Ditylenchus destructor and Ditylenchus dipsaci

Abstract

EPPO BulletinVolume 38, Issue 3 p. 363-373 Free Access Ditylenchus destructor and Ditylenchus dipsaci Correction(s) for this article Erratum Volume 42Issue 2EPPO Bulletin pages: 344-344 First Published online: August 7, 2012 Erratum Volume 39Issue 1EPPO Bulletin pages: 84-84 First Published online: March 11, 2009 First published: 11 November 2008 https://doi.org/10.1111/j.1365-2338.2008.01247.xCitations: 21 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 onFacebookTwitterLinkedInRedditWechat PM 7/87 (1) Specific scope This standard describes a diagnostic protocol for Ditylenchus destructor and Ditylenchus dipsaci1. Specific approval and amendment Approved in 2008-09. Introduction Among the more than 80 species presently recognized in the genus Ditylenchus Filipjev, 1936, only a few are parasites of higher plants, while the majority of species is mycophagous. Ditylenchus destructor is one of the few plant-parasites within the genus Ditylenchus Filipjev, 1936. The species is recorded from all continents, mainly from temperate regions. The known host range comprises more than 100 species of plants from a wide variety of families. Economically important crops are potato Solanum tuberosum, Iris spp., Tulipa spp., Dahlia spp., Gladiolus spp. Rheum rhabarbarum, Trifolium spp. and Daucus carota. Some weeds (e.g. Cirsium arvense, Mentha arvensis, Potentilla anserine, Rumex acetosella and Stachys palustris (Andersson, 1971)) and sugar beet (Beta vulgaris) can also be hosts and can act as sources of infection to crop plants. D. destructor is also capable of reproducing on the mycelium of many fungi. The nematode attacks subterranean parts of plants (tubers, stolons, bulbs, rhizomes, roots), but may occasionally also invade above-ground parts, mainly the base of stem). D. destructor is unable to withstand excessive desiccation unlike D. dipsaci. D. dipsaci is among the plant-parasitic nematodes of greatest economic impact worldwide and widely distributed mainly in temperate areas. Almost 500 different plant species are known as hosts for D. dipsaci but the different biological races of this nematode each have limited host-ranges. D. dipsaci lives mostly as an endoparasite in aerial parts of plants (stems, leaves, flowers), but also attacks bulbs, tubers and rhizomes. D. dipsaci readily withstands desiccation and can be isolated even from completely dry plant material after moistening (resistant stage = fourth-stage juveniles). Identity Name: Ditylenchus destructor Thorne, 1945 Synonyms: About 30 synonyms but none used in recent years (Sturhan & Brzeski, 1991) Taxonomic position: Nematoda: Tylenchida2: Anguinidae EPPO computer code: DITYDE Phytosanitary categorization: EU Annex designation: II/A2. Name: Ditylenchus dipsaci (Kühn, 1857) Filipjev, 1936 Synonyms: About 30 synonyms but none used in recent years (Sturhan & Brzeski, 1991) Taxonomic position: Nematoda: Tylenchida2: Anguinidae EPPO computer code: DITYDI Phytosanitary categorization: Ditylenchus dipsaci: EPPO A2 list no. 174, EU Annex designation: II/A2. Detection Symptoms caused by D. destructor: Common symptoms of infestation of D. destructor are discoloration and rotting of the plant tissue. Potatoes The first symptoms are white spots under the skin. Badly affected tubers have slightly sunken areas with cracked and papery skin (1, 2); the tissue below the skin is darkened and may have a mealy or spongy appearance. Symptoms may be more visible after storage. There is in general a secondary invasion of fungi, bacteria and free-living nematodes. Figure 1Open in figure viewerPowerPoint Ditylenchus destructor damage on potatoes (pictures Crown copyright; reproduced courtesy of the Central Science Laboratory, York, GB). Figure 2Open in figure viewerPowerPoint Ditylenchus destructor damage on potatoes (LNPV Nematology, Rennes, FR). Flower bulbs and corms Infestations usually begin at the base of the bulb and extend up to the fleshy scales with yellow to dark brown lesions. Secondary rotting may occur and the bulbs can be destroyed. Specimens of D. destructor are accumulated on the boundary between distinctly diseased parts and healthy sections, but are rarely isolated from completely decayed tissues. Carrots Damage to carrots appears as transverse cracks in the skin with white patches in the sub-cortical tissue (Fig. 3). The patches are easily seen in a transverse cut (Fig. 4). Infested areas are subject to secondary infections by fungi and bacteria resulting in decay and rot. Figure 3Open in figure viewerPowerPoint Carrots showing transverse cracks upon infection by Ditylenchus destructor. Figure 4Open in figure viewerPowerPoint Carrot disc with white sub-cortical patch caused by Ditylenchus destructor. Symptoms caused by D. dipsaci Common symptoms of infestation are swelling, distortion, discolouration and stunting of above-ground plant parts (Fig. 5), necrosis and rotting of bulbs and tubers. Figure 5Open in figure viewerPowerPoint Ditylenchus dipsaci damage on bean plants (pictures Crown copyright; reproduced courtesy of the Central Science Laboratory York, GB). Seeds Small seeds generally show no visible symptoms of infestation, but in larger seeds (e.g. Phaseolus vulgaris and Vicia faba) the skin may be shrunken and show discoloured spots (6, 7). Figure 6Open in figure viewerPowerPoint Ditylenchus dipsaci symptoms on Vicia faba seeds (Plant Protection Service, NL). Figure 7Open in figure viewerPowerPoint Ditylenchus dipsaci damage on Phaseolus vulgaris seeds. Bulbs, tubers (8, 9) Figure 8Open in figure viewerPowerPoint Ditylenchus dipsaci damage on onions. Figure 9Open in figure viewerPowerPoint Ditylenchus dipsaci damage on potatoes. Infested tissue is generally necrotic and bulbs show browning of the scales in concentric circles (seen in transverse sections, see Fig. 10); entire bulbs often become soft and, occasionally in the case of garlic, a strong smell may develop. Figure 10Open in figure viewerPowerPoint symptoms of D. dipsaci on Narcissus sp. (LNPV Nematology, Rennes, FR). Carrots and sugar beet On carrots early symptoms of D. dipsaci attacks are straddled leaves, multi-bud plant crowns and light discolorations of tap-root tops. The portion of the plant most affected by D. dipsaci is that 2–4 cm below and above ground. Severe symptoms of attacks by the nematode are leaf death and tap-root rot (Greco et al., 2002). These symptoms are similar to those described on sugar beet roots (Dunning, 1957) see Fig. 11. Figure 11Open in figure viewerPowerPoint Ditylenchus dipsaci symptoms on Beta vulgaris. Extraction of the nematodes Extraction from plant tissue D. destructor and D. dipsaci can be detected by placing plant tissue with suspected infestation into water. Any plant material to be tested is cut into pieces or sliced and placed on a Baermann funnel on a sieve covered with soft filter paper (e.g. cotton wool filter). These nematode species are very mobile and will usually emerge from the tissues within 2 to 4 h; the water from the bottom of the funnel can then checked by microscope for the presence of nematodes. Leaving the nematodes much longer in water will result in an increase in mortality. The use of mistifier extraction can provide active nematodes for a longer period. In tissues showing early symptoms of attack the nematodes can be detected by tissue dissection under a dissection microscope or by enzymatic destruction of the tissues, but this latter method is rather costly. Extraction from soil D. dipsaci and D. destructor can be extracted from soil by a suitable extraction method for nematodes of this size (see EPPO Standard PM 7/41: Diagnostic Protocol for Meloidogyne chitwoodi and Meloidogyne fallax Appendix 1). Nevertheless it should be noted that these nematodes are rarely found in soil unless the latter has been associated with an infested host. Extraction of D. dipsaci from seeds For extraction of D. dipsaci, seed samples are placed on a Baermann funnel (preferably on a sieve covered with soft filter paper) and covered by a small amount of water. The water is released from the bottom of the funnel after a period of 12 h to two days (or longer if the infestation is very low), and checked for the presence of nematodes using a microscope. Large seeds can be broken in a blender so that extraction time is reduced. Larger seed samples are best soaked in an abundance of water, which is poured off after one day (or longer) into a cylinder. The bottom layer is then examined for nematodes by microscope after sufficient time has been allowed for sedimentation to happen. It may be necessary to pour the water through 3 sieves of 50 µm or one sieve of 20 µm pore size. Recovery and identification of D. dipsaci from seed samples does not generally cause problems. When many nematode specimens of similar appearance are found (exclusively fourth-stage juveniles) the species is most probably D. dipsaci. Nevertheless identification should be confirmed by identification methods described below. Identification The identification of D. destructor and D. dipsaci should always be based on morphological methods first. Molecular methods have been developed for the identification of D. destructor and D. dipsaci and these methods may be used when there is a low level of infestation or when only juvenile stages are present. Morphological methods For identification, individual nematodes or the entire nematode suspensions are heated (to approximately 60°C) until the nematodes become immobile. The body of D. destructor killed by gentle heat is almost straight (never C-shaped, spiral or with posterior end markedly bent to the ventral side). Generally all developmental stages (females, males, 3 juvenile stages, eggs) can be isolated from infested plant tissues. Contamination of the extract by free-living mycophagous and bacteriophagous nematodes is common, in particular in decaying plant material (e.g. potato tubers). The body of D. dipsaci killed by gentle heat is straight or almost so (never C-shaped, spiral or posterior end markedly bent to the ventral side). In seed samples, nematodes other than D. dipsaci will rarely be found but contamination by other nematodes may occur particularly with samples containing other plant debris. From seeds, generally only fourth-stage juveniles of D. dipsaci can be isolated (no adults or early juvenile stages) and the specimens have a similar appearance. In bulbs, tubers, rhizomes etc. a variety of nematodes may be present, which invaded the plant tissues from the soil. In rotting tissue bacteriophagous or mycophagous nematodes are often dominant. D. dipsaci is mainly found in plant tissues which are still viable. All stages of D. dipsaci can be isolated from bulbs etc. (females, males, 3 juvenile stages, eggs), but fourth-stage juveniles often prevail. Microscopical examination has to concentrate on stylet-bearing nematodes (stylet delicate) with slender and (almost) straight body and a conical pointed tail. To distinguish Ditylenchus spp. from other tylenchid genera see Table 1. For precise identification, examination of the specific morphological characters at high microscopical magnification is necessary. Table 1. Key to distinguish Ditylenchus spp. from other tylenchid genera (modified from Brzeski, 1998) 1 Females mobile 2 Females swollen, globose or lemon-shaped Other genera 2 Female gonads prodelphic and outstretched 3 Female gonads paired or reflex when prodelphic Other genera 3 Pharyngeal glands offset from intestine or slightly overlapping it 4 Pharyngeal glands distinctly overlapping intestine Other genera 4 Metacorpus offset from procorpus, metacorporeal plates short or absent 5 Procorpus gradually expands into large metacorpus, metacorporeal plates long Other genera 5 Sperm large, head usually low, spermatheca not off-set. Ditylenchus Sperm small, head usually high, spermatheca off-set in most genera Other genera In non-related nematode genera the tail ranges from filiform to dome-shaped, the body of heat-relaxed specimens from straight to C-shaped and spiral with the posterior end often ventrally curved, a stylet may be absent, the pharynx distinctly off-set from intestine, the vulva located at mid-body, the male tail lacks a bursa, etc. Specimens of Aphelenchoides spp. are mostly easily distinguished by their well-demarcated rounded median pharyngeal bulb, long overlap of the pharynx over the intestine, presence of a mucro on the tail tip and lack of a bursa on male tail. Discriminating morphological characteristics of D. destructor, D. dipsaci, D. convallariae and D. myceliophagus are presented in Table 2; see also 14, 12. Table 2. Discriminating morphological characteristics of D. destructor, D. dipsaci, D. convallariae and D. myceliophagus (after Decker, 1969) D. destructor D. dipsaci D. convallariae D. myceliophagus A ratio ♀ 32 (18–41) 37 (36–40) 42 (32–54) 30 (23–44) Body length ♀ (mm) 1.0 (0.8–1.4) 1.1 (0.9–1.3)* 1.1 (0.9–1.3) 0.9 (0.6–1.0) Stylet length (µm) 10–12 11–13 11–13 7–10 Posterior bulb short, dorsally overlapping not overlapping not overlapping short, dorsally overlapping Number of lateral lines 6 4 6 6 Vulva position (%) 80 (78–83) 82 (79–82) 77 (74–79) 82.5 (74–90) Post-vulval sac length 2/3–3/4 of vulva-anus distance 1/2 of vulva-anus distance 1/4–1/2 of vulva-anus distance 2–2 1/4 vulva-anus distance Vulva-anus length 1 3/4–2 1/3 tail length 1 3/4–2 1/4 tail length 2–2 1/4 tail length 2–2 1/4 tail length Tail shape conoid, usually slightly bent to ventral side in posterior part conoid conoid broadly conoid Tail tip finely rounded sharply pointed sharply pointed finely rounded Spiculum length (µm) 9–12 10–12 8–11 9 Length of cone/total stylet length About 50% About 50% << 50% << 50% *The ‘giant’ race of D. dipsaci in Faba bean can be up to 2.0 mm long. Figure 14Open in figure viewerPowerPoint The amplification of two products (198 bp and 242 bp) in multiplex PCR using SCAR primers D09, D10, H05, and H06 for 8 giant and 10 normal populations of D. dipsaci (after Esquibet et al., 2003). Figure 12Open in figure viewerPowerPoint Ditylenchus destructor (A) Female, pharyngeal region. (B) Head of female. (C) Male, spicule region. (D) End of female tail. (E) Posterior portion of female. (F) Lateral field at midbody. Each unit on bars = 10 µm (After Sturhan & Brzeski, 1991). D. dipsaci and D. detructor have a stylet with a cone of the length of about 50% of total stylet length, whereas most other fungivorus species have stylets with cones shorter than 50% of the total stylet length (mainly about 1/3 of its length). In most species similar to D. dipsaci, the lateral fields have four incisures, the basal pharyngeal bulb is distinctly off-set from the intestine and the tail terminus is sharply pointed. A list of Ditylenchus species with specific characters has been published by Brzeski (1991) and Sturhan & Brzeski (1991). Molecular methods Several molecular techniques have been developed and are in use now for diagnostics of Ditylenchus species and are described in Appendices: A PCR-RFLP of the ITS-rRNA is described in Appendix 1 (this method is the only one that allows the identification of both D. destructor and D. dispaci). PCR with species specific primers have been developed for diagnostics of normal and giant races of Ditylenchus dipsaci. The use of PCR with specific primers constitutes a major development in DNA diagnostics and enables the detection of the stem nematode in a mixture with other soil-inhabiting organisms by a single PCR test, decreasing diagnostic time and costs compared with PCR-RFLP. Esquibet et al. (2003) developed a multiplex PCR with SCAR (sequence characterized amplified region) primers for identification of the giant and the normal races of D. dipsaci (Table 4 in Appendix 2, Fig. 10) (Appendix 2). Subbotin et al. (2005) described specific primers and PCR protocol for detection of D. dipsaci sensu stricto (Appendix 3). Several other specific primers have been proposed for diagnostics of D. dipsaci sensu lato (Marek et al., 2005 (Appendix 4); Kerkoud et al., 2007 (Appendix 5); Zouhar et al., 2007). Table 4. SCAR-PCR patterns of D. dipsaci, D. desructor and D. myceliophagus (Esquibet et al., 2003) D. dipsaci (host races) D. dipsaci (giant race) D. myceliophagus H05-H06 242 bp − − D09-D10 − 198 bp − Reference material Julius Kühn Institute (JKI), Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Toppheideweg 88, 48161 Münster (DE). Pest and Disease Identification Team, Central Science Laboratory, Sand Hutton, York YO41 1LZ (GB). Plant Protection Service Servizio fitosanitario regionale Via di Saliceto, n.81, 40128 Bologna (IT). Plant protection Service, P.O. Box 9102, 6700 HC Wageningen (NL). Department of Plant Protection Biology – Nematology, Swedish University of Agricultural Sciences, Alnarp (SE). 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: Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Toppheideweg 88, 48161 Münster (DE); Plant Protection Service, Diagnostic Centre, P.O. Box 9102, 6700 HC Wageningen (NL); Istituto per la Protezione delle Piante, Sezione di Bari, C.N.R., Via G. Amendola, 165/A, 70126 Bari (IT); Pest and Disease Identification Team, Central Science Laboratory, Sand Hutton, York YO41 1LZ (GB); Department of Plant Protection Biology – Nematology, Swedish University of Agricultural Sciences, Alnarp (SE). 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: D. Sturhan, retired from formerly Federal Biological Research Centre for Agriculture and Forestry, now Julius Kühn Institute (DE). The molecular part has been added by 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 Andersson S (1971) [The potato rot nematode, Ditylenchus destructor Thorne, as parasite in potatoes] In Swedish with English summary. Dissertation. Agricultural College of Sweden, Uppsala (SE). Brzeski MW (1991) Review of the genus Ditylenchus Filipjev, 1936 (Nematoda: Anguinidae). Revue de Nematologie 14, 9– 59. Brzeski MW (1998) Nematodes of Tylenchina in Poland and temperate Europe. Muzeum i Instytut Zoologii Polska Akademia Nauk, Warsaw (PL). 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. R Cook & DJ Hunt), pp. 633– 653. E.J. Brill, Leiden (NL). Decker H (1969) Phytonematologie. VEB Deutscher Landwirtschaftsverlag, Berlin (DE). Dunning RA (1957). Stem eelworm invasion of seedling sugar beet and development of crown canker. Nematologica 2 (Suppl.), 362– 8. Esquibet M, Bekal S, Castagnone-Sereno P, Gauthier JP, Rivoal R & Caubel G (1998). Differentiation of normal and giant Vicia faba populations of the stem nematode Ditylenchus dipsaci: agreement between RAPD and phenotypic characteristics. Heredity 81, 291– 298. Esquibet M, Grenier E, Plantard O, Andaloussi A & Caubel G (2003) DNA polymorphism in the stem nematode Ditylenchus dipsaci: Development of diagnostic markers for normal and giant races. Genome 46, 1077– 1083. Greco N, Brandonisio A & Boncoraglio P. (2002). Investigations on Ditylenchus dipsaci damaging carrot in Italy. Fourth International Congress of Nematology , 2002-06-8/13, Tenerife, Canary Islands, Spain. Nematology 4, 210 (abstract). Kerkoud M, Esquibet M, Plantard O, Avrillon M, Guimier C, Franck M et al . (2007). Identification of Ditylenchus species associated with Fabaceae seeds based on a specific polymerase chain reaction of ribosomal DNA-ITS regions. European Journal of Plant Pathology 118, 323– 332. Marek M, Zouhar M, Rysanek P & Havranek P (2005). Analysis of ITS sequences of nuclear rDNA and development of a PCR-based assay for the rapid identification of the stem nematode Ditylenchus dipsaci (Nematoda: Anguinidae) in plant tissues. Helminthologia 42, 49– 56. Sambrook J, Fritsch EF & Maniatis T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (US). Sturhan D & Brzeski MW (1991) Stem and bulb nematodes, Ditylenchus spp. In: Manual of Agricultural Nematology (Ed. WR Nickle), pp. 423– 464. Marcel Dekker, Inc., New York (US). Subbotin SA, Madani M, Krall E, Sturhan D & Moens M. (2005). Molecular diagnosis, taxonomy, and phylogeny of the stem nematode Ditylenchus dipsaci species complex based on the sequences of the internal transcribed spacer-rDNA. Phytopathology 95, 1308– 1315. Vrain TS, Wakarchuk DA, Levesque AC & Hamilton RI (1992) Interspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fundamental and Applied Nematology 16, 563– 573. Webster JM, Anderson RV, Baillie DL, Beckenbach K, Curran J & Rutherford T (1990). DNA probes for differentiating isolates of the pinewood nematode species complex. Revue de Nématologie 13, 255– 263. Wendt KR, Vrain TC & Webster JM (1993). Separation of three species of Ditylenchus and some host races of D. dipsaci by restriction fragment length polymorphism. Journal of Nematology 25, 555– 563. Zouhar M, Marek M, Douda O, Mazáková J & Ryšánek P (2007). Conversion of sequence-characterized amplified region (SCAR) bands into high-throughput DNA markers based on RAPD technique for detection of the stem nematode Ditylenchus dipsaci in crucial plant hosts. Plant Soil and Environment 53, 97– 104. Appendix 1 ITS-RFLP analysis according to Wendt et al. (1993) This protocol is a quite old and some of the reagents and equipments may have changed. It is strongly recommended to validate this protocol in the operator's laboratory conditions. 1. General information 1.1 Protocol developed by Wendt et al. (1993) 1.2 Individuals from D. dipsaci (nomal races and giant race), D. destructor and D. myceliophagous are extracted from plant material 1.3 The targeted region is the ITS region (including ITS1, ITS2 and 5.8S rDNA gene). They are not specific regions 1.4 Oligonucleotides: ITS–specific universal primers described by Vrain et al. (1992): 18S (5′-TTG ATT ACG TCC CTG CCC TTT-3′) and 26S (5′-GGA ATC ATT GCC GCT CAC TTT-3′). The amplicon is approximately 900 bp for D. dipsaci and D. myceliophagus, and 1200 bp for D. destructor 1.5 Amplification mix is provided in a kit containing Taq DNA Polymerase, nucleotides and reaction buffer (Perkin Elmer) 1.6 Amplification is performed in a thermocycler, e.g. Twin Block System EC Cycler (Ericomp). 2. Methods 2.1 Nucleic acid Extraction and Purification 2.1.1 Nematodes that had migrated to the lid of a Petri dish were rinsed off with 0.05 M NaCl, and concentrated by centrifugation at 2000 rpm for 2 min at room temperature. The NaCl solution was discarded and the nematodes were resuspended in seven volumes of Proteinase K buffer (0.1 M Tris pH = 8.0, 0.05 M EDTA, 0.2 M NaCl and 1% SDS) containing 1.0 mg mL−1 proteinase K. The solution containing the nematodes was frozen in liquid nitrogen, transferred to a mortar and ground into a fine powder. After thawing the solution was transferred to a 50 mL Falcon tube and was extracted three times with TE (10 mM Tris pH = 8.0 and 1.0 mM EDTA) saturated phenol pH 8.0 and twice with 24:1 chloroform, iso-amyl alcohol. The clean DNA was precipitated by two volumes of 95% ethanol, pelleted, dried and redissolved in TE with 10 µg mL−1 RNAse A (Webster et al., 1990) 2.1.2 Store overnight at 4°C or at −20°C for longer periods 2.2 Polymerase Chain reaction 2.2.1 Master mix (provided within kit), 1.5 µM of each Vrain primers and 20 µg of DNA 2.2.2 PCR cycling parameters 1 cycle of 1.5 min 94°C, 30 s at 50°C, 4 min at 72°C; 40 cycles of 45 s at 96°C, 30 s at 50°C, 4 min at 72°C; 1 cycle of 45 s at 96°C, 30 s at 50°C, and a final extension of 10 min at 72°C 2.3 Restriction of PCR amplicon 2.3.1 Restriction conditions According to supplier's conditions (Gibco Co., BRL). 3. Essential Procedural Information 3.1 Analysis of DNA fragments: DNA fragments are separated by electrophoresis on agarose gel and visualized under UV light according to standard procedures (e.g. Sambrook et al., 1989) 3.2 Identification of species. Restriction patterns for different species are presented in Table 3. Table 3. Restriction patterns of D. dipsaci (host races and giant race), D. destructor and D. myceliophagus with different restriction enzymes (−: data not available) (length of restriction bands in bp) D. dipsaci (normal races*) D. dipsaci (giant race) D. destructor D. myceliophagus Unrestricted PCR product 900 900 1200 900 AccI 900 − 1200 900 AluI 900 − 370, 290 900 BamHI 340, 220, 180 − 1000 900 DdeI 310, 290, 200 310, 290, 200 670, 570 300, 250, 130 DraI 340, 250 − 1200 900 HaeIII 900 800, 200 450, 170 450, 200 HincII 800 800 900, 250 900 HinfI 440, 350, 150 350, 150 780, 180 630, 310 HpaII 320, 200, 180 600, 200 1000 900 NsiI 900 − 1200 900 PstI 650, 400 650, 400 850, 400 620, 400 RsaI 450, 250, 140 490, 450 600, 250, 170 900 Sau3A 340, 260, 200, 110, 100 − 540, 400, 180 440, 100 TaqI 340, 230, 130 − 640, 200, 150 320, 260, 160 *Referred to as «host race» in Wendt et al. (1993). 3.3 A negative control (no DNA target) should be included in every experiment to test for contamination as well as a positive control (DNA from a reference strain of the pathogen) 3.4 The method can only be used on nematodes morphologically identified as Ditylenchus sp., as the primers are not specific for Ditylenchus spp. Appendix 2 Specific SCAR-PCR according to Esquibet et al. (2003) 1. General information 1.1 Protocol developed by Esquibet et al. (2003) 1.2 Individuals from D. dipsaci (host races and giant race) and D. myceliophagous are extracted from plant material 1.3 The targeted regions are unknown as the primers were Sequence Characterized Amplified Region (SCAR) 1.4 Oligonucleotides: for D. dipsaci (host races) H05 (5′-TCA AGG TAA TCT TTT TCC CCA CT-3′) and H06 (5′-CAA CTG CTA ATG CGT GCT CT-3′); for D. dipsaci (giant race) D09 (5′-CAA AGT GTT TGA TCG ACT GGA-3′) and D10 (5′-CAT CCC AAA ACA AAG AAA GG-3′). The amplicon is approximately 242 bp for D. dipsaci (host race) and 198 bp for D. dipsaci (giant race) 1.5 Taq DNA Polymerase 5 U µL−1 (Promega) is used for PCR amplification 1.6 Nucleotides are used at a final concentration of 0.25 mM each 1.7 MgCl2 final concentration is 1.5 mM 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. 2. Methods 2.1 Nucleic acid Extraction and Purification (Esquibet et al., 1998) 2.1.1 DNA was extracted from 2500–25 000 frozen mixed larvae and adults prepared using a phenol-chloroform procedure: the nematodes were rinsed in a microcentrifuge and ground in a pestle. Total genomic DNA was extracted according to the phenol-chloroforme procedure (Sambrook et al., 1989). Following ethanol precipitation, DNA was resuspended in TE buffer [0.01 M Tris (pH 8.0), 0.001 M EDTA] 2.1.2 Store overnight at 4°C or at −20°C for longer periods 2.2 Polymerase Chain reaction 2.2.1 Master mix include: 1.5 mM MgCl2 250 µM each dNTP 690 nM each primer for duplex PCR (H05-H06) or (D09-D10) or 500 nM each primer for multiplex PCR (H05-H06-D09-D10) 0.5 U Taq DNA polymerase (Promega) 2.2.2 PCR cycling parameters: 30 cycles of 1 min at 94°C, 1 min at 59°C, 1 min at 72°C. 3. Essential Procedural Information 3.1 Analysis of DNA fragments: DNA fragments are separated