Showing papers on "Globodera rostochiensis published in 2022"

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

Wrap-and-plant technology to manage sustainably potato cyst nematodes in East Africa

Abstract: Abstract Renewable eco-friendly options for crop protection are fundamental in achieving sustainable agriculture. Here, we demonstrate the use of a biodegradable lignocellulosic banana-paper matrix as a seed wrap for the protection of potato plants against potato cyst nematode (PCN), Globodera rostochiensis . Potato cyst nematodes are devastating quarantine pests of potato globally. In East Africa, G. rostochiensis has recently emerged as a serious threat to potato production. Wrapping seed potatoes within the lignocellulose banana-paper matrix substantially reduced G. rostochiensis field inoculum and increased potato yields by up to fivefold in Kenya, relative to farmer practice, whether or not impregnated with ultra-low doses of the nematicide abamectin (ABM). Markedly, ABM-treated banana paper at ~1,000 times lower than conventional recommendations reduced PCN inoculum. Assays and analyses revealed that the lignocellulose matrix disrupts parasite–host chemical signalling by adsorbing critical PCN hatching and infective juvenile host location chemicals present in potato root exudate. Recovery experiments confirmed adsorption of these host location chemicals. Our study demonstrates the use of waste organic material to sustainably manage PCN, and potentially other crop root pests, while increasing potato yields.

The Impact of Management Strategies on the Development and Status of Potato Cyst Nematode Populations in Switzerland: An Overview from 1958 to Present

Abstract: Globodera rostochiensis and G. pallida are some of the most successful and highly specialized plant parasitic nematodes and among the most regulated quarantine pests globally. In Switzerland, they have been monitored by annual surveys since their first detection in Swiss soil in 1958. The dataset created was reviewed to produce an overview of the development and actual status of potato cyst nematodes (PCNs) in Switzerland. Positive fields represent 0.2% of all the samples analyzed, and their distribution is limited to central-west and western Switzerland, suggesting that new introduction of PCNs and the spread of the initial introduced PCN populations did not occur. In this way, the integrated management used in Switzerland appears to be effective. However, the increasing availability of potato varieties with resistance to G. rostochiensis and the limited availability of varieties with resistance to G. pallida, together with other biotic and abiotic factors, have promoted changes in the dominance of either species. Consequently, an extended monitoring program is of interest to Swiss farmers, to avoid favoring virulent traits that could be present in Swiss Globodera populations.

Predicting potential global distribution and risk regions for potato cyst nematodes (Globodera rostochiensis and Globodera pallida)

Abstract: Potato cyst nematodes (PCNs), golden (yellow) cyst nematode (Globodera rostochiensis, gPCN) and pale (white) cyst nematode (G. pallida, pPCN), are important invasive pests in many countries and regions where they can cause significant yield and economic loss for agriculture. Prediction and identification of habitats suitable for PCNs are critical for developing biosecurity strategies, both pre and post border, to maximise the potential for early elimination should an incursion occur. To date, the potential global distribution of PCNs has not been thoroughly studied. Therefore, this study conducted a species distribution model to illustrate the potential global distribution of PCNs and risk regions. In this study, the Maximum Entropy Model (Maxent) associated with the Geographic Information System (GIS) was employed to reveal the potential distribution of the gPCN and pPCN. In addition to bioclimate, soil quality was also included in the model. The global cultivated lands, whether the susceptible hosts were present or not, were used to assess the maximum potential risk regions. The limitation factors for PCNs distribution were also assessed. Results showed that 66% of the global land surface was suitable for gPCN or pPCN or both, and both species can colonise more than 75% of the global cultivated lands. The coldest quarter's mean temperature and precipitation were critical limitations in unsuitable regions. In summary, the global risk maps of PCNs contribute valuable additional information that complements previous national/regional distribution predictions. The results of this distribution research will contribute practical support for decision-makers and practitioners to implement biosecurity strategies from a global perspective, that incorporate prevention or promptly enforce control practices to limit the damage caused by future incursions.

Belowground Chemical Interactions: An Insight Into Host-Specific Behavior of Globodera spp. Hatched in Root Exudates From Potato and Its Wild Relative, Solanum sisymbriifolium

Abstract: Understanding belowground chemical interactions between plant roots and plant-parasitic nematodes is immensely important for sustainable crop production and soilborne pest management. Due to metabolic diversity and ever-changing dynamics of root exudate composition, the impact of only certain molecules, such as nematode hatching factors, repellents, and attractants, has been examined in detail. Root exudates are a rich source of biologically active compounds, which plants use to shape their ecological interactions. However, the impact of these compounds on nematode parasitic behavior is poorly understood. In this study, we specifically address this knowledge gap in two cyst nematodes, Globodera pallida, a potato cyst nematode and the newly described species, Globodera ellingtonae. Globodera pallida is a devastating pest of potato (Solanum tuberosum) worldwide, whereas potato is a host for G. ellingtonae, but its pathogenicity remains to be determined. We compared the behavior of juveniles (J2s) hatched in response to root exudates from a susceptible potato cv. Desirée, a resistant potato cv. Innovator, and an immune trap crop Solanum sisymbriifolium (litchi tomato – a wild potato relative). Root secretions from S. sisymbriifolium greatly reduced the infection rate on a susceptible host for both Globodera spp. Juvenile motility was also significantly influenced in a host-dependent manner. However, reproduction on a susceptible host from juveniles hatched in S. sisymbriifolium root exudates was not affected, nor was the number of encysted eggs from progeny cysts. Transcriptome analysis by using RNA-sequencing (RNA-seq) revealed the molecular basis of root exudate-mediated modulation of nematode behavior. Differentially expressed genes are grouped into two major categories: genes showing characteristics of effectors and genes involved in stress responses and xenobiotic metabolism. To our knowledge, this is the first study that shows genome-wide root exudate-specific transcriptional changes in hatched preparasitic juveniles of plant-parasitic nematodes. This research provides a better understanding of the correlation between exudates from different plants and their impact on nematode behavior prior to the root invasion and supports the hypothesis that root exudates play an important role in plant-nematode interactions.

Sustainable management of the potato cyst nematode, Globodera rostochiensis, with two microbial fermentation products

Abstract: Potato cyst nematodes (PCN) cause an overall 9% yield loss of total potato production worldwide. Research on sustainable management of PCN is still under progress. Two microbial fermentation products (MFPs) from Alltech, a proprietary blend formulated with a bacterial fermentation media and a Cu component (MFP5075), and a microbial based product (MFP3048), were evaluated against the PCN Globodera rostochiensis. In laboratory tests, effectiveness of the MFPs was recorded in terms of PCN juveniles (J2) hatching from cysts, J2 mortality and their attraction toward potato roots using pluronic gel. Greenhouse trials were conducted to study the effect of the products on PCN infestation in potato plants and a pilot scale experiment was conducted to study the impact of these MFPs on nematode biodiversity in garden soil. All treatments were performed within a concentration range of 0, 0.5, 1, and 2% (v/v) MFP5075 and 2, 6, 10, and 20 g/10 ml (w/v) MFP3048. The attraction assay, juvenile hatching and the PCN infestation in plants results were compared with those in an untreated control and a commercial nematicide (Nemguard™) treatment. After 24 h of treatment with 0.5 and 1% MFP5075, a 13-fold and 43-fold reduction, respectively, relative to J2 survival was recorded compared to that of untreated control. However, no J2 survived at 2% and above concentration of the MFP5075 treatment. Treatment with MFP3048 was effective in causing mortality of J2 only after 48-h. In the attraction assay, a 20-fold and 8-fold reduction in number of J2 attracted toward potato roots was observed, when treated with MFP5075, compared to the untreated and the Nemguard™ treatment, respectively. Subsequently, 30–35 PCN cysts were treated with both products dissolved in potato root diffusate and the results were recorded in terms of number of J2 hatched in each treatment after 10 days. No J2 hatched in the MFP5075 treatment, whereas mean numbers (±SE) of 243 ± 11.5, 30 ± 2.5, and 1.3 ± 0.6 J2 were noted in the untreated control, MFP3048, and the Nemguard™ treatment, respectively. The treatment with the MFPs compromised the integrity of the unhatched J2, which looked granular, whereas the internal organs of the unhatched J2 could be clearly identified in the untreated control. In plant infestation studies, treatment with MFP3048 and MFP5075 caused 90.6 and 84.9 percent reduction in PCN infestation, respectively, in terms of cysts developed on roots compared to untreated control. Overall, results indicate that the MFPs could potentially provide a promising alternative for sustainable PCN management.

Resistance Evaluation for Native Potato Accessions against Late Blight Disease and Potato Cyst Nematodes by Molecular Markers and Phenotypic Screening in India

Abstract: The potato originated in southern Peru and north-western Bolivia (South America). However, native accessions have also been cultivated in India for many years. Late blight, caused by the fungus Phytophthora infestans, is the most devastating potato disease, while potato cyst nematode (Globodera spp.) (PCN) is another economically significant quarantine-requiring pest in India. In this study, we have generated a new Indian native collection of 94 potato accessions collected from different parts India. These accessions were screened against late blight and potato cyst nematode resistance by using gene-based molecular markers and phenotypic screening methods. Marker assisted selection using R1 gene-specific marker CosA210 revealed a late blight resistance gene in 11 accessions. PCN resistance bands were found in 3 accessions with marker TG689141, 5 accessions with marker 57R452, and 1 accession having Gro1-4-1602 marker for G. rostochiensis (Ro1,4), while 64 accessions amplified marker HC276 indicating G. pallida (Pa2,3) resistance gene (GpaVvrn QTL). On the other hand, phenotypic screening against late blight resistance under natural epiphytic conditions (hot-spot) revealed three accessions with high resistance, while others were resistant (1 accession), moderately resistant (5 accessions), susceptible (29 accessions), and highly susceptible (56 accessions). For G. rostochiensis (golden cyst nematode) and G. pallida (white cyst nematode) resistance, accessions were grouped into highly resistant (3, 3), resistant (0, 2), moderately resistant (6, 29), susceptible (32, 30), and highly susceptible (53, 30), respectively, for the two PCN species. Collectively, we identified promising accessions with high resistance to late blight (JG-1, Kanpuria Safed, and Rangpuria), and also highly resistant to both Globodera species (Garlentic, Jeevan Jyoti, and JG-1). Our findings suggested that these accessions would be useful for late blight and PCN resistance breeding, as well as future molecular studies in potatoes.

RANGE DYNAMICS OF POTATO NEMATODE &lt;i&gt;GLOBODERA ROSTOCHIENSIS&lt;/i&gt; (WOLLENWEBER, 1923) SKARBILOVICH, 1959 UNDER CONDITIONS OF GLOBAL CLIMATE CHANGE IN RUSSIA

Abstract: Globodera rostochiensis is one of the 100 most dangerous invasive species in Russia, causing significant damage to agriculture. In Russia, this nematode was first founded in Kaliningrad Region in 1949. In this study, we used ensemble modeling (ESDM) methods to predict the potential distribution of G. rostochiensis in Russia and found that with changes in global climate and land use in the future, there would be a tendency to expand the range in two directions - from the south to the north and from the west to the east. The history of the distribution of the species on the territory of Russia, the current and potential ranges of the species from 2020 to 2100 with a step of 20 years in the implementation of various models and scenarios of climate change and land use are presented. Information on native range, features of biology, signs of host plant damage and injuriousness of G. rostochiensis , methods of pathotypes identification, invasion vectors, and control measures are shown. The predicted ranges of the species are important for the development of measures to minimize future invasion of G. rostochiensis and their negative consequences

Rapid Diagnosis and Visual Detection of Potato Cyst Nematode (Globodera rostochiensis) Using Recombinase Polymerase Amplification Combination with Lateral Flow Assay Method (RPA-LFA)

Abstract: Globodera rostochiensis is an important quarantine pest, it causes serious potato yield losses annually. Reliable and rapid molecular detection of G. rostochiensis is pivotal to effective early disease diagnosis and managements. Herein, recombinase polymerase amplification integrated with lateral flow assays method (RPA-LFA) was developed to target the internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) of the golden cyst nematode (G. rostochiensis), which allowed for the rapid diagnosis and detection of this nematode from crude extracts of cysts and juveniles within 30 min. Sensitivity test results showed that 10−1 single juvenile and 10−3 single cyst can be reliably detected. Moreover, the RPA-LFA method can directly diagnose and detect G. rostochiensis from infested field soil. This is the first RPA-LFA method for diagnosis G. rostochiensis, it is a fast, accurate, and sensitive detection method and can be developed for detection of G. rostochiensis in fields and laboratories lacking large instrument and equipment.

A Novel Rhabdovirus Associated with the Idaho Population of Potato Cyst Nematode Globodera pallida

Abstract: Globodera pallida, a potato cyst nematode (PCN), is a quarantine endoparasitic pest of potato (Solanum tuberosum) in the US due to its effects on yield and quality of potato tubers. A new rhabdovirus, named potato cyst nematode rhabdovirus (PcRV), was revealed and characterized in the G. pallida populations collected in Idaho through use of high-throughput sequencing (HTS) and RT-PCR and found to be most closely related to soybean cyst nematode rhabdovirus (ScRV). PcRV has a 13,604 bp long, single-stranded RNA genome encoding five open reading frames, including four rhabdovirus-specific genes, N, P, G, and L, and one unknown gene. PcRV was found present in eggs, invasive second-stage juveniles, and parasitic females of G. pallida, implying a vertical transmission mode. RT-PCR and partial sequencing of PcRV in laboratory-reared G. pallida populations maintained over five years suggested that the virus is highly persistent and genetically stable. Two other Globodera spp. reproducing on potato and reported in the US, G. rostochiensis and G. ellingtonae, tested negative for PcRV presence. To the best of our knowledge, PcRV is the first virus experimentally found infecting G. pallida. Based on their similar genome organizations, the phylogeny of their RNA-dependent RNA polymerase domains (L gene), and relatively high identity levels in their protein products, PcRV and ScRV are proposed to form a new genus, provisionally named “Gammanemrhavirus”, within the family Rhabdoviridae.

Characterization and response of two potato receptor-like kinases to cyst nematode infection

Abstract: ABSTRACT Plant-parasitic cyst nematodes (Heterodera and Globodera spp.) secrete CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) effector proteins, which act as ligand mimics of plant CLE peptides to promote successful nematode infection. Previous studies of the Arabidopsis-beet cyst nematode (BCN; H. schachtii) pathosystem showed that Arabidopsis CLE receptors including CLAVATA1 (CLV1), CLV2, and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) are required for BCN CLE signaling. Studies further revealed that nematode CLE signaling through GmCLV2 and StCLV2, an Arabidopsis CLV2 orthologue from soybean (Glycines max) and potato (Solanum tuberosum), respectively, is required for the soybean cyst nematode (SCN; H. glycines) and the potato cyst nematode (PCN; G. rostochiensis) to induce disease in their respective host plant. In this study, we identified and characterized two additional potato receptors, StRPK2 and StCLV1, homologues of Arabidopsis RPK2 and CLV1, for a role in PCN parasitism. Using promoter-reporter lines we showed that both StRPK2 and StCLV1 are expressed in the potato root but vary in their spatial expression patterns. Interestingly, StRPK2 but not StCLV1 was found to be expressed and upregulated at PCN infection sites. Nematode infection assays on StRPK2-knockdown lines revealed a decrease in nematode infection. Collectively, our results suggest that parallel CLE signaling pathways involving StCLV2 and StRPK2 are important for PCN parasitism and that manipulation of nematode CLE signaling may represent a viable means to engineer nematode resistance in crop plants including potato.

Tare Soil Disinfestation from Cyst Nematodes Using Inundation

Abstract: The dissemination of soil tares in the potato and sugar beet processing industry is one of the main paths for the spread of potato cyst nematodes (PCN), a severe quarantine pest. Efficient measures for the disinfestation of tare soil from PCN, but also from beet cyst nematodes (BCN), are needed. In our study, Globodera pallida (a PCN) and Heterodera schachtii (a BCN) cysts were sealed in gauze bags and imbedded in sedimentation basins. The cysts were either placed (a) in a presedimentation basin (Brukner basin) for three days, (b) in the presedimentation basin for three days and subsequently in sedimentation basins for nine weeks or (c) in sedimentation basins for nine weeks (without presedimentation). We tested the viability of the eggs and juveniles by hatching assays and using the reproduction rates in bioassays. We demonstrated that PCN and BCN imbedded in a sedimentation basin were only still showing some hatching activity after 2.5 weeks, while no hatching was observed when an additional Brukner basin treatment was conducted before sedimentation.

Globodera rostochiensis (yellow potato cyst nematode)

Abstract: This datasheet on Globodera rostochiensis covers Identity, Overview, Distribution, Dispersal, Hosts/Species Affected, Diagnosis, Biology & Ecology, Environmental Requirements, Natural Enemies, Impacts, Uses, Prevention/Control, Further Information.

Globodera pallida (white potato cyst nematode)

Abstract: This datasheet on Globodera pallida covers Identity, Overview, Distribution, Dispersal, Hosts/Species Affected, Diagnosis, Biology & Ecology, Environmental Requirements, Natural Enemies, Impacts, Uses, Prevention/Control, Further Information.

Management of Potato Cyst Nematodes (Globodera Spp.) Using Biotechnological Approaches

Abstract: Potato cyst nematodes (PCN) (Globodera spp.) are economically important worldwide. Delivering double-stranded RNA (dsRNA) as sprays on the potato crop holds considerable promise and potential against the cyst nematode. This method is cost-effective and accrues multiple advantages to the growers. However, this method requires low-cost production of dsRNA sequences and should be stable under field conditions. Leaves should be able to uptake dsRNA, and it should have systemic distribution in the plant. In potato cyst nematode case, chemical mutagens have not yielded results. The combined applications of RNAi and CRISPR/Cas9 appears to be an alternative approach for the management of potato cyst nematodes.

Sensitivity of potato cyst nematodes to fluazaindolizine

Abstract: Potato cyst nematodes (PCN), Globodera spp., cause damage to potatoes in more than 60 countries and several management strategies, including the application of chemical nematicides, are commonly used for their control. However, due to stringent regulations in Europe several nematicides have been, or are being, removed from the market due to their potential toxic effects on the environment and human health. New solutions and nematode management strategies are being sought to control these challenging and economically important nematodes. In this study, the effects of Salibro™, a novel sulfonamide nematicide based on the active ingredient fluazaindolizine (Reklemel™ active), were evaluated on the hatching, motility, infectivity and reproduction of PCN in the laboratory. Depending upon the duration of nematode pre-exposure, Salibro™ at concentrations of 5-250 mg fluazaindolizine (active substance) (a.s.) kg−1 (equivalent to 5-250 ppm a.s.) affected hatching, motility and infectivity of second-stage juveniles of G. pallida and G. rostochiensis, whereas the reproduction of G. pallida was only influenced at 5-50 mg a.s. kg−1. Salibro™, under laboratory conditions, has intrinsic activity against both PCN species and could be a promising additional tool for the integrated management of PCN. Further studies are needed to demonstrate Salibro™ efficacy under field conditions.

The efficacy of cost-effective bionematicide against potato cyst nematode globodera rostochiensis

Abstract: