Bulletin OEPP/EPPO Bulletin (2017) 47 (2), 174–197 ISSN 0250-8052. DOI: 10.1111/epp.12391

European and Mediterranean Plant Protection Organization Organisation ´enne et Me´diterrane´enne pour la Protection des Plantes PM 7/40 (4)

Diagnostics Diagnostic

PM 7/40 (4) and

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 or new infestations, identification by morpholog- Globodera rostochiensis and Globodera pallida ( 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: rostochiensis, Wollenweber, 1923; let. Eventually it begins feeding on cells in the pericycle, Heterodera schachtii solani Zimmerman, 1927; Heterodera cortex or endodermis. The 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.

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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 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 450 lm, and Within the genus Globodera, body length ranges from 445 with a tanned brown skin (Fig. 6A). The cuticle surface has to 510 lm, stylet length is 18–29 lm, tail length 37–55 lm a zigzag pattern of ridges; a distinct D-layer is present. The and the hyaline tail part 21–31 lm. perineal area (Figs 5 and 7A) consists of a single circum- Juveniles of cyst nematodes can be distinguished from fenestration around the vulval slit, with tubercules on cres- juveniles of root-knot nematodes (Meloidogyne spp.) by a cents near the vulva. The anus is subterminal without more heavily sclerotized lip region, relatively strong stylet, fenestra, the vulva is in a vulval basin; underbridge and shape of the tail and more robust appearance (Fig. 8). In bullae are rarely present (Fleming & Powers, 1998), and in such cases it is advised to perform a cyst extraction or to particular not present in G. rostochiensis and G. pallida. perform a molecular test on the juveniles. Eggs retained in cyst, no egg-mass present. 4.1.2. Identification to species level 4.1.1.2 Juveniles. In addition to the juveniles in cysts, The identification of Globodera to species level based on juveniles of cyst nematodes may be found incidentally in morphology can be difficult because of the observed

1- Spherical (Globodera) 2- Ovoid (Punctodera) 3- Lemonshape with reduced cone (some Heterodera or Cactodera) 4- Lemonshape including prominent cone (most Heterodera)

a : Anal region fenestrate, vulva region circumfenestrate (Punctodera) b : Anal region non fenestrate, vulva region circumfenestrate, (Globodera & Cactodera) c : Anal region non fenestrate, vulva region semi fenestrate-bifenestrate (Heterodera) d : Anal region non fenestrate, vulva region semi Fig. 4 Form of cysts and characteristics of fenestrate-ambifenestrate (Heterodera) the vulval-anal region. (After Baldwin & Vulva Mundo-Ocampo (1991).)

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Globodera spp. Heterodera spp. Punctodera spp.

Fig. 6 Heteroderidae cysts. Scale bar = 350 lm. (Courtesy NAK, NL.)

Fig. 7 Perineal region. Green arrows indicate the vulva, black arrows the anus. Globodera spp. vulval fenestra/anal region non- Globodera sp. vulval fenestra/anal region Punctodera sp. vulval fenestra/anal fenestrate. Punctodera spp. vulval fenestra/ non fenestrate region fenestrate anal region fenestrate. (Courtesy NAK, NL.)

Table 1. 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 lm 3 Mean length of J2 stylet ≥27 lm G. zelandica 3 Mean length of J2 stylet <19 lm G. leptonepia Mean length of J2 stylet ≥19 lm 4 4 Hyaline region of J2 > 31 lm G. bravoae Hyaline region of J2 ≤ 31 lm 5 5 Mean Granek’s ratio usually >2, mostly parasites of 6 Mean Granek’s ratio ≤2, mostly parasites of Asteraceae 11 6 Combination of: mean J2 DGO ≥5.5 lm; mean Granek’s ratio <3; J2 lip region with 4–6 7 annules, stylet knobs rounded to slightly anteriorly projected Not with the above combination of all characters; mean J2 DGO <5.5 lm8 7 Cyst wall lacking a network-like pattern, ridges close; mean number of cuticular ridges = 13 (10–18); ♂ G. ellingtonae spicules with a pointed, thorn-like tip Cyst wall exhibiting network-like or maze-like patterns; mean number of cuticular ridges = 7–8(5–15); G. tabacum ♂ spicules with a finely rounded tip 8 Cysts with prominent bullae in the terminal region of most specimens; J2 lip region with 3 annules, G. capensis mean hyaline region >28 lm Cyst abullate, at most with small vulval bodies in some specimens; J2 lip region with 4–6 annules, 9 mean hyaline region <28 lm 9 J2 stylet knobs distinctly anteriorly directed to flattened anteriorly; mean J2 stylet length >23 lm; Granek’s ratio <310 J2 stylet knobs rounded to flattened anteriorly; mean J2 stylet length <23 lm; 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 lminJ2,♂ gubernaculum = 11.2–12.9 lm G. millefolii Mean stylet <25 lminJ2,♂ gubernaculum = 6.0–9.9 lm G. artemisiae

The morphological key to Globodera species presented above has used the mean average of morphometric characters to assist with differentiation, due 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 2.

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G. pallida tail G. pallida habitus G. pallida anterior

20 µm 100 µm 20 µm

M. hapla tail M. hapla habitus M. hapla anterior

Fig. 8 Difference between Meloidogynidae and Heteroderidae juveniles. Comparison between Meloidogyne hapla and G. pallida. (Courtesy Fera.)

variability of key characteristics. Therefore the use of a G. rostochiensis (Fig. 9A) and G. pallida (Fig 9B). For combination of cyst and second-stage juvenile characteris- cysts, the most important diagnostic differences are in the tics is recommended for reliable identification. First the perineal area, i.e. the number of cuticular ridges between the nematodes should be identified with the key presented in vulva and anus and Granek’s ratio (Fig. 10A,B). The second- Table 1. If the nematodes are identified as PCN species, stage juvenile characteristics are stylet length and stylet knob species identification should be performed using the mor- shape (Table 1, Fig. 10C). As the range of values for each of phological and morphometric characters presented in these characteristics can overlap between species care is Table 2. needed. In such cases, confirmation with molecular tech- Globodera rostochiensis and G. pallida are morphologi- niques is recommended. It should also be noted that this data cally and morphometrically closely related (Stone, 1973a,b). is for specific populations described in the publications and Figure 9 presents some drawings of different stages of that natural deviations from the range may occur.

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 174–197 180 Diagnostics

When cysts without live content, meaning they do not G. 1.7) 1.9) – – contain viable eggs or second stage juveniles, are found, 2.8) 3) 3) < < > species identification is not possible4. 4.2 ( 3.5 ( 9.5 ( The three other Globodera species which could cause con- – – – : ‘it may be fusion during identification of PCNs in Europe are Globodera millefolii (Kirjanova & Krall, 1965) Behrens, 5 Globodera artemisiae

will not be used and 1975 , (Eroshenko & Kazachenko,

G. achilleae 1972) Behrens, 1975, and Globodera tabacum sensu lato. These first two species are not parasitic on potato but have been recorded on Achillea millefolium and Artemisia G. achilleae vulgaris, respectively, in comparable agricultural areas. The G. tabacum species complex (G. tabacum tabacum (Lowns- bery & Lownsbery, 1954) Skarbilovich, 1959; G. tabacum solanacearum (Miller & Gray, 1972) Behrens, 1975, and (2010).) G. tabacum virginiae (Miller & Gray, 1972) Behrens, 1975)

et al. is found in North and Central America. Globodera tabacum tabacum is also present in Southern Europe. It parasitizes Nicotiana tabacum () and some other solanaceous plants (but not potato). Table 1 and Fig. 7 provide a morpho- 10) 1 10) 1.0 (0.8 14) 1.2 10) 1.6 (1.3 14)metric 1.3 and morphological comparison between the PCNs < < < < > G. millefolii5, G. artemisiae and G. tabacum. See also Bald- m. (After Subbotin 15 ( 16 ( 20 ( 11 ( 31 ( l – – – – – win & Mundo-Ocampo (1991), Mota & Eisenback (1993), Brzeski (1998) Wouts & Baldwin (1998) and Subbotin et al. . So from this point onwards the species name (2010) for more detailed information on other members of 26) 5 29) 5 24) 8 26) 4 23) 16 – – – – – the Heteroderidae and identification keys. Two new Globodera species have recently been G. millefolii described, Globodera ellingtonae, detected on potato in Oregon, USA (Handoo et al., 2012) and in Argentina (Lax

, as the description was based on a single female. Brzeski (1998) reported on et al., 2014) and Globodera capensis detected in a potato field in South (Knoetze et al., 2013). The differences

species, range and mean values in between this species and PCN species are minute and only molecular methods can allow a reliable distinction. The

is a junior synonym of species are only locally present in the USA, Argentina and species inquirenda

Globodera South Africa and have not been detected in Europe so far.

G. achilleae 4.2. Molecular methods As G. rostochiensis and G. pallida are morphologically clo- sely related, several polymerase chain reaction (PCR)-based

, 2010, 2011 tests have been developed to separate the two PCN species. Recommended molecular tests are described in Appen- et al. dices 3–8. It should be noted that many tests that were devel- oped especially to distinguish G. rostochiensis from 5 Rounded to slightly anteriorly projected 24 (22 5 Rounded to anteriorly flattened 23 (18 5 Distinct forward projections 23.8 (22 5 Rounded to anteriorly projected 25 (24 4 Rounded to anteriorly flattened 21.8 (19 – – – – – G. pallida have not been tested so far against species such as J2 styletKnob width Knob shape Stylet length Number ofG. cuticular ridges between anus and millefolii vulval basin Granek’s ratio , G. Cyst measurements tabacum or G. mexicana. This limitation (Kirjanova & Krall, 1965) Behrens, 1975 as

4 527) 4 490) 3 525) 4 515) 4 505) 3 It should be noted that under European conditions, especially when – – – – –

’. According to Subbotin cysts without live content have been detected in fields used for the pro-

G. millefolii duction of potato in the past, it is highly probable that these cysts 477 (410 413 (357 484 (440 492 (472 468 (425 belong to either one of the PCN species G. rostochiensis or G. pallida. 5Krall (1978) considered G. millefolii (Kirjanova & Krall, 1965) Beh-

G. millefolii rens, 1975 as species inquirenda, as the description was based on a sin- gle female. Brzeski (1998) reported on Globodera achilleae: ‘it may be 1 Morphological and morphometric characters useful for identification of instead. 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 Krall (1978) considered millefolii Table 2. 1 Species J2 body length conspecific with G. millefolii G. tabacum G. artemisiae G. pallida G. rostochiensis G. millefolii will be used instead.

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 174–197 PM 7/40 (4) Globodera rostochiensis and G. pallida 181

Fig. 9 Illustrations on the left-hand side of 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) tailof 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 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).) should be noted. Tests that were developed after 2000 gener- methods, especially when new introductions are sus- ally do not have these shortcomings. Specific identification pected. of G. achilleae from G. rostochiensis and G. pallida is pos- sible following the PCR restriction fragment length polymor- 4.2.1. PCR tests phism (RFLP) test developed by Sirca et al. (2010). There The following PCR tests are recommended for the identifi- are also differences between European and non-European cation of isolated cysts or individuals from G. rostochiensis populations of the two species, which might be made visible and G. pallida: with sequencing techniques (Hockland et al., 2012). As performance characteristics of the different tests pre- DNA barcoding can also be used to support identification. sented below vary (in particular with regard to their analyti- Identification of G. rostochiensis and G. pallida cal specificity) the choice of test should be made according should preferably combine morphological and molecular to the circumstances of use.

Test Appendix

Bulman & Marshall (1997): a multiplex PCR test using species-specific primers based on ribosomal 18S and ITS1 sequences 3 Thiery & Mugniery (1996): an internal transcribed spacer (ITS)-RFLP PCR test based on primers described by Vrain et al. (1992) 4 Real-time PCR tests for species specific identification as well as detection of G. rostochiensis, G. pallida and G. tabacum 5 (based on LSU rDNA) available as an all-inclusive real-time PCR kit (http://www. cleardetections.com) A Taqman real-time PCR targeting the internal transcribed spacer I (ITSI) gene (Fera) 6 High-throughput diagnosis of PCNs (Globodera spp.) in soil samples using real-time PCR (Reid et al., 2015) 7 Quantification of viable eggs of the PCNs (Globodera spp.) using either trehalose or RNA-specific real-time PCR 8

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 174–197 182 Diagnostics

Appendix 2 describes nucleic acid isolation. G. rostocheinsis. Sequences are available in databases including Q-bank (http://www.q-bank.eu/Nematodes/). 4.2.2. DNA barcoding A protocol for DNA barcoding based on COI, 18S rDNA 4.3. Pathotypes and 28S rDNA is described in Appendix 5 of PM 7/129 DNA barcoding as an identification tool for a number of The term ‘pathotype’ is used by the International PCN regulated pests: DNA barcoding nematodes (EPPO, 2016) Pathotype Scheme proposed by Kort et al. (1977) but is and can support the identification of G. pallida and now considered too general. Many PCN populations

Fig. 10 (A) Perineal measurements for Globodera identification. (B) Vulval-anal ridge patterns for four Globodera species. (C) Stylets from four species of Globodera. See footnote 6 (section 4.1.2) for G. achilleae. (After Fleming & Powers (1998).)

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 174–197 PM 7/40 (4) Globodera rostochiensis and G. pallida 183 cannot conclusively be assigned to the pathotypes 5. Reference material described in this scheme. There are differences in viru- lence between the two PCN species, in particular Reference material can be obtained from: between populations of G. pallida, and they are of National Plant Protection Organization, National Reference utmost importance in populations from , Laboratory, PO Box 9102, 6700 HC Wageningen (NL). but identification at this level is not adequate at the Food and Environmental Research Agency (Fera), Sand moment and it is time-consuming, expensive and requires Hutton, York YO41 1LZ (GB). specific analysis (Hockland et al., 2012). Any population Julius Kuhn-Institut€ (JKI), Federal Research Centre for Culti- showing signs of a new or unusual virulence (i.e. over- vated Plants, Messeweg 11–12, 38104 Braunschweig (DE). coming the resistance currently available in potato culti- French National Institute for Agricultural Research (INRA) vars in Europe) should be tested as soon as possible. In Biology of Organisms and Populations for Plant Protection practice, the virulence of populations can be tested on a Domaine de la Motte, BP 35327, 35653 Le Rheu Cedex (FR). set of cultivars used in each country. An EPPO Standard, PM 3/68 Testing of potato varieties to assess resistance 6. Reporting and documentation to Globodera rostochiensis and Globodera pallida was adopted in 2006. Guidance on reporting and documentation is given in EPPO Standard PM 7/77 Documentation and reporting on a diagnosis. 4.4. Testing the viability of eggs and juveniles

Testing of the viability of the eggs and juveniles may be 7. Performance criteria required for regulatory purposes. This can be done by dif- ferent methods. When performance criteria are available, these are provided (1) Visual morphological determination of viability (a with the description of the test. Validation data is also table with descriptions and figures is provided in available in the EPPO Database on Diagnostic Expertise Appendix 9). These observations require trained per- (http://dc.eppo.int), and it is recommended to consult this sonnel. database as additional information may be available there (2) Determination of viability with a bioassay. Two (e.g. more detailed information on analytical specificity, full tests are described in Appendix 1. Such tests validation reports, etc.). require more time to perform than visual morpho- logical determination of viability and generally 8. Further information more time than determination of viability by hatch- ing tests. Dormancy might play a role and should Further information on this organism can be obtained from: be lifted. An additional aspect of bioassays is the L den Nijs and G Karssen, National Plant Protection Orga- possibility of false negative results due to a very nization, National Reference Laboratory, PO Box 9102, low cyst content. 6700 HC Wageningen (NL). E-mail: l.j.m.f.den- (3) Determination of viability by hatching tests. Three [email protected] or [email protected] tests are described in Appendix 10. Such tests require more time to perform than visual morpho- 9. Feedback on this Diagnostic Protocol logical determination of viability. When determin- ing the viability with a hatching test, it should be If you have any feedback concerning this Diagnostic Proto- noted that cysts which have formed recently may col, or any of the tests included, or if you can provide addi- be dormant (e.g. when sampling is performed in tional validation data for tests included in this protocol that the autumn after potato harvest). To break the dor- you wish to share please contact: [email protected]. mancy cysts should be exposed to +4°C for at least 4 months. 10. Protocol revision (4) Determination of the viability of eggs using tre- halose. The test is described in Appendix 11, An annual review process is in place to identify the need for based on the publications by van den Elsen et al. revision of diagnostic protocols. Protocols identified as need- (2012) and Ebrahimi et al. (2015) ing revision are marked as such on the EPPO website. (5) Determination of viability and identification on the When errata and corrigenda are in press, this will also be basis of RNA. The test is described in Appendix 8, marked on the website. based on the publication by Beniers et al. (2014). Morphological determination of viability of eggs by Acknowledgements staining with Meldola’s Blue is also possible but the chemi- cal is not easily available so this technique is not described This Standard was originally developed under the EU in this Protocol. DIAGPRO Project (SMT 4-CT98-2252) by a partnership of

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 174–197 184 Diagnostics contractor laboratories and intercomparison laboratories in Hockland S, Niere B, Grenier E, Blok V, Phillips M, den Nijs L et al. European countries. The first draft was prepared by: L den (2012) An evaluation of the implications of virulence in non- Nijs and G Karssen, National Plant Protection Organization, European populations of Globodera pallida and G. rostochiensis for potato cultivation in Europe. Nematology, 14,1–13. National Reference Laboratory, PO Box 9102, 6700 HC Ibrahim SK, Perry RN, Burrows PR & Hooper DJ (1994) Wageningen (NL). It was revised by G Anthoine (Anses, Differentiation of species and populations of Aphelenchoides and of Plant Health Laboratory, Angers, FR), L Kox, G Karssen, angustus using a fragment of ribosomal DNA. Journal B van den Vossenberg and L den Nijs (National Plant Pro- of Nematology, 26, 412–421. tection Organization, National Reference Laboratory, Knoetze R, Swart A & Lowwrens RT (2013) Description of Globodera Wageningen, NL). capensis n. sp. (Nematoda: Heteroderidae) from South Africa. – Methods performed in and Austria provided by Nematology, 15, 233 250. € Kort J, Ross H, Rumpenhorst HJ & Stone AR (1977) An international D Kaemmerer, Bayerische Landesanstalt fur Landwirtschaft. scheme for identifying and classifying pathotypes of potato cyst- Methods performed in Norway provided by C Magnusson. nematodes Globodera rostochiensis and G. pallida. Nematologica 23, Methods performed in Sweden provided by S Manduric. 333–339. The Standard was reviewed by the Panel on Diagnostics Krall E (1978) Compendium of cyst nematodes in the USSR. in Nematology. Nematologica 23, 311–332. Lax P, Rondan Duenas~ JC, Franco-Ponce J, Gardenai CN & Doucet ME (2014) Morphology and DNA sequence data reveal the presence References of Globodera ellingtonae in the Andean region. Contributions to Zoology 83, 227–243. Anthoine G & Chappe A-M (2010) From soil to species: is there a Mota MM & Eisenback JD (1993) Morphology of females and cysts of procedure for molecular detection and identification of potato cyst Globodera tabacum tabacum, G. t. virginiae, and G. t. solanacearum nematodes? Aspects of Applied Biology 103,97–104. (Nemata: ). Journal of Nematology 25, 136–147. Baldwin JG & Mundo-Ocampo M (1991) Heteroderinae, cyst- and non- Muhammad Z (1996) the effect of storage conditions on subsequent cyst-forming nematodes. In: Manual of Agricultural Nematology (Ed. hatching of Globodera pallida (Nematoda, ). Pedobiologia Nickle WR), pp. 275–362. Marcel Dekker, New York,NY, (US). 40, 342–351. Beniers JE, Been TH, Mendes O, van Gent-Pelzer MPE & van der Lee Phillips MS, Forrest JMS & Wilson LA (1980) Screening for resistance TAJ (2014) Quantification of viable eggs of the potato cyst to using closed containers.Annals of Applied nematodes (Globodera spp.) using either trehalose or RNA-specific Biology 96, 317–322. Real-Time PCR. Nematology, 16, 1219–1232. Reid A, Evans FF, Mulholland V, Cole Y & Pickup J (2015) High- Brzeski MW (1998) Nematodes of Tylenchina in Poland and throughput diagnosis of potato cyst nematodes in soil samples. Plant Temperate Europe. Muzeum i Instytut Zoologii PAN, Warsaw (PL). Pathology: Techniques and Protocols 1302, 137–148. Bulman SR & Marshall JW (1997) Differentiation of Australasian van Riel HR & Mulder A (1998) Potato cyst nematodes (Globodera potato cyst nematode (PCN) populations using the polymerase chain species) in western Europe. In: Potato Cyst Nematodes: Biology, reaction (PCR). New Zealand Journal of Crop and Horticultural Distribution and Control (Eds Marks RJ & Brodie BB), pp. 271–298. Science 25, 123–129. CAB International, Wallingford (GB). De Ley P & Blaxter ML (2004) A new system for Nematoda: Sharma SB & Sharma R (1998) Hatch and emergence. In: The Cyst combining morphological characters with molecular trees, and Nematodes (Ed. Sharma SB), pp. 191–216. Kluwer Academic translating clades into ranks and taxa. In: Proceedings of the Fourth Publishers, Dordrecht (NL). International Congress of Nematology, 8–13 June 2002 Tenerife (ES) Siddiqi MR (2000) Key to genera of Heteroderinae. In: Tylenchida: (Eds Cook R & Hunt DJ), pp. 865. Brill, Leiden (NL). Parasites of Plants and Insects (Ed. Siddiqi MR), pp. 395–396. Ebrahimi N, Viaene N & Moens M (2015) Optimizing trehalose-based CABI Publishing, Wallingford, Oxon (UK). quantification of live eggs in potato cyst nematodes (Globodera Sirca S, Geric Stare B, Strajnar P & Urek G (2010) PCR-RFLP rostochiensis and G. pallida). Plant Disease 99, 947–953. diagnostic method for identifying Globodera species in Slovenia. EPPO (2016) PM 7/129 (1) DNA barcoding as an identification tool for Phytopathologia Mediterranea 49, 361–369. a number of regulated pests. EPPO Bulletin 46, 501–537. Stone AR (1972) Heterodera pallida n. sp. (Nematoda: Heteroderidae) a van den Elsen S, Ave M, Schoenmakers N, Landeweert R, Bakker J & second species of potato cyst nematode. Nematologica 18, 591–606. Helder J (2012) A rapid, sensitive and cost-efficient assay to estimate Stone AR (1973a) CIH Descriptions of Plant-Parasitic Nematodes No viability of potato cyst nematodes. Phytopathology 102, 140–146. 16 Globodera rostochiensis. CAB International, Wallingford (GB). EU (2007) Council Directive 2007/33/EC of 11 June 2007 on the Stone AR (1973b) CIH Descriptions of Plant-Parasitic Nematodes No control of potato cyst nematode and repealing Directive 69/465/EEC. 15 Globodera pallida. CAB International, Wallingford (GB). Official Journal of the European Communities 156,12–22. Subbotin SA, Cid del Prado Vera I, Mundo-Ocampo M & Baldwin JG Fleming CC & Powers TO (1998) Potato cyst nematode diagnostics: (2011) Identification, phylogeny and phylogeography of morphology, differential hosts and biochemical techniques. In: Marks circumfenestrate cyst nematodes (Nematoda: Heteroderidae) as RJ & Brodie BB (Eds), Potato Cyst Nematodes, Biology, Distribution interfered from analysis of ITS-rDNA. Nematolog 13, 805–824. and Control, pp. 91–114. CAB International, Wallingford (GB). Subbotin SA, Mundo-Ocampo M & Baldwin GB (2010) Systematics of Handoo ZA, Carta LK, Skantar AM & Chitwood DJ (2012) Cyst Nematodes (Nematoda: Heteroderinae), Nematology Monographs Description of Globodera ellingtonae n. sp. (Nematoda: & Perspectives, Vol. 8A. Brill, Leiden (NL), pp. 107–177. Heteroderidae) from Oregon. Journal of Nematology 44,40–57. Thiery M & Mugniery D (1996) Interspecific rDNA restriction fragment Hesling JJ (1978) Cyst nematodes: morphology and identification of length polymorphism in Globodera species, parasites of solanaceous Heterodera, Globodera and Punctodera. In: “Plant Nematology” plants. Fundamental and Applied Nematology 19, 471–479. (Ed. Southey JF), pp. 125–155. Ministry of Agriculture, Food and Vrain TS, Wakarchuk DA, Levesque AC & Hamilton RI (1992) Fisheries, London (UK). Interspecific rDNA restriction fragment length polymorphism in the

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Xiphinema americanum group. Fundamental and Applied after removal from the container and to extract cysts from Nematology 16, 563–573. the soil when no infection is detected visually. Wouts WM & Baldwin JG (1998) and identification. In: This can also be performed in closed containers kept in a The Cyst Nematodes (Ed. Sharma SB), pp. 83–122. Kluwer, dark room (Phillips et al., 1980). Dordrecht (NL).

Appendix 1 – Bioassays Test B Test of reproduction (method performed in Norway) The infective success of PCNs is tested on potato plants in Test A Bioassay (method performed in Germany and 500-mL pots with sand, using nylon bags with cysts (up to 20) Austria) as inoculation units. It is recommended to treat tubers with This method relies on the principle that if PCNs are present gibberellic acid in order to induce and synchronize the germi- in a soil sample (even in very low numbers) they will mul- nation. Each pot is filled with 1/3 of the total soil volume and tiply when given access to the roots of growing potato a nylon bag with cysts is placed below one potato tuber and plantlets in a small container. The presence of developing then filled up with sand. The pots are placed in a randomized cysts on the roots can then be observed through the trans- fashion in a growth cabinet with approximate day/night tem- parent walls of the special containers used6 peratures of 20°C/16°C and an 18-h light period. The pots Depending on the size of the container about 100– should receive mineral nutrients and water as required. After 200 mL of soil from the sample should be put into each 3 months the shoots are cut and the soil and roots are air- container, ensuring that the soil remains suitably moist. Pre- dried. The newly formed cysts are extracted from soil (for pare as many containers as needed to process the entire sam- instance by the Fenwick can), and collected and counted. ple. Eyes are cut from well-chitted certified tubers with a Each new cyst represents a successful infection and hence is a circular blade (diameter approximately 3 cm) and placed in measure of the infection potential of the population. the containers. Bioassay in autumn/winter requires chitting of tubers (through fumigation or treatment with gibberellic Appendix 2 – Extraction of nucleic acid acid). To avoid growth of fungi, eye cuttings should be left to dry for half a day at room temperature before being Many methods or kits can be used for extraction of nucleic placed on the soil samples in the containers (eyes upwards) acids from juveniles or cysts. The methods/kit described and covered with nematode-free soil. Control containers below have been evaluated in combination with the PCR with known infestations are used in each test. tests described in Appendices 3 and 4. The square containers are placed close together on a This paragraph only concerns DNA extraction. RNA planting table, shading each other to prevent the growth of extraction is described with each specific test. algae on the transparent walls. To allow optimal host–para- site interaction air temperature in the glasshouse is ideally 1. Manual DNA extraction with lysis buffer maintained at 22/16°C (day/night) and always kept below 1.1 Tissue source 25°C (possibly giving additional light in winter) and above This procedure can be applied to either isolated adults or 13°C. Containers should be watered moderately to achieve juveniles, even one individual, but also to Globodera cysts optimal root penetration of the soil. Watering may be done (maximum of 10 cysts pooled together) (Anthoine & manually or by trickle irrigation. Surplus irrigation water Chappe, 2010). can run off through a hole in the bottom of the containers. 1.2 The DNA extraction procedure includes chemical The risk of contaminating healthy samples by means of treatment with a lysis buffer (Tris 10 mM pH = 8, adjacent infested samples has been shown to be unlikely. It EDTA 1 mM, Nonidet P40 1%, proteinase K 100 lg might be necessary to take measures against foliar blight – mL 1) (Ibrahim et al., 1994) and a mechanical action. during the course of the bioassay. If an individual plant For Globodera juveniles or adults, one or several nema- should die, the soil in the container should be tested for todes are picked up and transferred to a microtube contain- cysts using the Fenwick can or a related method. ing 100 lL of lysis buffer. Visual observation of females and cysts is done when For Globodera cysts, one to a maximum of 10 cysts are cysts are observed in the control containers, generally after transferred into a microtube containing 1 mL of lysis buffer. 6–10 weeks of cultivation. Before counting of females and In both cases, glass beads (one 3-mm diameter and about cysts, potato leaves are cut with pruning shears. New cysts 50 of 1-mm diameter, Sigma) are added to the microtube are visible on the roots through the transparent walls of the and the nematode crushed by shaking the microtube [e.g. containers when infection levels are high. To detect low using a Tissulyser II (Qiagen ) at 30 beats per second for levels of infestation, it is advised to inspect roots and soil 40 s]. Alternatively the nematodes can be crushed with a hand pestle, which is either for unique use or disinfected 6 – They can be obtained from Ritter GmbH, Schwabenstraße 50 54, between uses. D-86836 Untermeitingen (DE).

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Microtubes are then incubated at 55°C for about 1 h and Table (continued) at 95°C for 10 min. The DNA supernatant obtained is transferred into a new microtube. Volume 1.3 No further cleanup of the extracted DNA is needed. Working per reaction Final 1.4 The crude DNA extract is stored at 20°C until use. Reagent concentration (lL) concentration

MgCl2 (Life Technologies) 25 mM 2 2 mM 2. Qiagen DNA extraction dNTPs (Life Technologies) 10 mM each 0.4 0.16 mM l l † Forward primer ITS5 10 M 0.625 0.25 M DNA extraction using a Kit QIAamp DNA mini kit (Qia- Reverse primer PITSp4 10 lM 0.625 0.25 lM† gen) is performed according to the manufacturer’s instruc- Reverse primer PITSr3 10 lM 0.625 0.25 lM† tions on Globodera individuals or cysts (up to 10 cysts Taq DNA polymerase 5U/lL 0.12 0.6 U pooled together). (Life Technologies) Subtotal 24 Genomic DNA extract or Appendix 3 – Multiplex PCR test (Bulman & cDNA Marshall, 1997) Dilution of the amplicons 1 derived from a first PCR if appropriate 1. General information Total 25 1.1 Identification of Globodera using the protocol *Molecular grade water should be used preferably or prepared l developed by Bulman & Marshall (1997). purified (deionised or distilled), sterile (autoclaved or 0.45 M 1.2 Different G. pallida and G. rostochiensis popula- filtered) and nuclease-free. † l tions, from different pathotypes and geographical 250 M of each primer is mentioned in the original publica- origins, were used. The nucleic acid source is full tion but laboratories performing the test use a final concentra- tion ranging from 0.15 to 1 lM. cysts. 1.3 The test is designed to the 18S rRNA gene and the 2.2.2 PCR cycling parameters internal transcribed spacer ITS1 region. 2 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 60°C and 1.4 The PCR product of the reaction with the universal 30 s at 72°C, 5 min 72°C primer ITS5 and G. pallida-specific primer PITSp4 is 265 bp. 3. Essential procedural information The PCR product of the reaction with the universal 3.1 Controls primer ITS5 and G. rostochiensis-specific primer For a reliable test result to be obtained, the following (ex- PITSr3 is 434 bp. 0 0 ternal) controls should be included for each series of 1.5 ITS5 5 -GGAAGTAAAAGTCGTAACAAGG-3 nucleic acid isolation and amplification of G. pallida and PITSp4: 50-ACAACAGCAATCGTCGAG-30 0 0 G. rostochiensis and nucleic acid, respectively. PITSr3: 5 -AGCGCAGACATGCCGCAA-3 • Negative isolation control (NIC) to monitor contamination 1.6 Amplification is performed in a Peltier-type thermo- during nucleic acid extraction: it can be obtained by perform- cycler with a heated lid (e.g. Bio-Rad C1000). ing DNA extraction of the solution/buffer used to collect nematode specimens (e.g. DNA extraction buffer alone). 2. Methods • Positive isolation control (PIC) to ensure that nucleic acid 2.1 Nucleic acid extraction and purification of sufficient quantity and quality is isolated: DNA extract 2.1.1 DNA is extracted from cysts or juveniles obtained from extraction solution/buffer spiked with 2.1.2 For the DNA extraction procedure see Appendix 2. appropriate number of individuals confirmed as being 2.1.3 Either use extracted DNA immediately or store from either G. pallida or G. rostochiensis. This control is overnight at 4°Corat20°C for longer periods optional as long as this test is applied on isolated nema- 2.2 Polymerase chain reaction todes (not on bulk solutions or as a screening test). 2.2.1 Master mix • Negative amplification control (NAC): to rule out false posi- tives due to contamination during the preparation of the Volume reaction mix – amplification of molecular-grade water that Working per reaction Final is used to prepare the reaction mix instead of DNA extract. Reagent concentration (lL) concentration • Positive amplification control (PAC) to monitor the effi- Molecular-grade water* N.A. 16.1 N.A. ciency of the amplification: amplification of nucleic acid Tris HCl (pH 8.3) 500 mM 1 20 mM of the target organism – amplification of genomic DNA KCl 500 mM 2.5 50 mM of individuals from both species G. pallida and G. rostochiensis; the identity of the individuals or the (continued) genomic solutions used must have been confirmed.

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Interpretation of results: in order to assigning results from primer 26S is 1200 bp for Globodera spp. The pri- PCR-based test the following criteria should be followed. mers are described by Vrain et al. (1992). Taq Verification of the controls: restriction. • NIC and NAC should produce no amplicons. 1.4 18S 50-TTG ATT ACG TCC CTG CCC TTT-30 • PAC, and if relevant PIC, should produce amplicons of 26S 50-TTT CAC TCG CCG TTA CTA AGG-30 the expected sizes (265 bp for G. pallida, and 434 bp for Note: 18S and 26S are also referred to in publications as G. rostochiensis). 5367 and 5368, respectively. When these conditions are met: 1.5 Amplification is performed in a Peltier-type thermo- • A test will be considered positive if amplicons of 265 bp cycler with heated lid, e.g. Bio-Rad C1000. for G. pallida and 434 bp G. rostochiensis are produced. • A test will be considered negative if it produces no band 2. Methods or a band of a different size. • Tests should be repeated if any contradictory or unclear 2.1 Nucleic acid extraction and purification results are obtained. 2.1.1 DNA is extracted from cysts or juveniles. 3.2 Note: the test can only be used on nematodes mor- 2.1.2 Genomic DNA is isolated as described in phologically identified as Globodera spp., as the Appendix 2A. primers are not specific for Globodera spp. (cross- 2.1.3 Either use extracted DNA immediately or reaction with Heteroderidae cysts containing eggs or store overnight at 4°Corat20°C for longer juvenilles may be observed). periods. 2.2 Polymerase chain reaction 2.2.1 Master mix 4. Performance criteria available The following performance criteria were provided by Anses Plant Health Laboratory (FR) in July 2010 with the following Working Volume per Final l adaptations of the master mix: primer concentration 0.64 lM Reagent concentration reaction ( L) concentration and dNTP 0.25 mM each. The DNA extraction was per- Molecular-grade water* N.A. 30.4 N.A. formed with lysis buffer (Tris 10 mM pH = 8, EDTA 1 mM, Taq DNA polymerase 109 519 – Nonidet P40 1%, proteinase K 100 lgmL 1) and mechanical buffer (MP Biomedicals) disruption of cuticle [use of Tissulyser II (Qiagen) with glass MgCl2 (if not included in 25 mM 4 2 mM beads]. See the validation report for additional information. the Taq DNA buffer) dNTPs 10 lM each 0.5 100 lM 4.1 Analytical sensitivity data: one J2. Forward primer 18S 10 lM 2.5 0.5 lM 4.2 Analytical specificity data: the study included 11 Reverse primer 26S 10 lM 2.5 0.5 lM populations of G. pallida, 4 populations of Taq DNA Polymerase 5UlL–1 0.1 0.5 U G. rostochiensis, 5 populations of G. tabacum, one (MP Biomedicals, population each of G. mexicana and G. artemisiae. ex. Appligene Oncor) The populations cover different geographical areas. Subtotal 45 4.3 Data on repeatability: 100% for G. pallida, 100% Genomic DNA extract or cDNA for G. rostochensis. Dilution of the amplicons 5 4.4 Data on reproducibility: 96% (1 J2) for G. pallida, derived from a first 100% (1 J2) for G. rostochiensis. PCR if appropriate 4.5 Diagnostic specificity data: 91% for G. pallida (the Total 50 test cross-reacted with G. tabacum ‘virginae’), *Molecular grade water should be used preferably or prepared purified 100% for G. rostochensis. (deionised or distilled), sterile (autoclaved or 0.45 lM filtered) and nuclease-free. Appendix 4 – ITS PCR-RFLP test (Thiery & Mugniery, 1996) 2.2.2 PCR cycling parameters 2 min at 94°C, 30 cycles of 1 min at 94°C, 50 s at 60°C, 1 min at 72°C. 1. General information 2.3 Restriction fragment length polymorphism (RFLP) reaction 1.1 Identification of Globodera spp. using a protocol 2.3.1 If required store products at 4°C before analysis developed by Thiery & Mugniery (1996) 2.3.2 Master mix: according to the supplier’s 1.2 The test is designed for the internal transcribed instructions. spacer (ITS) region. 2.3.3 Incubation temperature, time: incubation time/ 1.3 The PCR product of the reaction with the ITS-spe- temperature for digestion – overnight at the cific universal forward primer 18S and reverse

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recommended temperature (see supplier’s Table 3. Sizes of RFLP fragments (Thiery & Mugniery, 1996) instructions). Species Bsh1236I RFLP pattern

G. rostochiensis (European populations) 900, 190, 110 3. Essential procedural information G. pallida (European populations) 500, 400, 350, 190, 110 G. ‘mexicana’ 500, 400, 190, 110 3.1 Controls G. tabacum tabacum 445, 400, 190, 110 For a reliable test result to be obtained the following (exter- G. tabacum virginae 445, 400, 190, 110 nal) controls should be included for each series of nucleic G. tabacum solanacearum 445, 400, 190, 110 acid isolation and amplification of G. pallida and G. rostochiensis and nucleic acid, respectively. 4.1 Analytical sensitivity data: one J2. • Negative isolation control (NIC) to monitor contamination 4.2 Analytical specificity data: The study included 11 during nucleic acid extraction: this can be obtained by per- populations of G. pallida, 4 populations of forming DNA extraction of the solution/buffer used to col- G. rostochiensis, 5 populations of G. tabacum, one lect nematode specimens (e.g DNA extraction buffer alone). population of G. mexicana and G. artemisiae. The • Positive isolation control (PIC) to ensure that nucleic acid populations cover different geographical areas. of sufficient quantity and quality is isolated: DNA extract 4.3 Data on repeatability: 100% for G. pallida, 100% obtained from extraction solution/buffer spiked with an for G. rostochensis. appropriate number of individuals confirmed as being 4.4 Data on reproducibility: 90% (1 J2) for G. pallida, from either G. pallida or G. rostochiensis. This control is 93% (1 J2) for G. rostochiensis. optional as long as this test is applied on isolated nema- 4.5 Diagnostic specificity: 91% for G. pallida (1 non- todes (not on bulk solutions or as a screening test). target species detected in 11), 100% for • Negative amplification control (NAC): to rule out false G. rostochiensis. positives due to contamination during the preparation of the reaction mix – amplification of molecular-grade water that is used to prepare the reaction mix should be used Appendix 5 – Real-time PCR tests for the identification of G. rostochiensis, G. pallida instead of DNA extract. G. tabacum • Positive amplification control (PAC) to monitor the effi- and based on LSU rDNA ciency of the amplification: amplification of nucleic acid (ClearDetections Kit) of the target organism – amplification of genomic DNA of individuals from both species G. pallida and 1. General information G. rostochiensis, the identity of the individuals or the genomic solutions used must have been confirmed. 1.1 Scope of the tests: identification of G. rostochiensis, 3.2 Interpretation of results: in order to assign results G. pallida and G. tabacum juveniles and cysts and from PCR-based test the following criteria should detection of G. rostochiensis, G. pallida and be followed. G. tabacum juveniles and cysts in cyst mixtures Verification of the controls: (complex DNA background) by real-time PCR. • NIC and NAC should produce no amplicons. 1.2 The tests target the LSU (28S) rDNA gene. • PAC, and if relevant PIC, should produce amplicons of 1.3 Amplicon sizes: G. rostochiensis 448 bp, G. pallida the expected sizes, namely 1200 bp for Globodera spe- 86 bp, G. tabacum 482 bp. cies. 1.4 Oligonucleotide sequences are not disclosed. These When these conditions are met: tests are available as all-inclusive real-time PCR kit • A test will be considered positive if it produces the (ClearDetections, The Netherlands, http://www.clea restriction fragment lengths as given in Table 3. rdetections.com). • A test will be considered negative if it produces no band or a band of a different size. 2. Methods • Tests should be repeated if any contradictory or unclear results are obtained. 2.1 Nucleic acid extraction 3.3 Note: The method can only be used on nematodes These real-time PCR tests can be combined with any nema- morphologically identified as Globodera spp., as the tode DNA extraction method delivering target DNA. Valida- primers are not specific for Globodera spp. tion was performed with the ‘Nematode DNA extraction and purification kit’ from ClearDetections. When using these tests for nematode quantification purposes it is highly recom- 4. Performance criteria available mended to include an (internal or external) DNA standard in The following performance criteria were provided by Anses the extraction procedure to correct for potential DNA losses Plant Health Laboratory (FR), July 2010: during the DNA extraction and purification process.

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2.2 Real-time PCR with the combination of a Bio-Rad CFX Connect PCR The real-time PCR kit includes target and general nematode machine and ClearDetections PCR mix equal 86.0°C for DNA real-time PCR primer sets, positive amplification con- G. rostochienis, 85.5°C for G. pallida and 89.5°C for trol(s) (PACs) and PCR mix with fluorescent DNA-binding G. tabacum. dye. • Tests should be repeated if any contradictory or unclear 2.2.1 PCR cycling conditions results are obtained. Enzyme activation: 3 min at 95°C. Amplification: 35 cycles • The real-time PCR primer set included in the kit for the of 10 s at 95°C, 1 min at 63°C, 30 s at 72°C. Melt curve: detection of ‘nematode DNA’ can be used when in doubt 0.2–0.5°C steps 72°C ? 95°C. about the presence of nematode DNA in a DNA sample (check for possible false negatives).

3. Essential procedural information 4. Performance criteria available 3.1 Controls For a reliable test result to be obtained the following con- These real-time PCR tests are validated in line with PM 7/ trols should be included for each series of nucleic acid 98. extraction and amplification of the target organism and tar- 4.1 Analytical sensitivity: one single juvenile or egg, get nucleic acid, respectively. against a background of 1000 juveniles or eggs of • Negative isolation control (NIC) to monitor contamina- non-target cyst nematodes. tion during nucleic acid extraction: nucleic acid extraction 4.2 Diagnostic sensitivity: 100%. and subsequent amplification, preferably clean extraction 4.3 Analytical specificity: 100% (when using the kit for buffer. the three species on one sample). • Positive isolation control (PIC) to ensure that nucleic acid Number of populations of target organisms tested: of sufficient quantity and quality is isolated: nucleic acid 4 G. rostochiensis populations, 3 G. pallida popula- extraction and subsequent amplification of the target tions and 2 G. tabacum populations (for details see organism. Alternatively, the all-inclusive real-time PCR full validation report Table 6 in the database on kit contains a separate real-time PCR primer set for the diagnostic expertise). detection of ‘nematode DNA’, which can be used to Number of non-target organisms tested: G. achilleae, check for the presence and quantity of nematode DNA in G. artemisiae, G. mexicana, Heterodera goettingiana, the nucleic acid sample. Heterodera schachtii, Heterodera betae, Punctodera • Negative amplification control (NAC) to rule out false stonei. positives due to contamination during the preparation of Several target and non-target species (from differ- the reaction mix: amplification of molecular grade water ent origins) were tested and no cross-reactions that was used to prepare the reaction mix. were noted for the G. tabacum real-time PCR test. • Positive amplification controls (PACs, e.g. included in the The G. pallida real-time PCR test is specific for kit) to monitor the efficiency of the amplification: the G. pallida populations tested, including one amplification of nucleic acid of the target organism. This from South America. In addition, it picks up its can include genomic DNA extracted from the target close relative G. mexicana. The real-time PCR test organism, a cloned PCR product (plasmid DNA) or syn- for G. rostochiensis is specific for G. rostochiensis thetic DNA. populations, including South American populations, 3.2 Interpretation of results: in order to assign results and G. tabacum. These results demonstrate that in from PCR-based tests the following criteria should all cases where G. rostochiensis and G. tabacum be followed: cysts may be jointly found in samples and positive Verification of the controls: real-time PCR signals are found for • The PAC and PIC amplification curves should be expo- G. rostochiensis, the real-time PCR test for nential. G. tabacum must be used to verify possible false • NIC and NAC should give no amplification. positive results. When these conditions are met: 4.4 Diagnostic specificity: 100% (when using the kit for • A test will be considered positive if it produces an expo- the three species on one sample). nential amplification curve. 4.5 Reproducibility: 100%. • A test will be considered negative if it does not produce 4.6 Repeatability: 100%. an amplification curve or if it produces a curve which is 4.7 Accuracy: 100%. not exponential. 4.8 Dynamic range: between 10–100 and 0.1 billion • A melt curve analyses is performed and the obtained copies of target DNA.

melting temperature (Tm) value equals the Tm value of 4.9 Selectivity: 100%. the PAC (1°C). Tm values may vary depending on the 4.10 Robustness: no real-time PCR failure is observed PCR machine and PCR mix used. Tm values obtained when the primer combinations are exposed to a

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temperature gradient. With a deviation in annealing 2. Methods temperature (Ta)of1.0°C from the normal Ta (63°C), all ΔCt values are <1. The real-time PCR 2.1 Nucleic acid extraction and purification tests for the detection of G. pallida, 2.1.1 Single cysts of Globodera spp., the anterior G. rostochiensis and G. tabacum are robust. half of a single cyst containing eggs, or single juveniles are used for DNA extraction. Prior to Appendix 6 – Diagnosis of G. pallida and DNA extraction each cyst is individually dis- G. rostochiensis PCNs using Taqman real- sected in water. Homogenization of the cyst time PCR developed by Fera, GB should be performed using a sterile micropestle in 180 lL of ATL buffer until the cyst can no longer be seen. Care should be taken when 1. General Information removing the pestle to ensure that cyst frag- 1.1 The test was developed by Fera, York, GB follow- ments remain in the tube. ing on from the Potato Council Project R287 ‘Vali- 2.1.2 DNA extractions are performed using a Qiagen dation of quantitative DNA detection systems for DNeasy Blood and Tissue Kit, following the man- PCN’ and was finalized in October 2009. ufacturer’s protocol for tissues with the 1.2 DNA was typically extracted from either a single cyst following modifications. The sample is placed in or the anterior half of a single cyst of Globodera spp. a 1.5-mL Eppendorf tube and homogenized with Egg and/or juvenile number varies based on the given a micropestle in 180 lL of ATL buffer. 20 lLof cyst being tested. A typical sample of half a single Proteinase K is added, the sample vortexed to cyst would contain approximately 100 eggs (based on mix and centrifuged briefly to pool. The samples 315 cyst counts by Fera). DNA can also be extracted are incubated at 56°C and 100 r.p.m. for at least from single juveniles. 3 h or overnight. The manufacturer’s protocol is 1.3 The real-time PCR test targets the internal tran- continued from step 3, and for elution (steps 7 scribed spacer I (ITSI) gene, accession numbers and8)eachtime60lL of AE buffer is used, giv- AF016871 for G. pallida and EF622531 for ing DNA in a total volume of 120 lL. G. rostochiensis. 2.1.3 No nucleic acid cleanup procedure is required. 1.4 The forward primer is located at base 521 of 2.1.4 Extracted DNA should be stored between 20 G. pallida AF016871 and base 356 of and 30°C prior to use, and numerous freeze– G. rostochiensis EF622531. thaw cycles avoided. 1.5 The amplicons length including primers is 71 bp. 2.2 Real-time PCR 1.6 Oligonucleotides: forward primer Glob 531F, 50-T Applied Biosystems TaqMan Universal Master Mix II, GT-AGG-CTG-CTA-YTC-CAT-GTY-GT-30;reverse with no UNG (4440043); primer Glob 601R, 50-CCA-CGG-ACG-TAG-CAC- ACA-AG-30; and the two probes GP LNA, 50 TGC- Working Volume per Final l CGT-ACC-(C)(A)G-CGG-CAT-30 labelled with the Reagent concentration reaction ( L) concentration reporter FAM and the quencher BHQ-1 and GR PCR-grade water N.A. 6.75 lL N.A. 0 0 LNA, 5 -GCC-GTA-CC(T)-(T)GC-GGC-AT-3 TaqMan Universal 29 10 lL19 labelled with the reporter TET and the quencher Master BHQ-1, where DNA bases surrounded with brackets Mix II, no UNG are locked nucleic acid (LNA) bases. Dual-labelled (Applied Biosystems) Glob 531F 7.5 lM 0.375 lL 112.5 nM probes with incorporated LNA bases can be ordered Glob 601R 7.5 lM 0.375 lL 112.5 nM from Sigma Genosys. l l GR LNA 5 M 1.0 L 200 nM 1.7 Applied Biosystems TaqMan Universal Master GP LNA 5 lM 0.5 lL 100 nM Mix II, without uracil-N glycoslyase (UNG) Subtotal 19 lL (4440043). The enzyme used is AmpliTaq Gold DNA As extracted 1 lL As extracted Ultra Pure DNA polymerase (Applied Biosystems), Total 20 lL with the mix used at a final concentration of 19. 1.8 No reaction additives are used. 2.3 PCR cycling parameters: 50°C for 2 min; initial 1.9 Sterile water diethylpyrocarbonate (DEPC)-treated, denaturation 95°C for 10 min; cycling denaturation molecular biology grade (Severn Biotech Ltd). 95°C for 15 s; cycling annealing and extension 1.10 Applied Biosystems ABI Prism 7900HT. 60°C for 1 min; heating and cooling ramp at 100%; 1.11 Data analysis using Applied Biosystems sequence run for 40 cycles; fluorescence capture at all steps detection system, software versions 2.0, 2.2 and 2.4. and cycles.

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This cross-reaction is only observed when a sample 3. Essential procedural information is positive for G. rostochiensis. 3.1 Controls ○ 2. The G. pallida test cross-reacts with high concentra- For a reliable test result to be obtained, the following (ex- tions of DNA of G. tabacum; however, this generates a ternal) controls should be included for each series of non-exponential amplification profile and can be differ- nucleic acid isolation and amplification of the target organ- entiated from G. pallida and G. rostochiensis. ism and target nucleic acid, respectively. Glodobera tabacum is not known to be present in the • Negative isolation control (NIC) to monitor cross-reac- UK; however, if G. tabacum is suspected then a G. tions with the host tissue and/or contamination during tabacum-positive control should be run with each test nucleic acid extraction: nucleic acid extraction and to aid interpretation of the results, and any inconclusive subsequent amplification of a sample of clean extrac- results followed up with testing by another method. tion buffer. It is recommended to perform NIC several ○ 3. The test G. pallida test cross-reacts with Punctodera times in a series of extracts, for example one per five spp., generating a Ct >37; however, firstly Punctodera samples. cysts have a distinct morphology compared with • Positive isolation control (PIC) to ensure that nucleic acid Glodobera and would not be selected for molecular of sufficient quantity and quality is isolated: nucleic acid testing and secondly this Ct value would instigate extraction and subsequent amplification of a whole cyst further testing. of either G. pallida or G. rostochiensis. • Negative amplification control (NAC) to rule out false 4. Performance criteria available positives due to contamination during the preparation of the reaction mix: amplification of PCR-grade water that Validation data is available in the EPPO database on diag- was used to prepare the reaction mix, in place of DNA. nostic expertise (http://dc.eppo.int/validationlist.php) • Positive amplification control (PAC) to monitor the effi- 4.1 Analytical sensitivity data ciency of the amplification: amplification of nucleic acid A typical sample would be half a cyst, containing approxi- of the target organisms, from both G. pallida and mately 100 eggs; however, the test is able to detect a single G. rostochiensis separately. juvenile. A 100% success rate is achieved for single juve- 3.2 Interpretation of results niles. Numerous half cysts with variable egg numbers have Verification of the controls: been tested. DNA from a single cyst is detectable at or at • The PIC and PAC amplification curves should be expo- greater than a 1000 fold dilution. nential. 4.2 Analytical specificity data • NIC and NAC should give no amplification. Target organisms tested: 100+ populations of G. pallida When these conditions are met: from 26 counties (see validation report); 30+ populations of • A test will be considered positive if it produces an expo- G. rostochiensis from 10 counties (see validation report). nential amplification curve. Non-target organisms tested: populations of G. tabacum • A test will be considered negative if it does not produce (see validation report); populations of G. achillae/millefolii an amplification curve or if it produces a curve which is (see validation report). not exponential. The G. pallida test cross-reacts with G. tabacum; however, • From the validation data, it was stated that a test is con- this gives a non-exponential profile and can be differentiated sidered as positive if the Ct value is below 37 (due to for G. pallida and G. rostochiensis.TheG. pallida test cross- cross-reactions). This threshold should be re-evaluated reacts with Punctodera spp.; however, firstly Punctodera cysts by the laboratory at the limit of detection. For reference, have a distinct morphology compared with Glodobera and one single juvenile produces an average Ct of 30 for would not be selected for molecular testing and secondly this both species; therefore this is greater than the limit of species generates a Ct >37 which would instigate further inves- detection. tigation. • A Ct between 37 and 40 for either test is considered an 4.3 Data on repeatability inconclusive result and requires further investigation, such Extractions were tested at both neat and 103 dilutions with as repeating the DNA extraction and PCR and/or testing 8 replicates. Repeatability was 100%. the sample by an alternative method. 4.4 Data on reproducibility • Tests should be repeated if any contradictory or unclear Extractions obtained were tested at neat concentration by results are obtained. two different operators across two 7900 machines available • Additional considerations: in the laboratory. Reproducibility was 100%. ○ 1. The G. pallida probe is known to cross-react 4.5 Diagnostic sensitivity slightly with G. rostochiensis DNA. The cross-reac- The test was compared with the UKAS-accredited conven- tion will show as a slight increase in DRn in the tional PCR test of Bulman & Marshall (1997) using 149 FAM channel (G. pallida) as the DRn increases samples. This resulted in a 100% diagnostic sensitivity for exponentially in the TET channel (G. rostochiensis). both G. pallida and G. rostochiensis.

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4.6 Diagnostic specificity Technologies). 5 S-blocks (1–5) and a microplate (MP) are The test was compared with the UKAS-accredited conven- used in the sample processing, containing: tional PCR test of Bulman & Marshall (1997) using 149 S-block 1, 400 lL isopropanol and 30 lL MagAttract Sus- samples. The diagnostic specificity was G. pallida 87.1% pension G; and 93.8% for G. rostochiensis. The testing gave no false S-block 2, 400 lL RPW buffer; negatives for either species. S-block 3, 400 lL 96% ethanol; S-block 4, 400 lL 96% ethanol; Appendix 7 – High-throughput diagnosis of S-block 5, 500 lL sterile PCR-grade water (Sigma) contain- PCNs (Globodera spp.) in soil samples using ing 0.02% (v/v) Tween 20; real-time PCR (Reid et al., 2015). MP containing 200 lL sterile PCR-grade water; The DNA sample contained in the MP can be either pro- 1. General information cessed immediately or sealed with an adhesive sealing sheet and stored at 20°C. 1.1 Scope of the test: detection and identification of 2.2 Real-time PCR G. rostochiensis, and G. pallida in soil samples 2.2.1 Master mix using real-time PCR. 1.2 Test developed by Reid et al. (2015). 1.3 The test targets the internal transcribed spacer 1 Volume Working per Final region of the ribosomal DNA repeat unit (ITS1), Reagent concentration reaction (lL) concentration accession number FJ212165 for G. pallida. 1.4 Oligonucleotides: forward primer G. pallida and PCR-grade water (Sigma) N.A. 4.6 N.A. G. rostochiensis:50 CGTTTGTTGTTGACGGACAY TaqMan Environmental 29 15 19 A30; reverse primer G. pallida and G. rostochiensis:50 Master Mix 2.0 0 (Life Technologies) GGCGCTGTCCRTACATTGTTG 3 ;andtwoprobes 9 9 0 0 TaqMan Exogenous 10 1.5 0.5 G. pallida 5 CCGCTATGTTTGGGC 3 and Internal 0 0 G. rostochiensis 5 CCGCTGTGTATKGGC 3 Positive Control labelled with the reporter FAM and the quencher Primer Mix MGBNFQ (minor groove binding non-fluorescent (Life Technologies) 9 9 quencher). TaqMan Exogenous 50 0.15 0.25 1.5 7900HT Fast Real-Time PCR System (Life Tech- Internal Positive Control DNA nologies). (Life Technologies) 1.6 The real-time PCR tests are run using the SDS soft- Forward primer 5 lM 1.25 0.25 lM ware supplied with the 7900HT machine. Analysis (50 CGTTTGTTGTTGA settings are set to automatic. CGGACAYA 30) Reverse primer 5 lM 1.25 0.25 lM 0 2. Methods (5 GGCGCTGTCCRT ACATTGTTG 30) 2.1 Nucleic acid extraction and purification pallida probe 5 lM 0.625 0.125 lM 0 The following method is for a float size of approximately (5 CCGCTATGTTT 0 1 mL. For larger floats the sample should be split into sev- GGGC 3 ) rostochiensis probe 5 lM 0.625 0.125 lM eral tubes. (50 CCGCTGTGTA The dry float is manually scraped off the filter paper into a TKGGC 30) Fast Funnel MINI placed over a 2-mL Safe-Lock tube and 8 Subtotal 25 tungsten carbide beads are added to the tube. Batches of 48 DNA dilution 5 tubes are disrupted in the TissueLyser II set at 30 Hz for 30 s Total 30 and 1.5 mL of AP1 buffer is added prior to another round of shaking at 30 Hz for 30 s. Tubes are centrifuged at 1180 g 2.2.2 PCR conditions: for 5 min and a minimum of 400 lL of the supernatant is transferred using a pipette fitted with a wide-bore tip to an Step Time Temperature individual well of a 96-well S-block containing 5 lLof RNase A. The S-block is incubated at 65°C for 10 min and Pre-incubation 2 min 50°C centrifuged at 3000 g for 5 min. Enzyme activation 10 min 95°C 340 lL of supernatant of each sample is transferred in S- Amplification ° block 1 (see below) and processed using the BS96 DNA (40 cycles) DNA denaturation 15 s 95 C Primer annealing/extension 60 s 60°C Plant program of a MagMAX Express-96 (Life

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2.3 Real-time PCR ‘identification test’ organisms, whole-genome amplified DNA or a synthetic 2.3.1 Master mix (either pallida,orrostochiensis) control (e.g. cloned PCR products). The PAC should preferably be near to the limit of detection for more appropriate control of the reaction. Volume As an alternative (or in addition) to the external positive Working per Final Reagent concentration reaction (lL) concentration controls (PIC and PAC), internal positive controls (IPC) can be used to monitor each individual sample separately. PCR-grade water (Sigma) N.A. 6.25 N.A. Positive internal controls can either be genes present in the TaqMan Environmental 29 15 19 matrix DNA or added to the DNA solutions. Master Mix 2.0 Alternative internal positive controls can include: (Life Technologies) • Forward primer 5 lM 1.25 0.25 lM Specific amplification or co-amplification of endogenous (50 CGTTTGTTGT nucleic acid, using conserved primers that amplify con- TGACGGACAYA 30) served non-pest target nucleic acid that is also present in Reverse primer 5 lM 1.25 0.25 lM the sample (e.g. plant cytochrome oxidase gene or 0 (5 GGCGCTGTC eukaryotic 18S rDNA). 0 CRTACATTGTTG 3 ) • Amplification of samples spiked with exogenous nucleic Probe: either pallida 5 lM 1.25 0.25 lM (control sequence) acid that has no relation to the target (50 CCGCTAT GTTTGGGC 30)or nucleic acid (e.g. synthetic internal amplification controls) rostochiensis or amplification of a duplicate sample spiked with the tar- (50 CCGCTGTGT get nucleic acid. ATKGGC 30) 3.2 Interpretation of results: in order to assign results Subtotal 25 from the PCR-based test the following criteria DNA dilution 5 should be followed. Total 30 Verification of the controls: • The PIC and PAC (as well as IC and IPC as applicable) 2.3.2 PCR conditions: amplification curves should be exponential. • 2 min at 50°C, 10 min at 95°C, 40 cycles at 15 s 95°C, NIC and NAC should give no amplification. 60 s 60°C. When these conditions are met: • A test will be considered positive if it produces an expo- 3. Essential procedural information nential amplification curve. • A test will be considered negative if it does not produce 3.1 Controls an amplification curve or if it produces a curve which is For a reliable test result to be obtained, the following (ex- not exponential. ternal) controls should be included for each series of • Additionally for SYBR Green-based real-time PCR tests nucleic acid extraction and amplification of the target the Tm value should be as expected. organism and target nucleic acid, respectively. • Tests should be repeated if any contradictory or unclear • Negative isolation control (NIC) to monitor contamina- results are obtained. tion during nucleic acid extraction: nucleic acid extrac- tion and subsequent amplification preferably of a 4. Performance criteria available sample of uninfected matrix (soil confirmed to be free from Globodera) or if not available clean extraction SASA (GB) performed the following validation: buffer. 4.1 Analytical sensitivity data • Positive isolation control (PIC) to ensure that nucleic 0.1 pg of PCN DNA of either G. pallida or acid of sufficient quantity and quality is isolated: nucleic G. rostochiensis. This level allows detection of 1 viable acid extraction and subsequent amplification of a matrix cyst of either G. pallida or G. rostochiensis, i.e. a cyst con- sample that contains the target Globodera species (e.g. a taining a minimum of 1 live juvenile nematode. The test is float spiked with at least one of the target species, but able to detect 1 cyst of G. rostochiensis among 100 of ideally both). G. pallida and vice versa. • Negative amplification control (NAC) to rule out false 4.2 Analytical specificity data positives due to contamination during the preparation of Inclusivity: 641 G. pallida populations and 531 the reaction mix: amplification of molecular-grade water G. rostochiensis populations have been tested from across that is used to prepare the reaction mix. Scotland. • Positive amplification control (PAC) to monitor the effi- Exclusivity: primers were designed to take account of ciency of the amplification: amplification of nucleic acid non-target local species of cyst nematodes including solution from G. pallida and G. rostochiensis. This can Heterodera avenae (2 populations), Puntodera punctipenis include the use of nucleic acid extracted from the target (3 populations) and Globodera achillaea (6 populations), as

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 174–197 194 Diagnostics these are the other cyst nematodes present in the UK. No EF_Gpal_probe (50-Yakimo Yellow- CGAAGA{A} cross reactivity with any of these species occurred. No {T}GA cross reactions were observed with G. artemisia (3 popula- CCCGGC- BHQ1-30) and p_EF-Gros_2LNA (50-6 tions), and G. hypolysi (1 population) which are not consid- FAM-CTCGAAGAG{C}GAC{C}CTG-BHQ1-30). ered to be present in the UK. Cross-reactions occur with DNA bases surrounded with brackets are LNA bases. G. mexicana (3 populations), G. tabacum (12 populations) 1.7 Reverse transcription and real-time PCR are per- and G. ellingtonae (1 population), but none of these species formed within one step using the One Step Prime- have been detected so far in the UK. Sequencing of posi- ScriptTM RT-PCR (TaKaRa RR064A). tive results at the end of each testing season allows evalua- 1.8 No reaction additives are used. tion of whether any cross-reacting species were responsible 1.9 RNAse-free reagents, including molecular-grade water. for positive test results. No cross-reactions were observed. 1.10 Applied Biosystems ABI Prism 7500. 4.3 Data on repeatability 1.11 Data analysis with the 7500 Fast System Software Not available. Some information included under 4.5. version 1.4 4.4 Data on reproducibility Not available. Some information included under 4.5. 2. Methods 4.5 Other information Testing of soil samples from Scottish fields during 2008–10 2.1 Nucleic acid extraction using morphological identification (of PCNs from a float 2.1.1 Extraction of RNA is performed using a Master- extracted using a Fenwick can) to species level as the diag- PureTM Complete DNA and RNA Purification Kit nostic method provided a rate of 2.0% of samples testing (Epicentre) following the manufacturer’s proto- positive for PCN. The first 3 years using the PCR diagnos- col for tissue with the following modifications: tic (2011–13) provided a similar rate of 2.0% of samples 2.1.2 1–50 cysts of Globodera spp, are used for testing positive. (It should be noted that these were differ- nucleic acids extraction. ent samples taken at a different time period.) Collect the cysts in a 1.5 mL tube. Add 100 lL of tap water. Add a stainless steel ball (3 mm) or a glass 2008–10 2011–13 bead and crush using the Retsch MM301/ MM400 for 5 min at 30 Hz. Mean no. of samples tested per annum 5777 16 052 Samples with only dead PCNs 5.1% N/A Spin off the tubes. Samples with live PCNs 2.0% 2.0% Then follow the manufacturer’s protocol. Samples with live G. rostochiensis 1.1% 1.0% 2.2 Real-time RT-PCR Samples with live G. pallida 1.0% 1.1% A single-step real-time RT-PCR is performed as described below and using the One step Prime ScriptTM RT-PCR (TaKaRa RR064A) for a final volume of 25 lL. Appendix 8 – Qualitative detection of viability and identification of PCNs Volume (Globodera spp.) using RNA-specific real- per time RT-PCR Working reaction Final Reagent concentration (lL) concentration

PCR-grade water 2.25 1. General information One Step RT-PCR 29 12.5 19 1.1 The test was developed for the detection of viability Buffer III (TaKaRa) l l and identification of Globodera spp. by real-time Primer Fw_EF_Grp_mRNA 10 M 0.75 0.3 M Primer Rv_EF_Gp_mRNA-1 10 lM 0.75 0.3 lM RT-PCR. This protocol is based on the article of Probe EF_Gpal_probe 5 lM 2 0.5 lM Beniers et al. (2014). Probe p_EF-Gros_2LNA 5 lM 0.5 0.1 lM 1.2 Total nucleic acids are extracted from a sample con- Prime Script TM RT N.A. 0.5 N.A. taining from 1 to 50 Globodera cysts. Enzyme Mix II (TaKaRa) 1.3 The real-time PCR test targets the elongation factor TaKaRa TaqTM 5U/lL 0.5 2.5 U 1-a gene (EF1-a). HS (Takara) ROXTM Reference 509 0.25 0.59 1.4 No data on primer position. Dye II* 1.5 No data on amplicon size. Subtotal 20 lL 1.6 Oligonucleotides: the primer set is composed of the DNA As extracted 5 lL As extracted 0 forward primer Fw_EF_Grp_mRNA (5 -ACAA- Total 25 lL GATCGGAGGTATCG-30) and the reverse primer * Rv_EF_Gp_mRNA-1 (50-GTGGTTCATGATGATGA Linked to the cycling machine used; consult the manufacturer’s instructions; N.A.: Not applicable. CCTG-30). One probe is used for each species:

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2.3 PCR cycling parameters: 42°C for 5 min; initial Appendix 9 – Visual determination7 denaturation 95°C for 10 s; cycling denaturation 95°C for 10 s; cycling annealing and extension Characteristics of live and dead eggs and juveniles of the 60°C for 1 min; run for 40 cycles; fluorescence cap- potato cyst nematodes: ture at all steps and cycles.

Live eggs (Figs. 11 & 12) Dead eggs (Figs. 16 & 17) a. Whole egg is intact a. Egg may be damaged/broken 3. Controls and empty 3.1 Controls b. Egg shell is smooth b. Egg shell often not smooth For a reliable test result to be obtained the following con- c. Egg is clear/transparent with c. Contents have black/grey granular distinct contents or a dark line appearance with no structure trols should be included for each series: • down the middle of the egg Negative isolation control (NIC) to monitor contamina- d. Curled juvenile fill up against d. Shrivelled disintegrated juvenile tion during nucleic acid extraction: nucleic acid extraction the egg shell in egg and subsequent amplification preferably of clean extrac- e. Sometimes clear lip region e. No clear lip region or stylet tion buffer/collecting solution or a Heterodera cyst in and stylet present present 100 lL of tap water. Live juveniles (Figs. 13, 14 & 15) Dead juveniles (Figs. 18, 19 & 20) • Positive isolation control (PIC) to ensure that nucleic acid a. Juvenile has clear lip region, a. No clear lip region, partly or stylet visible completely grey/black structure of sufficient quantity and quality is isolated: nucleic acid b. Juvenile has strong smooth b. Cuticle shrivelled or not intact extraction and subsequent amplification of a matrix sam- cuticle ple that contains viable cysts of target Globodera spp. c. Intestine is filled with grey c. Transparent, body with clear (e.g. suspension of viable eggs of each species G. pallida granular structure, solid patches or completely transparent and G. rostochiensis). d. Clear lopsided distinction d. No clear lopsided distinction • Negative amplification control (NAC) to rule out false between pharynx and intestine between pharynx and intestine e. Juvenile sharply bent at an angle positives due to contamination during the preparation of or lying in half circle the reaction mix: amplification of RNase-free molecu- Included in counts: heads Not included in counts: tails, empty lar-grade water that is used to prepare the reaction shells mix. • Positive amplification control (PAC) to monitor the efficiency of the cDNA amplification: amplification of nucleic acid solution from G. pallida and Appendix 10 – Hatching test G. rostochiensis. (A) Hatching test (method performed in Norway) 3.2 Interpretation of results The hatching medium, potato root diffusate (PRD), is The PIC and PAC should have an exponential curve. obtained by passing 1000 mL of tap water through a 500- The NIC and NAC control should give no amplification. mL pot containing a 3-week-old potato plant growing in When these conditions are met: + ° • sand. After filtering, the PRD is stored at 3 C in the dark A test will be considered positive if it produces an expo- until needed. The PRD is used without dilution in closed nential amplification curve. = • glass vials (Ø 23.5 mm, height 34 mm) functioning as A test will be considered negative if it does not produce hatching units. Each vial contains one cyst bag made of an amplification curve. • nylon net with 20 cysts, which is completely covered by Tests should be repeated if any contradictory or unclear PRD. Cysts collected in the autumn need to be exposed results are obtained. +4°C for 4 months to break dormancy. After exposure to the root diffusate a high hatching frequency is reached after only 2 weeks. Each week the cyst bags are transferred to 4. Validation new hatching units with fresh PRD. The number of hatched The real-time RT-PCR has been validated according to PM juveniles is counted weekly and accumulated to form the 7/98. total hatch. At the end of the test the juveniles remaining in 4.1 Analytical sensitivity: 1 viable juvenile or egg. the cysts are counted, so the hatching can be expressed as a 4.2 Diagnostic sensitivity: 100%. percentage of the total cyst content (Fig. 21). 4.3 Diagnostic specificity: Globodera spp. 68.2%, (B) Hatching test (methods performed in Sweden) G. pallida: 72.2%, G. rostochiensis 86.7%. Prior to the exposure to the hatching stimulus, cysts that 4.4 Analytical specificity: 100%, no cross-reactions with have been stored dry should be pre-soaked in water in Petri other organisms (H. betae, H. glycines, H. schachtii, dishes, staining blocks or other suitable containers for 4– H trifolii, G. tabacum, Cactodera cacti) 4.5 Reproducibility: 100% 7Based on a test performance study between laboratories in The Nether- 4.6 Repeatability: 100% lands (L. den Nijs pers. comm.)

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Fig. 11–20 Characteristics of live and dead eggs and juveniles of the potato cyst nematodes.

5 days. Whereas non-hydrated cysts trend to float, hydrated sand substrate in a greenhouse. Three-week-old plants are ones sink to the bottom of the Petri dish, which gives a removed from the substrate and their roots are rinsed in good indication of a cyst’s hydration level. During this water, after which the plants are transferred one by one into hydration phase, the water in the containers is renewed 200-mL beakers filled with tap water and incubated at room daily to prevent bacterial and fungal growth. Repeated up temperature under aeration by an aquarium air pump (only and down pipetting of the cysts helps to get rid of the fungi the root system is immersed in the water). and favours the hydration. Diffusate is collected after 24 h and filtered and is then Potato plants for production of the hatching medium (i.e. ready to use. Hydrated cysts (up to 100, depending on the PRD) are grown in small (200 mL) clay pots with silver number of cysts found in the sample) are soaked in the

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tested. 20 cysts of G. pallida and G. rostochiensis are put respectively in two sieves as positive controls. All samples and controls are left at room temperature in the dark until they are checked for hatching. Each sample and the con- trols are checked for hatching every 10 days. If juveniles have hatched, the test is considered positive and the result is that the cysts are viable. New PRD is added every 10 days if necessary. If no hatching occurs after 30 days, the cysts are crushed and the viability of juveniles is assessed by visual examina- tion (Appendix 9). If viable juveniles are detected, the result of the test is positive, otherwise it is negative. If pos- itive controls do not hatch the viability tests are not consid- ered valid.

Fig. 21 Cumulative hatch of G. rostochiensis in potato root diffusate from 3-week-old plants (n = 4). Appendix 11 – Viability test by trehalose (van den Elsen et al., 2012; Ebrahimi et al., 2015) undiluted PRD. The PRD is replaced daily with a fresh sample. Trehalose, a disaccharide, is present in high concentrations The test lasts until juveniles start to hatch or for a total in the perivitelline fluid of eggs in cysts and can be used as – of 8 weeks. viability marker. Pre-soaked cysts (1 2 days) are boiled to (C) Hatching test (methods performed in France) free the trehalose that was present in the live eggs within This test is shorter and follows the procedure described the cysts. The boiling of the cysts breaks down the mem- in test B with the following modifications: brane and releases the trehalose into the solution. The pres- PRD production: sprout tubers are placed on a funnel put ence of trehalose can be verified directly using a simple on a plastic (transparent) beaker filled up with tap water. This detection kit; the disaccharide is hydrolysed into two glu- assemblage is stored at room temperature (around 18–19°C) cose molecules, and subsequently the glucose can be in the dark for 4 weeks. The water with PRD is filtered, detected. Cutting the cysts before or after boiling facilitates divided into aliquot parts and frozen until use (20°C). After the release of the trehalose into the solution. The latter is freezing for 48 h, the PRD is evaluated with the previous necessary when little living content is expected. When spe- batch of PRD (previous production) and reference G. pallida cies identification is to be performed after the viability test, and G. rostochiensis populations. If the test is satisfactory, lysis buffer should be added after the trehalose measure- the PRD can be used for the hatching test. ment. Extraction of DNA of this solution can take place Hatching test: instead of being rehydrated, cysts are and a subsequent PCR test can be performed (see Appen- – deposited in a fine sieve (250 lm) placed on a small dish dices 2 8). filled with PRD. One sieve is prepared per sample to be

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 174–197