PM 7/40 (5) <i>Globodera rostochiensis</i> and <i>Globodera pallida</i>

EPPO BulletinEarly View STANDARD ON DIAGNOSTICSFree Access PM 7/40 (5) Globodera rostochiensis and pallida First published: 09 May 2022 https://doi.org/10.1111/epp.12836AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text full-text accessPlease review our Terms Conditions of Use check box below share version article.I have read accept the Wiley Online Library UseShareable LinkUse link a this article with your friends colleagues. Learn more.Copy URL Share linkShare onFacebookTwitterLinked InRedditWechat Specific scope: This Standard describes Diagnostic Protocol for pallida.11 brand names chemicals or equipment in these Standards implies no approval them exclusion others that may also be suitable. The terms used are those Pictorial Glossary Morphological Nematology.22 http://www.eppo.int/QUARANTINE/diag_activities/EPPO_TD_1056_Glossary.pdf. should conjunction 7/76 diagnostic protocols. Authors contributors given Acknowledgements section amendment: Approved as an 2003-09. Revisions approved 2009-09, 2012-09 2017-02. Fourth revision 2021-10. 1 INTRODUCTION (potato cyst nematodes, PCNs) cause major losses Solanum tuberosum (potato) crops (van Riel & Mulder, 1998). main route spread nematodes is movement infested soil (e.g. on farm machinery, adhering tubers). Infestation occurs when second-stage juvenile hatches from egg enters root near growing tip by puncturing epidermal cell walls, then internal its stylet. Eventually it begins feeding cells pericycle, cortex endodermis. nematode induces enlargement breakdown their walls form large, syncytial transfer cell. syncytium provides nutrients nematode. Infested potato plants reduced system and, because decreased water uptake, death plant can eventually occur. In different tests detection identification presented which depending circumstances. some countries, official control place routine testing required. For such country itself molecular techniques very useful. other situations, imported material potential quarantine damaging new infestations, morphological methods performed experienced nematologists more suitable (PM protocols). A flow diagram describing procedure G. Figure 1. FIGURE 1Open figure viewerPowerPoint Flow-diagram 2 IDENTITY Name: (Wollenweber, 1923), Skarbilovich, 1959. Synonyms: Heterodera rostochiensis, Wollenweber, 1923; schachtii solani Zimmerman, 1927; (Wollenweber) Kemner, 1929; Taxonomic position: Nematoda, Tylenchida,33 Developments combining classification based data analysis refer ‘Tylenchomorpha’ (De Ley Blaxter, 2004). Heteroderidae. Code: HETDRO. Phytosanitary categorization: A2 List no. 125, Quarantine pest (Annex II B). (Stone, 1973). Tylenchida,3 HETDPA. 124, Note taxonomy: noted recent study, Thevenoux et al., 2020, has shown presence larger genetic diversity than previously known, suggesting species south Peru. 3 DETECTION 3.1 Symptoms Above-ground symptoms due PCNs not specific often go undetected. General include patches poor growth crop, sometimes showing yellowing, wilting foliage; tuber size roots extensively branched stuck them. However, there many causes symptoms. Plants therefore lifted visual cysts young females roots, sample taken testing. Young just visible naked eye tiny white, yellow brown pin-heads surface (Figures 3). Detection lifting only possible short time mature into easily lost at lifting, method time-consuming. Soil best way determine PCNs. 2Open Potato infected rostochiensis. (Courtesy: NRC-NPPO, Netherlands.) 3Open Broken eggs pallida. 3.2 Statutory sampling procedures Recommendations found Council Directive 2007/33/EC 11 June 2007 PCN Repealing 69/465/EEC (EU, 2007). 3.3 Extraction There various processes extracting soil. Simple flotation good elutriation. described 7/119 Nematode extraction (EPPO, 2013). generally round, distinguishes most types cysts. Prior identification, need removed floats. process usually requires examination float staff trained separating similar globular bodies It time-consuming, upon efficiency whether any further clean-up been used, e.g. acetone flotation. critical diagnosis false-negative results result if missed stage. distinction between morphology reliably experts. When moist samples immediately processed viability envisaged, they stored above zero 5°C temperature influences hatching behaviour (Muhammad, 1996; Sharma Sharma, dried higher approximately 35°C might influence viability. Educational videos available website European Union Reference Laboratory Plant Parasitic Nematodes (https://sitesv2.anses.fr/en/minisite/plant-parasitic-nematodes/videos-media). 3.4 Bioassay Another detecting bioassay (Appendix 1, test A). 3.5 Direct extracts following Appendix nucleic acid extraction. Test Multiplex real-time PCR (Gamel 2017) High-throughput (Globodera spp.) using (Reid 2015) 4 Real-time species-specific well tabacum (based LSU rDNA), all-inclusive kit (http://www.cleardetections.com) 5 IDENTIFICATION stages, highly recommended combine methods, especially introductions suspected. 4.1 Identification basis features examination, juveniles obtained soil, tubers. colour female appropriate stage development indication species: changes during maturation white while one directly Differential interference contrast identifying specimens mounted microscope slides. 4.1.1 genus level 4.1.1.1 Cysts Heteroderidae characteristics vulval–anal region (Table Figures 4-7). Further information provided keys Brzeski (1998), Baldwin Mundo-Ocampo (1991), Wouts Siddiqi (2000) Subbotin al. (2010). Table Dichotomous key Lemon-shaped Not Round oval Two separated fenestrae equal Punctodera One large vulval fenestra 4Open Form region. (After Mundo-Ocampo, 1991.) 5Open perineal (Hesling, 1978) 6Open Scale bar =350 μm. (Courtesy NAK, 7Open Perineal Green arrows indicate vulva black anus. spp. fenestra/anal non-fenestrate. fenestrate. present characteristics: smoothly rounded small projecting neck, terminal cone, diameter ±450 μm, tanned skin (Figure 6a). cuticle zigzag pattern ridges. area 7a) consists single circumfenestration around slit, tubercules crescents vulva. anus subterminal without fenestra, basin; underbridge bullae rarely (Fleming Powers, 1998), particular Eggs retained cyst, egg-mass present. 4.1.1.2 Juveniles addition cysts, incidentally after non-sedentary stages nematodes. Distinction difficult; cases strongly advised perform where (see Section 4.2) proceed Protocol. Some information, however, below. mobile vermiform annulated, taper head tail regions. Within Globodera, body length ranges 445 510 stylet 18–29 37–55 μm hyaline part 21–31 distinguished root-knot (Meloidogyne heavily sclerotized lip region, relatively strong stylet, shape robust appearance 8). juveniles. 8Open Difference Meloidogynidae Comparison Meloidogyne hapla FERA, GB) mean average morphometric characters assist differentiation, owing overlap ranges. If population carried out only, compare taxonomic descriptions 3. stated above, 2. (after (2010)) Cuticle thin, transparent mali thick, dark Mean J2 ≤26 ≥27 zelandica <19 leptonepia ≥19 Hyaline >31 bravoae ≤31 Granek’s ratio >2, mostly parasites Solanaceae 6 ≤2, Asteraceae Combination of: DGO ≥5.5 μm; <3; 4–6 annules, knobs slightly anteriorly projected 7 combination all characters; <5.5 8 Cyst wall lacking network-like pattern, ridges close; number cuticular = 13 (10–18); ♂ spicules pointed, thorn-like ellingtonae exhibiting maze-like patterns; 7–8 (5–15); finely sensu lato prominent specimens; >28 capensis abullate, <28 9 distinctly directed flattened anteriorly; >23 <3 10 <23 ≥3 2.1–2.5 2.8 mexicana 5–6 annules 12 ≥25 J2, gubernaculum 11.2–12.9 millefolii <25 6.0–9.9 artemisiae useful species, range values µm Lownsbery Lownsbery, 1954; Eroshenko Kazachenko 1972; Golden Klindic, 1973; Stone, 1973a b, 1991; Mota Eisenback, 1993; Brzeski, 1998; Flemming Manduric Anderson, 2004) Species measurements Knob width Stylet Number basin 468 (425–505) 3–4 Rounded Anteriorly 21.8 (19–23) 12–31b b From Powers (1998); (1998) refers 16–31. (usually >14) 1.3–9.5 (>3) 484 (440–525) 4–5 Distinct forward projections 23.8 (22–24) 8–20 <14) 1.2–3.5 (<3) 476 (410–527) 24 (22–26) 5–15 1–4.2 (<2.8) millefoliia Krall (1978) considered (Kirjanova Krall, 1965) Behrens, 1975 inquirenda, description was female. reported achilleae: ‘it conspecific millefolii’. According 2010, 2011 achilleae junior synonym millefolii. So point onwards name will instead. 492 (472–515) 25 (24–26) 4–11 1.6 (1.3–1.9) 413 (357–490) 3–5 22.6 (18–29) 5–16 1.0 (0.8–1.7) 4.1.2 difficult observed variability characteristics. Therefore, use reliable identification. identified morphologically morphometrically closely related 1973a,b). presents drawings 9a) 9b). important differences area, i.e. 10a,b). knob 3, 10c). As each care needed. cases, confirmation recommended. populations publications natural deviations 9Open Illustrations left-hand side plate (side labelled bold), rostochiensis: (a) entire juvenile; (b) (c) lateral field, mid-body; (d) pharyngeal (e) male; (f) (g) field male, (h) cysts; (i) neck female; (j) male. C.I.H. Descriptions Plant-Parasitic Nematodes, Set 2, No. 16.) right-hand B juvenile: entire; anterior; head; tail; mid-body region; face lips; base. Stone (1972).) 10Open Vulval–anal ridge patterns four species. Stylets Globodera. See footnote (Section 4.1.2) achilleae. Fleming 1998.) live content, meaning do contain viable juveniles, found, possible.44 under conditions, content detected fields production past, probable belong either An educational video (perineal features) three could confusion Europe 1975,55 conspecific but (Eroshenko Kazachenko, 1972) 1975, lato. first two parasitic recorded Achillea millefolium Artemisia vulgaris, respectively, comparable agricultural areas. complex (G. (Lownsbery 1954) 1959; solanacearum (Miller Gray, virginiae 1975) North Central America. Southern Europe. parasitizes Nicotiana (tobacco) solanaceous (but potato). provide comparison millefolii, tabacum. Eisenback (1993), (2010) detailed members keys. Additionally, described, ellingtonae, Oregon, USA (Handoo 2012) Argentina (Lax 2014), capensis, South Africa (Knoetze minute distinction. locally USA, so far. sandveldensis agulhasensis, both parasitizing non-Solanaceae plants, 2017a b) inclusion subsequent revision. 4.2 Molecular related, several polymerase chain reaction (PCR)-based developed separate Appendices 3–9. were distinguish specifically tested far against mexicana. limitation noted. Tests 2000 shortcomings. restriction fragment polymorphism (RFLP) Sirca non-European made sequencing (Hockland 2012). (Skantar 2007) allows DNA barcoding support preferably 4.2.1 isolated individuals pallida: performance vary (in regard analytical specificity) choice according circumstances use. 4. rDNA) multiplex primers ribosomal 18S ITS1 sequences Bulman Marshall (1997) transcribed spacer (ITS)-RFLP Vrain (1992) (Thiéry Mugniéry, 1996) Taqman® targeting I (ITSI) gene (Fera) RNA-specific RT-PCR (Beniers 2014) 4.2.2 protocol COI, rDNA 28S 7/129 tool regulated pests: 2016) Sequences databases including Q-bank (https://qbank.eppo.int/nematodes/). 4.3 Pathotypes term ‘pathotype’ International Pathotype Scheme proposed Kort (1977) now too general. Many cannot conclusively assigned pathotypes scheme. virulence pallida, utmost importance America, adequate moment time-consuming expensive Any signs unusual (i.e. overcoming resistance currently cultivars Europe) soon possible. practice, set country. Standard, 3/68 Testing varieties assess 2021). 4.4 required regulatory purposes. done methods. Visual determination (a table figures 10). These observations require personnel. Determination bioassay. Such tests. Dormancy play role lifted. additional aspect bioassays possibility low content. Three 11. determining test, formed recently dormant autumn harvest). To break dormancy, exposed +4 °C least months. trehalose. 12, van den Elsen (2012) Ebrahimi (2015) RNA. 9, publication Beniers (2014). staining Meldola’s Blue possible, chemical available, technique REFERENCE MATERIAL from: National Protection Organization, Centre, PO Box 9102, 6700 HC Wageningen (the Netherlands); Food Environmental Research Agency (Fera), Sand Hutton, York YO41 1LZ (GB); Julius Kühn-Institut (JKI), Federal Centre Cultivated Plants, Messeweg 11–12, 38104 Braunschweig (Germany). French Institute Agricultural (INRAe) Biology Organisms Populations Domaine de la Motte, BP 35327, 35653 Le Rheu Cedex (France). REPORTING AND DOCUMENTATION Guidance reporting documentation 7/77 Documentation diagnosis. PERFORMANCE CRITERIA criteria test. validation Database Expertise (http://dc.eppo.int), consult database specificity, reports, etc.). FURTHER INFORMATION organism E. Heese G Karssen,