Bulletin OEPP/EPPO Bulletin (2017) 47 (2), 174–197 ISSN 0250-8052. DOI: 10.1111/epp.12391 European and Mediterranean Plant Protection Organization Organisation Europe´enne et Me´diterrane´enne pour la Protection des Plantes PM 7/40 (4) Diagnostics Diagnostic PM 7/40 (4) Globodera rostochiensis and Globodera pallida Specific scope Specific approval and amendment This Standard describes a diagnostic protocol for Approved as an EPPO Standard in 2003-09. Globodera rostochiensis and Globodera pallida.1 Revisions approved in 2009-09, 2012-09 and 2017-02. Terms used are those in the EPPO Pictorial Glossary of Morphological Terms in Nematology.2 This Standard should be used in conjunction with PM 7/ 76 Use of EPPO diagnostic protocols. of imported material for potential quarantine or damaging 1. Introduction nematodes or new infestations, identification by morpholog- Globodera rostochiensis and Globodera pallida (potato cyst ical methods performed by experienced nematologists is nematodes, PCNs) cause major losses in Solanum more suitable (PM 7/76 Use of EPPO diagnostic tuberosum (potato) crops (van Riel & Mulder, 1998). The protocols). infective second-stage juveniles only move a maximum of A flow diagram describing the diagnostic procedure for about 1 m in the soil. Most movement to new localities is G. rostochiensis and G. pallida is presented in Fig. 1. by passive transport. The main routes of spread are infested seed potatoes and movement of soil (e.g. on farm machin- 2. Identity ery) from infested land to other areas. Infestation occurs when the second-stage juvenile hatches from the egg and Name: Globodera rostochiensis (Wollenweber, 1923), enters the root near the growing tip by puncturing the epi- Skarbilovich, 1959. dermal cell walls, and then internal cell walls, with its sty- Synonyms: Heterodera rostochiensis, Wollenweber, 1923; let. Eventually it begins feeding on cells in the pericycle, Heterodera schachtii solani Zimmerman, 1927; Heterodera cortex or endodermis. The nematode induces enlargement schachtii rostochiensis (Wollenweber) Kemner, 1929; of the root cells and breakdown of their walls to form a Taxonomic position: Nematoda, Tylenchida3, Heteroderi- large, syncytial transfer cell. This syncytium provides nutri- dae ents for the nematode. Infested potato plants have a reduced EPPO Code: HETDRO root system and, because of the decreased water uptake, Phytosanitary categorization: EPPO A2 List no. 125, EU death of the plant can eventually occur. Annex designation I/A2 In this Diagnostic Protocol different tests for detection Name: Globodera pallida (Stone, 1973). and identification are presented which can be used depend- Synonyms: Heterodera pallida Stone, 1973 ing on the circumstances. In some EPPO countries official Taxonomic position: Nematoda, Tylenchida3, Heteroderi- control is in place and routine testing is required. For such dae routine testing in the country itself molecular techniques EPPO Code: HETDPA can be very useful. In other situations, such as the testing Phytosanitary categorization: EPPO A2 List no. 124, EU Annex designation I/A2 1Use of brand names of chemicals or equipment in these EPPO Stan- dards implies no approval of them to the exclusion of others that may also be suitable. 3Developments combining a classification based on morphological data 2http://www.eppo.int/QUARANTINE/diag_activities/EPPO_TD_1056_ and molecular analysis refer to ‘Tylenchomorpha’ (De Ley & Blaxter, Glossary.pdf 2004). 174 ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 174–197 PM 7/40 (4) Globodera rostochiensis and G. pallida 175 Soil sample Extraction (cysts and debris) G. pallida or Isolation of cysts No G. rostochiensis not identified Morphological identification to genus level Molecular test for detection and/or identification Yes Globodera sp. identified No (for direct testing on floats see Appendix 7) Yes Yes Empty cyst? No Identification at species level not Morphological identification based possible on perineal patterns and juveniles (see Table 1) Negative uncertain (overlap of values) Positive Negative Molecular Positive detection (Appendices 3, 4, 5, 6, 8) G. pallida or Positive G. rostochiensis Negative not identified G. pallida or G. pallida or G. rostochiensis G. rostochiensis identified identified Fig. 1 Flow-diagram for identification of G. rostochiensis and G. pallida. Note that identification of G. rostochiensis and G. pallida should preferably combine morphological and molecular methods, especially when new introductions are suspected. ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 174–197 176 Diagnostics 3. Detection 3.1 Symptoms Above-ground symptoms due to PCNs are not specific and often go undetected. General symptoms include patches of poor growth in the crop, with plants sometimes showing yellowing, wilting or death of foliage; tuber size is reduced and roots are extensively branched with soil stuck to them. However, there are many other causes of these symptoms. Plants should therefore be lifted for a visual check for the presence of cysts and young females on the roots, or a soil sample should be taken for testing. Young females and cysts are just visible to the naked eye as tiny white, yellow Fig. 3 Broken cyst with eggs of G. pallida. (Courtesy: Plant Protection Service, NL.) or brown pin-heads on the root surface (Figs 2 and 3). Detection by lifting plants is only possible for a short time nematode cysts. Prior to identification, cysts need to be as females mature into cysts and then can easily be lost at removed from the floats. This process usually requires lifting, and it is time-consuming. Soil testing is therefore examination of the float by staff trained in separating nema- the best way to determine the presence of PCNs. tode cysts from similar globular bodies in the soil. It can be time-consuming, depending upon the efficiency of 3.2 Statutory sampling procedures extraction and whether any further clean-up has been used, e.g. acetone flotation. This process is critical to the effi- Recommendations on sampling can be found in Council ciency of the diagnosis because false negative results may Directive 2007/33/EC of 11 June 2007 on the control of result if any Globodera cysts are missed at this stage. The PCN and Repealing Directive 69/465/EEC (EU, 2007). distinction between PCNs and other cysts based on mor- phology can only be reliably performed by trained experts. 3.3 Extraction procedures When moist soil samples are not immediately processed and viability tests are envisaged, they should be stored above There are various processes for extracting cysts from the zero and below 5°C as temperature influences hatching beha- soil. Simple methods based on flotation can be as good as viour (Muhammad, 1996; Sharma & Sharma, 1998). Soil elutriation. Extraction methods are described in PM 7/119 samples should not be dried at a temperature higher than Nematode extraction. Globodera cysts are generally round, approximately 35°C as this might also influence the viability. which distinguishes them from most other types of 3.4 Bioassay Another procedure for detecting the nematodes is bioassay (Appendix 1, test A). 3.5 Direct testing of soil extracts A test for the direct detection and identification of G. rostochiensis and G. pallida in soil extracts/floats (cysts and debris) using real-time PCR has been developed by Reid et al. (2015) and is described in Appendix 7. 4. Identification Identification of G. rostochiensis and G. pallida should preferably combine morphological and molecular methods, especially when new introductions are suspected. For identification based on morphology, second-stage juveniles and cysts should be obtained from soil, plant roots or tubers. The colour of the female at the appropriate stage of development can be used as an indication of species: a Fig. 2 Potato roots infected by G. rostochiensis. (Courtesy: Plant female which changes during maturation from white to yel- Protection Service, NL.) low then into a brown cyst is G. rostochiensis, while one ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 174–197 PM 7/40 (4) Globodera rostochiensis and G. pallida 177 which changes from white directly to brown is G. pallida. Identification of cysts and other stages is in general based on a combination of morphological and morphometric char- acters and/or molecular methods. In well-defined circum- stances molecular tests alone can be applied. Differential interference contrast is highly recommended for identifying specimens mounted on microscope slides. 4.1 Identification on the basis of morphological features 4.1.1 Identification of cyst and juveniles to genus level 4.1.1.1 Cysts. Identification of Heteroderidae cysts to genus level is based on the form of the cysts and the char- acteristics of the vulval–anal region (Table 1 and Figs 4– Fig. 5 The perineal region of a Globodera cyst (Hesling, 1978). 7). Further information is provided by keys of Brzeski (1998), Baldwin & Mundo-Ocampo (1991), Wouts & Bald- soil extracts after extraction for the detection of non-seden- win (1998), Siddiqi (2000) and Subbotin et al. (2010). tary stages of nematodes. A dichotomous key to genus of Heteroderidae cysts is Distinction between juveniles of Globodera and other presented below: Heteroderidae is difficult; in such cases it is strongly 1 Lemon-shaped cyst Not Globodera advised to perform a cyst extraction where possible or to Round cyst 2 perform a molecular test on the juveniles (see section 4.2) 2 Two large separated fenestrae of equal size Not Globodera and to proceed with this Diagnostic Protocol. Some infor- One large vulval fenestra Globodera mation, however, is provided below. Globodera juveniles should present the following charac- Globodera cysts should present the following characteris- teristics: tics: The mobile second-stage juveniles of Globodera are ver- Cysts of Globodera are smoothly rounded with a small miform, annulated, and taper at head and tail regions. projecting neck, no terminal cone, diameter Æ 450 lm, and Within the genus Globodera, body length ranges from 445 with a tanned brown skin (Fig.