EPPO BulletinVolume 38, Issue 3 p. 379-389 Free Access Heterodera glycines First published: 11 November 2008 https://doi.org/10.1111/j.1365-2338.2008.01249.xCitations: 1 European and Mediterranean Plant Protection Organization Organisation Européenne et Méditerranéenne pour la Protection des Plantes AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat PM 7/89 (1) Specific scope This standard describes a diagnostic protocol for Heterodera glycines.11 Use of names of chemicals or equipment in these EPPO standards implies no approval of them to the exclusion of others that may also be suitable. Specific approval and amendment Approved in 2008-09. Introduction Heterodera glycines or soybean cyst nematode is of major economic importance on soybean (Glycine max L.). Soybean cyst nematode occurs in most countries of the world where soybean is produced. It is widely distributed in countries with large areas cropped with soybean: USA, Brazil, Argentina, South Korea, Iran, Canada and Russia. It has been also reported from Colombia, Indonesia, North Korea, Bolivia, India, Italy, Iran, Paraguay and Thailand (Baldwin & Mundo-Ocampo, 1991; Manachini, 2000, Riggs, 2004). Soybean cyst nematode occurs in 93.5% of the area where soybean is grown. The soybean cyst nematode has a broad host range particularly within the Leguminosae and has been detected on crops such as Glycine, Vicia, Trifolium, Phaseolus, Lespedeza & Pisum. However, damage of economic importance is mainly observed on soybean. It is also able to attack a number of non-legumes including some ornamentals such as Geranium and Papaver, but also many weeds of at least 23 families (Moore, 1984). Field populations of H. glycines exhibit diversity in their ability to develop on resistant soybean cultivars. Therefore, several race tests for H. glycines populations have been proposed (Golden et al., 1970; Riggs et al., 1981). As these tests proved not to be always reliable for race characterisation, a revised classification scheme for genetically diverse H. glycines populations was recently proposed (Niblack et al., 2002). Further information can be found in the EPPO data sheet on Heterodera glycines (EPPO/CABI, 1997). Identity Name: Heterodera glycines Ichinohe, 1952 Synonyms: none Taxonomic position: Nematoda: Tylenchina22 Recent development combining a classification based on morphological data and molecular analysis refer to ‘Tylenchomorpha’ (De Ley & Blaxter, 2004). Heteroderidae EPPO code: HETDGL Phytosanitary categorization: EPPO A2 list No. 167. Detection The symptoms of Heterodera glycines are not specific. General symptoms are, for example, patches of poor growth in a soybean crop. Sometimes the plants in these patches show yellowing, wilting or loss of leaves with reduced seed production. There is usually a sharp dividing line between affected and non-affected areas of the field. In the affected areas, rows are slow to close and may remain so throughout the season. The most severe damage is often in the centre of the affected area. Damaged areas are frequently located near the field entrance where machinery moves into the field or in areas where soil from another field is deposited by wind or water. Reduction in seed yield is usually the first sign that an infestation is present. Usually the combination of reduced growth and yellowing is named ‘yellow dwarf disease’ for soybean infested with H. glycines. Root infestation increases the number of lateral roots and reduces the number of Rhizobium nodules and nitrogen fixation. Young females and cysts appear white, yellow or brown, about the size of a pin-head just visible with the naked eye on the root-surface. They may be confused with soybean Rhizobium nodules. However, soil sampling for cysts is the best method for detecting the presence of the soybean cyst nematode; juveniles may be found in extractions for free-living nematodes. Identification Identification of cysts and second-stage juveniles is based in general on a combination of morphological & morphometrical characters and molecular techniques. For light microscopical identification, it is recommended to examine specimens mounted in fixative on microscope slides. Interference phase microscopy is recommended to observe juveniles. Extraction procedures They are numerous methods for extracting cysts from the soil; simple methods based on flotation can be as good as elutriation. A description of each method is given in Appendix 1. Morphology Baldwin & Mundo-Ocampo (1991) produced a useful key for the identification of cyst and non-cyst forming genera within the subfamily Heteroderinae. So far six cyst-forming genera have been described and these can be identified with keys from Wouts & Baldwin (1998) which include both cyst and juveniles. This key is also useful towards species level within the genus Heterodera (see Appendix 3). Heterodera glycines is a member of the schachtii-group, which includes more than 10 species. Characters of the schachtii-group are given below under section ‘cysts’. Some important members of this group are listed in Table 1 which also provides the most important diagnostic characteristics of their second-stage juvenile characters. Table 1. Several diagnostics characters for differentiation of the second-stage juvenile of H. glycines from some closely related species of the schachtii-group Species Body length (µm) Stylet length (µm) Stylet knobs Tail length (µm) Hyaline part of tail length (µm) References H. glycines 375–540 (470) 22.0–26.0 (24.0) Concave anteriorly 40.0–61.0 (47.0) 21.0–33.0 (27.0) Graney & Miller (1982); Burrows & Stone (1985) H. daverti 400–480 (457) 24.0–26.0 (25.0) Slightly concave 49.0–60.0 (55.0) 28.0–39.0 (33.3) Wouts & Sturhan (1977) H. betae 550–660 (595) 29.5–33 (31.0) Deeply concave 65.0–84.0 (71.0) 32.0–50.0 (39.0) Wouts et al. (2001) H. ciceri 440–585 (525) 27.0–30.0 (28.6) Concave anteriorly 53.0–72.0 (60.0) 31.0–42.0 (36.0) Vovlas et al. (1985) H. galeopsidis 495–620 (553) 25.9–28.2 (27.1) Concave anteriorly 60.5–75.1 (68.1) 33.2–44.7 (40.3) Hirschmann & Triantaphyllou (1979) H. lespedezae 401–531 (481) 23.4–25.8 (24.3) Concave anteriorly 45.7–60.7 (53.5) 20.4–37.8 (26.3) Hirschmann & Triantaphyllou (1979) H. medicaginis 420–510 (460) 24.0–26.0 (25.0) Concave anteriorly 41–60 (52.0) 22.0–33.0 (28.5) Gerber & Maas (1982) H. rosii 430–661 (549) 26.6–33.8 (31.3) Concave anteriorly 58.4–76.9 (65.9) 36.9–44.6 (40.4) Duggan & Brennan (1966) H. schachtii 390–550 (450) 23.0–29.0 (26.0) Concave anteriorly 38.0–60.0 (47.0) 20.0–32.0 (26.0) Graney & Miller (1982); Brzeski (1998) H. sonchophila 437–492 (469) 24.1–26.9 (25.7) Concave anteriorly 46.5–56 (51.9) 25.8–30.2 (28.2) Kirjanova, Krall & Krall (1976) H. trifolii 494–535 (517) 26.5–29.0 (28.0) Deeply concave 56–70 (65.3) 33–41 (37.5) Mulvey & Anderson (1974); Wouts & Sturhan, (1977) Drawings of male, female, cyst and second-stage juvenile are presented in Fig. 1. Figure 1Open in figure viewerPowerPoint Heterodera glycines. A–B, F–H. Second-stage juvenile: A: Body. B: Anterior part. F: Head region. G: Stylets. H: Tails. C: Male anterior part. D: Male Head. E: Tail. I: Cyst cone front view. J: Female development (A–H, J after Hirschmann, 1956; I after Ohshima et al., 1981). Morphological characteristics of Heterodera glycines Sedentary females Sedentary females are white to pale yellow, body swollen, lemon shaped with projecting neck. Body usually covered with reticulate ridges. Gelatinous matrix or egg sac present and containing up to 200 eggs. Sub-crystalline layer prominent. Stylet slender with posteriorly projecting (sloping) knobs. In young females the ovaries are paired and coiled, nearly filling the entire body and open posteriorly through the vulva. Vulval and anus present on a terminal cone-shaped projection. The vulva, a transverse slit, is surrounded by thin walled crescent shaped areas, the so called semifenestrae. The mature, pale yellow female body wall changes upon death to a brown tough-walled cyst. Cysts Cysts are lemon-shaped with a protruding neck and cone. Cyst ranges in length from 340 to 920 µm and width from 320 to 610 µm. Cuticle surface with a zig-zag pattern of ridges. Vulval region usually intact in young cysts; in older specimens, the thin walled cuticle of the cone is lost leaving an open fenestra crossed by the vulval bridge and dividing the fenestra in two semifenestrae (= ambifenestrate). The vulval bridge bears the vulval slit, this slit is wider than the fenestral width, respectively 50 (45–60) µm and 40 (32–50) µm. Bullae scattered just bellow the underbridge, prominent and elongated. Also the underbridge is well developed (see 2, 3). Figure 2Open in figure viewerPowerPoint Diagram showing terminal cone region structures of H. schachtii-group cyst (after Baldwin & Mundo-Ocompo, 1991). Figure 3Open in figure viewerPowerPoint Fenestral patterns of terminal cyst region of Heteroderinae. A) Ambifenestrate vulval region; anal region not fenestrate. B) Bifenestrate vulval region; anal region not fenestrate. C) Circumfenestrate vulval region; anal region not fenestrate. D) Circumfenestrate vulval region with separate anal fenestra (after Baldwin & Mundo-Ocompo, 1991). Males Males are usually present, with a morphology typical for the genus. Lateral field with 4 incisures. Head offset with 4–5 annules. Stylet robust, 27 (25.5–28.5) µm long with anteriorly protruding knobs. Dorsal pharyngeal gland opening close to stylet base (about 4 µm). Spicules robust, curved ventrally and 34 (33–37) µm in length. Second-stage juveniles Second-stage juveniles are vermiform, annulated and tapering at both ends. Body length ranging from 375–540 µm, head offset with 3 to 4 annules and four incisures in the lateral field present. Stylet robust, 24 (22–26) µm long with anteriorly concave knobs. Tail gradually tapering towards a finely rounded terminus, 47 (40–61) µm long with a hyaline tail part of 27 (21–33) µm in length. Several diagnostics characters for differentiation of the second-stage juvenile of H. glycines from some closely related species of the schachtii-group are presented in Table 1. The characteristics of both cysts and second-stage juveniles in the sample should be determined for a reliable identification. These two stages are normally present in infested soil samples, or second-stage juveniles can be obtained directly from cysts. For cysts, the vulval cone should be examined and fenestra shape, vulva width and presence of an underbridge and bullae should be noted. For second-stage juvenile stylet length, stylet knob shape and tail and hyaline tail length and body length should be determined. Molecular methods As Heterodera glycines can be hard to distinguish from other species in the schachtii group using morphological and morphometrical features, some molecular methods can be used for reliable diagnostics of this species. Several PCR-based methods of identification of the soybean cyst nematode have been developed during the last decade and are now in use. All these methods are based on detection of minor unique differences in the ITS-rRNA sequences of H. glycines. Szalanski et al. (1997) were the first who proposed to use a digestion with enzyme FokI for PCR products amplified ITS1-rRNA gene to separate the soybean cyst nematode from the sugar beet cyst nematode and the clover cyst nematodes. Another PCR-ITS-RFLP protocol was developed by Subbotin et al. (2000) and then tested with several H. glycines populations by Zheng et al. (2000) and Tanha Maafi et al. (2003). With this method, the PCR product amplified from the ITS1–5.8S-ITS2 rRNA gene should be digested with one of the following restriction enzymes: AvaI, CfoI, HpaII, MvaI, PstI, or RsaI. With a combination of RFLP profiles most populations of H. glycines studied so far can be distinguished from other cyst nematode species. Reliable detection of the second-stage juveniles of the soybean cyst nematodes even in samples mixed with other cyst and soil-inhabiting nematodes can be also achieved using PCR with species-specific primer methods (Subbotin et al., 2001; Ou et al., 2008). PCR with species-specific primers is quicker but there is a risk of false negatives if DNA is not extracted properly. The PCR-ITS-RFLP protocol is more informative but is more expensive and time consuming. Descriptions of protocols for the PCR-ITS-RFLP and PCR with species specific primers methods are given in detail in Appendix 2. Reference material Prof. Dr. Gerrit Karssen, Plant protection Service, National Reference Library, P.O. Box 9102, 6700 HC Wageningen (NL). Reporting and Documentation Guidance on reporting and documentation is given in EPPO Standard PM 7/77 (1) Documentation and reporting on a diagnosis. Further information Further information on this organism can be obtained from: Prof. Dr. Gerrit Karssen, Plant protection Service, National Reference Laboratory, P.O. Box 9102, 6700 HC Wageningen (NL); Prof. Dr. Sergei Subbotin, Plant Pest Diagnostic Center, California Department of Food and Agriculture, 3294 Meadowview road, Sacramento, CA 95832 (USA). Footnotes 1 Use of names of chemicals or equipment in these EPPO standards implies no approval of them to the exclusion of others that may also be suitable. 2 Recent development combining a classification based on morphological data and molecular analysis refer to ‘Tylenchomorpha’ (De Ley & Blaxter, 2004). Acknowledgements This protocol was originally drafted by: G Karssen, Plant protection Service, National Reference Laboratory, PO Box 9102, 6700 HC, Wageningen (NL); S Subbotin, Plant Pest Diagnostic Center, California Department of Food and Agriculture, 3294 Meadowview road, Sacramento, CA 95832 (USA) and G. Anthoine LNPV-Unité de Nematologie, Domaine de la Motte au Viconte BP 35327 Le Rheu (FR). References Al-Banna L, Williamson V & Gardner SL (1997) Phylogenetic analysis of nematodes of the genus Pratylenchus using nuclear 26S rDNA. Molecular-Phylogenetics and Evolution 7, 94– 102. CrossrefCASPubMedWeb of Science®Google Scholar Amiri S, Subbotin SA & Moens M (2003) Comparative morphometrics and RAPD studies of Heterodera schachtii and H. betae populations. Russian Journal of Nematology 11, 91– 99. Web of Science®Google Scholar Baldwin JG & Mundo-Ocampo M (1991) Heteroderinae, cyst- and non-cyst-forming nematodes. In: Manual of Agricultural Nematology (Ed. WR Nickle), pp. 275– 362. Marcel Dekker, Inc. New York (US). Web of Science®Google Scholar Brzeski MW (1998) Nematodes of Tylenchina in Poland and Temperate Europe. Muzeum i Instytut Zoologii PAN, Waszawa. 396 p. Google Scholar Burrows PR & Stone AR (1985) Heterodera glycines. C.I.H. Descriptions of plant-parasitic nematodes. Set 8, No. 118. CIP, St. Albans (GB). Google Scholar De Ley P & Blaxter M (2004) A new system for Nematoda: combining morphological characters with molecular trees, and translating clades into ranks and taxa. In: Nematology Monographs and Perspectives (Ed. Cook R & Hunt DT), pp. 633– 653. EJ Brill, Leiden (NL). Web of Science®Google Scholar Duggan JJ & Brennan PA (1966) Heterodera rosii (Heteroderidae), a new species of cyst-forming nematode from curled dock. Irish Journal of Agricultural Research 5, 113– 120. Web of Science®Google Scholar EPPO/CABI (1997) Data sheets on quarantine pests: Heterodera glycines. In: Quarantine Pests for Europe, 2nd edn (Ed. Smith IM et al .), pp. 607– 611. CAB International, Wallingford (GB). Google Scholar Gerber K & Maas PWTh (1982) A redescription of Heterodera medicaginis Kirjanova. Nematologica 28, 94– 100. CrossrefWeb of Science®Google Scholar Golden AM, Epps JM, Riggs RD, Duclos LA, Fox JA & Bernard RL (1970) Terminology and identity of infraspecific forms of the soybean cyst nematode (Heterodera glycines). Plant Disease Reporter 54, 544– 546. Google Scholar Graney LSO & Miller LI (1982) Comparative morphological studies of Heterodera schachtii and H. glycines. In Book: Nematology in the Southern Region of the United States. Southern Cooperative Series Bulletin 276, 96– 107. Google Scholar Hirschmann H (1956) Comparative morphological studies on the soybean cyst nematode, Heterodera glycines and the clover cyst nematode, H. trifolii (Nematoda: Heteroderidae). Proceedings Helminthological Society Washington 23, 140– 151. Google Scholar Hirschmann H & Triantaphyllou AC (1979) Morphological comparison of members of the Heterodera trifolii species complex. Nematologica 25, 458– 481. CrossrefWeb of Science®Google Scholar Ichinohe M (1952) On the soy bean nematode, Heterodera glycines n. sp. from Japan. Oyo-Dobutsugaku-Zasshi 17(1–2), 1– 4. Google Scholar Joyce SA, Reid A, Driver F & Curran J (1994) Application of polymerase chain reaction (PCR) methods to the identification of entomopathogenic nematodes. In: Genetics of entomopathogenic nematodes-bacterium complexes. Proceedings of symposium and workshop (Eds. Burnell AM, Ehlers RU & Masson JP), S. Patrick's college, Maynooth, Co. Kildrare, Ireland. EC DG XII, Luxembourg, pp. 178–1 87. Google Scholar Kirjanova ES, Krall EL & Krall H (1976) The sowthistle cyst-nematode Heterodera sonchophila n. sp. (Nematoda: Heteroderidae) from Estonia. Eesti NSV Teaduste Akademia Toimetised, Biologilini Seeria, 25, 305– 315. Google Scholar Manachini B (2000) First report of Heterodera glycines Ichinohe on soybean in Italy. Bolletino di Zoologia Agraria e di Bachicoltura, Serie II, 32, 261– 267. Google Scholar Moore WF (1984) Soybean Cyst Nematode. Mississippi Coop. Ext. Service (US). Google Scholar Mulvey RH & Anderson RV (1974) Heterodera trifolii. C.I.H. Descriptions of Plant Parasitic Nematodes set 4, no. 46, 4 p. Google Scholar Niblack TL, Arelli PR, Noel GR, Opperman CH, Orf JH, Schmitt DP et al . (2002) A revised classification scheme for genetically diverse populations of Heterodera glycines. Journal of Nematology 34, 279– 288. CASPubMedWeb of Science®Google Scholar Ohshima Y, Momota Y & Shimizu K (1981) Additional information on some morphological characteristics of Heterorodera glycines Ichinohe, 1952. Japanese Journal of Nematology 10, 52– 53. Google Scholar Ou S, Peng D, Liu X, Li Y & Moens M (2008) Identification of Heterodera glycines using PCR with sequence characterised amplified region (SCAR) primers. Nematology 10, 397– 403. CrossrefCASWeb of Science®Google Scholar Riggs RD (2004) History and distribution. In: Biology and Management of Soybean Cyst Nematode (Ed. DP Schmitt et al .), p. 262. Schmitts & Associates of Marceline, Marceline (US). Google Scholar Riggs RD, Hamblen ML & Rakes L (1981) Infra-species variation in reactions to hosts in populations. Journal of Nematology 13, 171– 179. PubMedWeb of Science®Google Scholar Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (US). Wiley Online LibraryGoogle Scholar Subbotin SA, Waeyenberge L & Moens M (2000) Identification of cyst forming nematodes of the genus Heterodera ( Nematoda: Heteroderidae) based on the ribosomal DNA-RFLP. Nematology 2, 153– 164. CrossrefCASWeb of Science®Google Scholar Subbotin SA, Peng D & Moens M (2001) A rapid method for the identification of the soybean cyst nematode Heterodera glycines using duplex PCR. Nematology 3, 365– 371. CrossrefCASWeb of Science®Google Scholar Szalanski A, Sui DD, Harris TS & Powers TO (1997) Identification of cyst nematodes of agronomic and regulatory concern with PCR-RFLP of ITS1. Journal of Nematology 29, 255– 267. CASPubMedWeb of Science®Google Scholar Tanha Maafi Z, Subbotin SA & Moens M (2003) Molecular identification of cyst-forming nematodes (Heteroderidae) from Iran and a phylogeny based on ITS-rDNA sequences. Nematology 5, 99– 111. CrossrefWeb of Science®Google Scholar Turner SJ (1998) Sample preparation, soil extraction and laboratory facilities for the detection of potato cyst nematodes. In: Potato Cyst Nematodes: Biology, Distribution, and Control (Ed. RJ Marks & BB Brodie), pp. 75– 90. CAB International, Wallingford (GB). Google Scholar Vovlas N, Greco N & Di Vito M (1985) Heterodera ciceri sp. n. (Nematoda: Heteroderidae) on Cicer arientinun L. from Northern Syria. Nematologica Mediterranea 13, 239– 252. Google Scholar Wouts WM & Baldwin JG (1998) Taxonomy and identification. In: The Cyst Nematodes (Ed. SB Sharma), pp. 83– 122. Kluwer Academic Publishers, Dordrecht (NL). CrossrefGoogle Scholar Wouts WM & Sturhan D (1977) The identity of Heterodera trifolii Goffart, 1932, and the description of H. daverti n. sp. (Nematoda: Tylenchida). Nematologica 24(1978), 121– 128. CrossrefWeb of Science®Google Scholar Wouts WM, Rumpenhorst HJ & Sturhan D. (2001) Heterodera betae sp. n., the yellow beet cyst nematode (Nematoda: Heteroderidae). Russian Journal of Nematology 9, 33– 42. Web of Science®Google Scholar Zheng J, Subbotin SA, Waeyenberge L & Moens M (2000) Molecular characterisation of Chinese Heterodera glycines and H. avenae populations based on RFLPs and sequences of rDNA-ITS regions. Russian Journal of Nematology 8, 109– 113. Web of Science®Google Scholar Appendix 1 Extraction procedures from soil Cyst extraction by flotation technique These methods are based on the characteristic that dried cysts float. The dried soil sample is added to a beaker, flask or white dish that is filled with water. The suspension is well stirred. After 30 s to a few minutes, depending on the soil type, the water is cleared and the liquid will only contain the floating organic debris and cysts. Adding a drop of detergent will cause the cysts to move to the edge and the cysts can be picked out by hand using a brush. Other techniques are carefully decanting, or using a paper strip around the beaker and raising the water level so cysts can adhere to it. A variety of methods to isolate the cysts from the debris are in use (Turner, 1998). One that should be mentioned is the Fenwick can, an apparatus that has been in use for many years. The apparatus is a brass container, tapering towards the top, with a sloping collar around the outside of the rim which collects overflow and directs it towards an outlet. The can has a sloping internal base with a drain plug at its lowest point. The can is first filled with water and the soil sample is added by washing through a 1-mm sieve supported on a long-stemmed funnel above the can. The organic matter will immediately rise and overflow onto the collar and be collected on two sieves of 840 µm and 250 µm. Most of the cysts in the soil sample will be collected at this stage. The funnel above the can is then removed and the soil at the base of the can is elutriated by means of water flowing rapidly through a long glass or metal tube. The tube is inserted deep into the can to stir the sediment and release any trapped cysts; this is continued for approximately 1 min. The cysts are collected on the 250-µm sieve for further processing. The Schuiling centrifuge is a semi-automatic extraction method. The air-dried, 200 mL soil sample is added to a transparent cylindrical container half-filled with water. The contents are swirled with a rotating two-pronged fork at 450–500 rev min−1, creating a vortex and causing cysts and similar-sized floating particles to be forced to the centre through a wire-mesh cylinder (1.5 mm aperture).The mesh cylinder is fixed above a tube of the same diameter leading down to an outlet. While swirling, more water is added around the inside of the main container washing off any adhering debris and cysts which are channelled to the outlet with the rest. The apparatus cleans itself after each sample processing. Further separation of cysts is by a special cleaning process involving the so-called Schuiling can and special sieves. In some laboratories the Schuiling units have been modified to suit different soils and conditions: the modifications include additional spinning and cleaning time, larger collecting sieves, an improved plastic cleaning ‘can’ for reducing the amount of debris and removal of the electrical parts from the apparatus to the wall above for safety reasons. Cysts are collected on a 250 µm pore sieve for further processing. Cysts and juveniles extraction by elutriator These methods are based on the difference in density of cyst in comparison to soil particles and can be used for wet soil. At the base of a (conical) column water enters through a perforated tube at a constant rate (minimal 0.6 L min−1). Soil is added into the column using a funnel. A small plate baffles the outlet of the funnel so that soil does not fall down the column too quickly. Via the overflow spout the cysts are collected on a pair of sieves (53 µm aperture). A variety of methods to isolate the cysts from the debris are in use (Turner, 1998). Appendix 2 Molecular detection of Heterodera glycinesPCR-ITS-RFLP protocol (Subbotin et al., 2000) 1. General information 1.1. Protocol developed by Subbotin et al. (2000) 1.2. Full cysts or second stage juveniles are nucleic acid source 1.3. The assay is designed to the internal trancribed spacer (ITS) region of the rDNA sequences of Heterodera spp. producing an amplicon of 1030 bp for H. glycines 1.4. Oligonucleotides: ITS–specific universal primers described by Joyce et al. (1994): forward primer AB28 (5′-ATA TGC TTA AGT TCA GCG GGT-3′) and reverse primer TW81 (5′-GTT TCC GTA GGT GAA CCT GC-3′) 1.5. Taq DNA Polymerase 5 U µL−1 (Qiagen) used for PCR amplification and enzymes AluI, AvaI, Bsh1236I, BsuRI, CfoI, MvaI, RsaI for amplicon restriction 1.6. Nucleotides (Qiagen) are used at a final concentration of 0.2 mM each 1.7. Buffers: 10 × PCR buffer containing 15 mM MgCl2 (Qiagen) 1.8. Molecular grade water (MGW) is used to make op reaction mixes; this should be purified (deionised or distilled), sterile (autoclaved or 0.45 µm filtered) and nuclease free 1.9. Amplification is performed in a thermal cycler, e.g. Gene-E (New Brunswick Scientific, Wezmbeck-Oppem, Belgium) 2. Methods 2.1. Nucleic acid Extraction and Purification 2.1.1. One to four cysts were transferred into 10 µL of double distilled water in an Eppendorf tube and crushed with a microhomogenisator. Eight µL of nematode lysis buffer (125 mM KCl; 25 mM Tris HCl, pH = 8.3; 3.75 mM MgCl2; 2.5 mM DTT; 1.125% Tween 20; 0.025% gelatine) and 2 µL proteinase K (600 µg µL−1) were added. The tubes were incubated at 65°C (1 h) and 95°C (10 min) consecutively 2.1.2. No DNA clean-up is required. Either use extracted DNA immediately, store overnight at –20°C for longer periods 2.2. Polymerase Chain reaction 2.2.1. Master mix (concentration per 100-µL single reaction). 10 µL 10 X reaction buffer (Qiagen), containing 15 mM MgCl2, final concentration 1 X; 1.5 mM MgCl2 20 µL 5X Q-solution (Qiagen) 200 µM dNTPs (Qiagen) 1.5 µM forward primer AB28 1.5 µM reverse primer TW81 0.8 U Taq DNA Polymerase (Qiagen, 5 U µL−1) 10 µL extracted DNA obtained as described above. 2.2.2. PCR cycling parameters. 4 min 94°C, 35 cycles of 1 min 94°C, 1.5 min 55°C, 2 min 72°C, final elongation 10 min 72°C. 2.3. Restriction of PCR amplicon 2.3.1. Restriction mix (concentration per 10-µL reaction). According to supplier's conditions (Gibco Co., BRL) with 5–7 µL of amplicon. 2.3.2. Incubation time/temperature for digestion. According to the supplier's conditions. 3. Essential Procedural Information 3.1. Analysis of DNA fragments: DNA fragments are separated by electrophoresis on agarose gel (1% for PCR, 1.5% for RFLP) and visualized under UV light according to standard procedures (e.g. Sambrook et al., 1989). 3.2. Identification of species. RFLP patterns for H. glycines and other Heterodera species populations are given in Table 2. Table 2. Approximate sizes of restriction fragments of rDNA ITS regions for cyst forming nematodes Species AluI AvaI Bsh1236I BsuRI CfoI MvaI RsaI H. avenae (type A) 1060 1060 880, ( 500, 380), 140 420, 360, 180, 50 750, 160, 110 400, 330, 290 1040 H. avenae (type B) 560, 500 1060 880, 140 420, 360, 180, 50 75