PM 7/40 (5) Globodera rostochiensis and Globodera pallida

Abstract

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