman, P. J. W., Rybarczyk, K. D., & Kiewnick, S. (2011). Barcoding quarantine nematodes and their close relatives: An update on the QBOL-project. Communications in Agricultural and Applied Biological Sciences, 76, 403–407.
Huang, D., & Yan, G. (2017). Specific detection of the root-lesion nematode Pratylenchus scribneri using conventional and real-time PCR. Plant Disease, 101, 359–365.
Hunt, D. J., & Handoo, Z. A. (2009). Taxonomy, identification and principal species. In R. N. Perry, M. Moens, & J. L. Starr (Eds.), Root-knot nematodes (pp. 55–97). Wallingford: CAB International.
Ibrahim, S., Davies, K., & Perry, R. (1996). Identification of the root-knot nematode, Meloidogyne incognita, using monoclonal antibodies raised to non-specific esterases. Physiological and Molecular Plant Pathology, 49, 79–88.
Jeszke, A., Dobosz, R., & Obrepalska-Steplowska, A. (2015). A fast and sensitive method for the simultaneous identification of three important nematode species of the genus Ditylenchus. Pest Management Science, 71, 243–249.
Jones, J. T., Haegeman, A., Danchin, E. G. J., Gaur, H. S., Helder, J., Jones, M. G. K., Kikuchi, T., Manzanilla-Lopez, R., Palomares-Rius, J. E., Wesemael, W. M. L., & Perry, R. N. (2013). Top 10 plant-parasitic nematodes in molecular plant pathology. Molecular Plant Pathology, 14, 946–961.
Karssen, G., Van Hoenselaar, T., Verkerk-Bakker, B., & Janssen, R. (1995). Species identification of cyst and root-knot nematodes from potato by electrophoresis of individual females. Electrophoresis, 16, 105–109.
Kiewnick, S., Wolf, S., Willareth, M., & Frey, J.-E. (2013). Identification of the tropical root-knot nematode species Meloidogyne incognita, M. javanica and M. arenaria using a multiplex PCR assay. Nematology, 15, 891–894.
Kiewnick, S., Holterman, M., van den Elsen, S., van Megen, H., Frey, J.-E., & Helder, H. (2014). Comparison of two short DNA barcoding loci (COI and COII) and two longer ribosomal DNA genes (SSU & LSU rRNA) for specimen identification among quarantine root-knot nematodes (Meloidogyne spp.) and their close relatives. European Journal of Plant Pathology, 140, 97–110.
Kiewnick, S., Frey, J.-E., & Braun-Kiewnick, A. (2015). Development and validation of LNA-based quantitative real-time PCR assays for detection and identification of the root-knot nematode Meloidogyne enterolobii in complex DNA backgrounds. Phytopathology, 105, 1245–1249.
Kiewnick, S., Bühlmann, A., & Frey, J.-E. (2016). DNA-barcoding of invertebrate plant pests. In N. Boonham, J. Tomlinson, & R. Mumford (Eds.), Molecular methods in plant disease diagnostics (p. 98). Wallingford: CAB International.
Kikuchi, T., Aikawa, T., Oeda, Y., Karim, N., & Kanzaki, N. (2009). A rapid and precise diagnostic method for detecting the pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification. Phytopathology, 99, 1365–1369.
Koyama, Y., Toyota, K., Miyamaru, N., Yoshida, K., & Uesugi, K. (2016). Development of a quantification method with real-time PCR for three Pratylenchus species causing damage to chrysanthemum in Japan. Nematology, 18, 687–695.
Kumari, S., & Subbotin, S. A. (2012). Molecular characterization and diagnostic of stubby root and virus vector nematodes of the family Trichodoridae (Nematode: Triplonchida) using ribosomal DNA genes. Plant Pathology, 61, 1021–1031.
Lambert, K., & Bekal, S. (2002). Introduction to plant parasitic nematodes. The Plant Health Instructor. https://doi.org/10.1094/PHI-I-2002-1218-01.
Leal, I., Green, M., Allen, E., Humbke, L., & Rott, M. (2007). Application of a real-time PCR method for the detection of pine wood nematode, Bursaphelenchus xylophilus, in wood samples from lodgepole pine. Nematology, 9, 351–362.
Leal, I., Allen, E., Foord, B., Anema, J., Reisle, C., Uzunovic, A., Varga, A., & James, D. (2015). Detection of living Bursaphelenchus xylophilus in wood, using reverse transcriptase loop-mediated isothermal amplification (RT-LAMP). Forest Pathology, 45, 134–148.
Lin, B. R., Wang, H. H., Zhuo, K., & Liao, J. L. (2016). Loop-mediated isothermal amplification for the detection of Tylenchulus semipenetrans in soil. Plant Disease, 100, 877–883.
Liu, X., Wang, H., Lin, B., Tao, Y., Zhuo, K., & Liao, J. (2017). Loop-mediated isothermal amplification based on the mitochondrial COI region to detect Pratylenchus zeae. European Journal of Plant Pathology, 148, 435–446.
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.
Madani, M., Ward, L. J., & De Boer, S. H. (2008). Multiplex real-time polymerase chain reaction for identifying potato cyst nematodes, Globodera pallida and Globodera rostochiensis, and the tobacco cyst nematode, Globodera tabacum. Canadian Journal of Plant Pathology, 30, 554–564.
Madani, M., Ward, L. J., & De Boer, S. H. (2011). Hsp90 gene, an additional target for discrimination between the potato cyst nematodes, Globodera rostochiensis and G. pallida, and the related species, G. tabacum tabacum. European Journal of Plant Pathology, 130, 271–285.
Marché, L., Valette, S., Grenier, E., & Mugniéry, D. (2001). Intra-species DNA polymorphism in the tobacco cyst nematode complex (Globodera tabacum) using AFLP. Genome, 44, 941–946.
Meng, Q. P., Long, H., & Xu, J. H. (2004). PCR assays for rapid and sensitive identification of three major root-knot nematodes, Meloidogyne incognita, M. javanica, and M. arenaria. Acta Phytopathologica Sinica, 34, 204–210.
Mokrini, F., Waeyenberge, L., Viaene, N., Andaloussi, F. A., & Moens, M. (2013). Quantitative detection of the root-lesion nematode, Pratylenchus penetrans, using qPCR. European Journal of Plant Pathology, 137, 403–413.
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. (2010). Multiplex real-time PCR assays for the identification of the potato cyst and tobacco cyst nematodes. Plant Disease, 94, 959–965.
Niu, J. H., Jian, H., Guo, Q. X., Chen, C. L., Wang, X. Y., Liu, Q., & Guo, Y. D. (2012). Evaluation of loop-mediated isothermal amplification (LAMP) assays based on 5S rDNA-IGS2 regions for detecting Meloidogyne enterolobii. Plant Pathology, 61, 809–819.
Notomi, T., Okayama, H., & Masubuchi, H. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28, E63.
Nowaczyk, K., Dobosz, R., Kornobis, S., & Obrepalska-Steplowska, A. (2008). TaqMan real-time PCR-based approach for differentiation between Globodera rostochiensis (golden nematode) and Globodera artemisiae species. Parasitology Research, 103, 577–581.
Oliveira, C. M. G. O., Blok, V. B., Neilson, R. N., Mróz, T. M., & Roberts, D. R. (2017). Hydrolysis probe-based PCR for detection of Pratylenchus crenatus, P. neglectus and P. penetrans. Nematology, 19, 81–91.
Papayiannis, L. C., Christoforou, M., Markou, Y. M., & Tsaltas, D. (2013). Molecular typing of cyst-forming nematodes Globodera pallida and G. rostochiensis, using real-time PCR and evaluation of five methods for template preparation. Journal of Phytopathology, 161, 459–469.
Peng, H., Peng, D. L., Hu, X. Q., He, X. F., Wang, Q., Huang, W. K., & He, W. T. (2012). Loop-mediated isothermal amplification for rapid and precise detection of the burrowing nematode, Radopholus similis, directly from diseased plant tissues. Nematology, 14, 977–986.
Perera, M. R., Taylor, S. P., Vanstone, V. A., & Jones, M. G. (2009). Protein biomarkers to distinguish oat and Lucerne races of the stem nematode, Ditylenchus dipsaci, with quarantine significance for Western Australia. Nematology, 11, 555–563.
Postnikova, O. A., Hult, M., Shao, J., Skantar, A., & Nemchinov, L. G. (2015). Transcriptome analysis of resistant and susceptible alfalfa cultivars infected with root-knot nematode Meloidogyne incognita. PLoS One, 10(2), e0118269.
Randig, O., Bongiovanni, M., Carneiro, R., & Castagnone-Sereno, P. (2002). Genetic diversity of root-knot nematodes from Brazil and development of SCAR markers specific for the coffee-damaging species. Genome, 45, 862–870.
Reid, A., Kenyon, D. M., Evans, F. F., Mulholland, V., Pickup, J., Blok, V. C., Paterson, A., & Phillips, M. S. (2010). Development of a high-throughput method for the detection and species determination of potato cyst nematodes. In Aspects of Applied Biology, 103, 3rd Symposium on Potato Cyst Nematodes (pp. 11–14). Newport: Harper Adams University College.
Reid, A., Evans, F., Mulholland, V., Cole, Y., & Pickup, J. (2015). High-throughput diagnosis of potato cyst nematodes in soil samples. In C. Lacomme (Ed.), Plant pathology: Techniques and protocols (pp. 137–148). New York: Springer. https://doi.org/10.1007/978-1-4939-2620-6_11.
Rybarczyk-Mydłowska, K., Mooyman, P., van Megen, H., van den Elsen, S., Vervoort, M., Veenhuizen, P., van Doorn, J., Dees, R., Karssen, G., Bakker, J., & Helder, H. (2012). Small subunit ribosomal DNA-based phylogenetic analysis of foliar nematodes (Aphelenchoides spp.) and their quantitative detection in complex DNA backgrounds. Phytopathology, 102, 1153–1160.
Sasser, J. N. (1990). Plant-parasitic Nematodes the Farmer’s Hidden Enemy (pp. 47–48). Raleigh: North Carolina State University Press.
Sayler, R. J., Walker, C., Goggin, F., Agudelo, P., & Kirkpatrick, T. (2012). Conventional PCR detection and real-time PCR quantification of reniform nematodes. Plant Disease, 96, 1757–1762.
Tastet, C., Val, F., Lesage, M., Renault, L., Marché, L., Bossis, M., & Mugniéry, D. (2001). Application of a putative fatty-acid binding protein to discriminate serologically the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from other Meloidogyne species. European Journal of Plant Pathology, 107, 821–832.
Tigano, M., De Siqueira, K., Castagnone-Sereno, P., Mulet, K., Queiroz, P., Dos Santos, M., & Carneiro, R. (2010). Genetic diversity of the root-knot nematode Meloidogyne enterolobii and development of a SCAR marker for this guava-damaging species. Plant Patholology, 59, 1054–1061.
Tomlinson, J. A., Barker, I., & Boonham, N. (2007). Faster, simpler, more specific methods for improved molecular detection of Phytophthora ramorum in the field. Applied and Environmental Microbiology, 73, 4040–4047.
Toumi, F., Waeyenberge, L., Viaene, N., Dababat, A. A., Nicol, J. M., Ogbonnaya, F. C., & Moens, M. (2015). Development of qPCR assays for quantitative detection of Heterodera avenae and H. latipons. European Journal of Plant Pathology, 10, 658–681.
Trudgill, D. L., & Blok, V. C. (2001). Apomictic, polyphagous root-knot nematodes: Exceptionally successful and damaging biotrophic root pathogens. Annual Review of Phytopathology, 39, 53–77.
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.
Vervoort, M. T. W., Vonk, J. A., Mooijman, P. J. W., Van den Elsen, S. J. J., Van Megen, H. H. B., Veenhuizen, P., Landeweert, R., Bakker, J., Mulder, C., & Helder, J. (2012). SSU ribosomal DNA-based monitoring of nematode assemblages reveals distinct seasonal fluctuations within evolutionary heterogeneous feeding guilds. PLoS One, 7(10), e47555. https://doi.org/10.1371/journal.-phone.0047555.
Wang, D. Y.-C., Kumar, S., & Hedges, B. S. (1999). Divergence time estimates for the early history of animal phyla and the origin of plants, animals and fungi. Proceedings of the Royal Society of London, 266, 163–171.
Wesemael, W. M. L., Viaene, N., & Moens, M. (2011). Root-knot nematodes (Meloidogyne spp.) in Europe. Nematology, 13, 3–16.
Xin, Z., Velten, J. P., Oliver, M. J., & Burke, J. J. (2003). High-throughput DNA extraction method suitable for PCR. BioTechniques, 34, 820–826.
Yan, G. P., 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.
Ye, W. (2011). Development of prime time-real-time PCR for species identification of soybean cyst nematode (Heterodera glycines Ichinohe, 1952) in North Carolina. Journal of Nematology, 44, 284–290.
Ye, W., & Giblin-Davis, R. M. (2013). Molecular characterization and development of real-time PCR assay for pine-wood nematode Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae). PLoS One, 8(11), e78804. https://doi.org/10.1371/journal.phone.0078804.
Zijlstra, C. (2000). Identification of Meloidogyne chitwoodi, M. fallax and M. hapla based on SCAR-PCR: A powerful way of enabling reliable identification of populations or individuals that share common traits. European Journal of Plant Pathology, 106, 283–290.
Zijlstra, C., & Van Hoof, R. A. (2006). A multiplex real-time polymerase chain reaction (TaqMan) assay for the simultaneous detection of Meloidogyne chitwoodi and M. fallax. Phytopathology, 96, 1255–1262.
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Braun-Kiewnick, A., Kiewnick, S. Real-time PCR, a great tool for fast identification, sensitive detection and quantification of important plant-parasitic nematodes. Eur J Plant Pathol 152, 271–283 (2018). https://doi.org/10.1007/s10658-018-1487-7
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DOI: https://doi.org/10.1007/s10658-018-1487-7