Abstract
Cyst nematodes from the genus Globodera are common, and widely distributed parasites of Solaraceae. Intact cysts persist in soil even up to 10 years without detriment. Out of two Globodera species occurring in Poland, Globodera rostochiensis is considered by European and Mediterranean Plant Protection Organization (EPPO) as a quarantine pest, while Globodera artemisiae is not. Therefore, the distinction between these two species is crucial. Classic methods of detection and differentiation are laborious and time-consuming. Instead, application of molecular biology techniques allows obtaining of rapid and reliable results. The aim of this study was to establish detection and differentiation method of two species, G. rostochiensis and G. artemisiae, based upon real-time polymerase chain reaction with the use of TaqMan probes. In reaction with primers and probes specific for the nematodes’ ribosomal DNA (rDNA), the samples used were DNAs isolated from the two species, alone or in mixture, as well as crushed single cysts. Applied probes enable not only to identify the species in DNA mixtures but also in a single cyst. The use of a crushed cyst eliminates long-lasting procedure of DNA isolation and reduces costs of analysis.
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References
Baldwin JG, Mundo-Ocampo M (1991) Heteroderidae, cyst- and non-cyst-forming nematodes. In: Nickle WR (ed) Manual and agricultural nematology. Marcel Dekker, New York, pp 275–362
Brzeski MW (1998) Nematodes of Tylenchina in Poland and Temperate Europe. Muzeum I Instytut Zoologii Polska Akademia Nauk, Warszawa
CEC Council (1969) CEC Council Directive 69/465/EEC of 8 December 1969 on control of potato golden nematode. Official Journal of the European Communities No. L323:3–4
Cheng PS, Zheng PS Ma CZ (1994) A new record of the genus Globodera (G. artemisiae) from Beijing, P. R. China. Sci Agric Sinica 27:89
Dobosz R, Obrępalska-Stęplowska A, Kornobis S (2006) Globodera artemisiae (Eroshenko et Kazachenko,1972) (Nematoda: Heteroderidae) from Poland. J Plant Prot Res 46(4):403–408
Hernandez M, Rodriguez-Lazaro D, Esteve T, Prat S, Pla M (2003) Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection. Anal Biochem 323(2):164–170
Hooper DJ (1985) Handling, fixing, staining and mounting nematodes. In: Southey JF (ed) Laboratory methods for work with plant and soil nematodes. Ministry of Agriculture, Fisheries and Food, London, pp 59–80
Horn A, Roux-Morabito G, Lieutier F, Kerdelhue C (2006) Phylogeographic structure and past history of the circum-Mediterranean species Tomicus destruens Woll. (Coleoptera: Scolytinae). Mol Ecol 15:1603–1615
Karnkowski W (2006) Nematodes being subject to pest control—detection and determination. Państwowa Inspekcja Ochrony Roślin i Nasiennictwa (in Polish)
Madani M, Subbotin SA, Moens M (2005) Quantitative detection of the potato cyst nematode, Heterodera schachtii, using Real-Time PCR with SYBR green I dye. Mol Cell Probes 19:81–86
Manduric S, Andersson S (2004) The identity of a Swedish Globodera (Nematoda: Heteroderidae) population, following comparison with known populations of G. artemisiae (Eroshenko et Kazachenko, 1972) Behrens, 1975. Russ J Nematol 12:39–44
Pogosjan ZE, Karapatjan DA (1975) Description of the male of Heterodera artemisiae from the Armenian SSR (Nematoda, Heteroderidae). Dokłady Akademii Nauk Armjanskoj SSR 61(5):306–311, (in Russian)
Sheppard SK, Bell J, Sunderland KD, Fenlon J, Skervin D, Symondson W (2005) Detection of secondary predation by PCR analyses of the gut contents of invertebrate generalist predators. Mol Ecol 14:4461–4468
Sturhan D (1988) First record of the mugwort cyst nematode Globodera artemisiae. Gesunde Pflanz 40:205–206, (in German)
Ward LN, Bej AK (2006) Detection of Vibrio parahaemolyticus in shellfish by use of multiplexed real-time PCR with TaqMan fluorescent probes. Appl Environ Microbiol 72:2031–2042
Watzinger F, Ebner K, Lion T (2006) Detection and monitoring of virus infections by real-time PCR. Mol Asp Med 27: 254–298
Wilski A (1956) Observations on the biology of the potato root eelworm (Heterodera rostochiensis Wr.) and experiments on its control by chemical means. Roczniki Nauk Rolniczych 73-A-2:245–288 (in Polish)
Zijlstra C, Lever AEM, Uenk BJ, van Silfhout CH (1995) Differences between ITS regions of isolates of root-knot nematodes Meloidogyne hapla and M. chitwoodi. Phytopathology 85:1231–1237
Zijlstra C, Uenk BJ, van Silfhout CH (1997) A reliable, precise method to differentiate species of root-knot nematodes in mixtures on the basis of ITS-RFLPs. Fundam Appl Nematol 20(1):59–63
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This study was supported by Polish Ministry of Science and Higher Education grant 2P06R08728. Performed experiments comply with the current law of Poland.
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Stefan Kornobis deceased.
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Nowaczyk, K., Dobosz, R., Kornobis, S. et al. TaqMan REAL-Time PCR-based approach for differentiation between Globodera rostochiensis (golden nematode) and Globodera artemisiae species. Parasitol Res 103, 577–581 (2008). https://doi.org/10.1007/s00436-008-1012-6
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DOI: https://doi.org/10.1007/s00436-008-1012-6