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Home , Cherry leaf roll virus, Cocoa necrosis virus, Comovirinae, EPPO Code, Grapevine fanleaf virus, Nepovirus

Bulletin OEPP/EPPO Bulletin (2017) 47 (2), 135–145 ISSN 0250-8052. DOI: 10.1111/epp.12376

European and Mediterranean Protection Organization Organisation Europe´enne et Me´diterrane´enne pour la Protection des Plantes PM 7/2 (2)

Diagnostics Diagnostic

PM 7/2 (2) Tobacco ringspot

Specific scope Specific approval and amendment

This Standard describes a diagnostic protocol for Tobacco First approved in 2000-09. ringspot virus1 Revision approved in 2017-03 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

A flow diagram describing the procedure for detection 1. Introduction and identification of TRSV is given in Fig. 1. (TRSV) is the type member of the within the family . It has isomet- 2. Identity ric particles of approximately 28 nm and is readily transmit- Name: Tobacco ringspot virus ted by sap inoculation. It is transmitted in nature by the Synonym: Anemone necrosis virus, blueberry necrotic ring- vector americanum and closely related spot virus Xiphinema . tabaci, mites, grasshoppers Taxonomic position: : Picornovirales: Secoviridae: and have also been reported as possible vectors (Stace- : Secoviridae: Nepovirus Smith, 1985) but their significance for the natural spread of EPPO Code: TRSV00 TRSV is currently unclear. The same holds true for Apis Phytosanitary categorization: EPPO A2 List no. 228; EU mellifera (the European honeybee), in which TRSV was Annex I/A1 found to systemically spread and propagate (Li et al., 2014). TRSV is transmitted by seed in several host . It naturally 3. Detection infects a wide range of herbaceous and woody hosts, causing many serious diseases in the regions of North America where 3.1 Symptoms the nematode vectors occur. The most significant crops affected by TRSV are (Glycine max), grapevine In addition to the crops already mentioned, TRSV has been ( vinifera) and blueberry (Vaccinium corymbosum) and, found in Anemone, Bacopa, Begonia semperflorens, to a lesser extent, tobacco ( tabacum), a number of annuum, Carica papaya, Citrillus lanatus, , ornamentals and Cucurbitaceae. Its host range is similar to Cucumis maxima, Cucumis melo, Cucumis pepo, that of ringspot virus (ToRSV), but it is less important Cucumis sativus, Daphne, , Gladiolus, on fruit crops generally; it does, however, pose a significant Hemerocallis, Hydrangea, walleriana, , risk to Vaccinium and Vitis within the EPPO region. TRSV Lobelia, , domestica, , , has been reported from many parts of the world. In the EPPO , , Portulaca, Phlox subulata, Prunus region it has been found in ornamentals, but most of these avium, Prunus incisa, Prunus persicae, Prunus serrula, reports were associated with movement of material originat- Prunus serrulata, Rubus fruticosus, Rubus sp., , ing from North America. In particular, it has been recorded in Solanum lycopersicum, Solanum melongena, Vaccinium several EPPO countries but these reports are either uncon- corymbosum, Vigna unguiculata and various weeds (Nemeth, firmed or concern detection in imported material. 1986; Stace-Smith, 1987; EPPO/CABI, 1997; NVWA, 2006, 2010; Coneva et al., 2008; Fuchs et al., 2010; Abdalla et al., 1Use of brand names of chemicals or equipment in this EPPO Standard 2012; Sneideris et al., 2012; Kundu et al., 2015). implies no approval of them to the exclusion of others that may also be In soybean, terminal buds are curved (bud blight), with suitable. other buds progressively becoming brown and brittle.

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 135–145 135 136 Diagnostic

Symptomatic or asymptomatic leaf samples

Detection by DAS-ELISA (Appendix 2) or Generic RT-PCR (Appendix 3) 1 or Bioassay (Appendix 1)

Test negative Test positive

Fig. 1 Possible combinations of tests for the detection and identification of TRSV. Since TRSV not detected Identification and confirmation by none of the molecular tests has been DAS-ELISA (Appendix 2) completely validated, the interpretation of test or results is critical and additional tests are Specific RT-PCR (Appendix 4-6) needed for confirmation. or 1Note that the generic RT-PCR has been Sequencing of amplicons of validated for detection. Therefore, sequence generic RT-PCR (Appendix 3) 1 analysis of the amplicon might not be sufficient for identification and confirmation by another test might be required in specific situations. Combine test results to decide whether the sample is 2This statement is made because of the lack TRSV positive, suspected but not confirmed, or negative 2 of validation data for the molecular tests and risks of cross-reactions.

Brown streaks may develop on the stems and petioles of and stunting of infected foliage (Stace-Smith, 1987). TRSV larger leaves and pods are underdeveloped and aborted. has only been found once in a cultivated Rubus species. Pods that set before infection may develop dark blotches. No definite symptoms have been associated with TRSV On grapevine, the new growth is weak and sparse, intern- in pelargonium. odes are shortened, leaves are small and distorted, and plants are stunted with very few berries produced and these 3.2 Sampling berries are deformed. It should be noted, that grapevines are often infected by several viruses at the same time, TRSV generally occurs in all plant parts (including roots which might influence the symptomatology. and seeds) of its host species. Detection is, however, easiest On blueberry, the symptoms include stunting, twig die- from soft tissues (e.g. young leaves and shoots). For this back, necrotic or chlorotic spots, and rings or line patterns reason, it is preferable to collect samples during an appro- on the foliage. priate period in the growing season of the host species con- On tobacco, TRSV causes ring and line patterns on the cerned to ensure an adequate concentration of virus. For foliage, and stunting. pelargonium, for example, tests are best done between In cucurbits, stunting with leaf mottle occurs and the November and April, or when temperatures are cooler. fruit is deformed. There is no experience in the region with testing of seeds On tomatoes, leaves of infected plants may display or pollen and for testing seeds these are germinated first. chlorosis and curling of the margins (Ontario CropIPM, 2009). In addition, discoloration and rugosity may be 3.3 Screening tests observed on the surface of and inside infected fruits. TRSV has only been seen a few times in cherry, in For detection of TRSV the following tests are recommended. which scattered young leaves throughout the crown show irregular blotching over the whole leaf blade and have 3.3.1 DAS-ELISA deformed margins. Double antibody sandwich (DAS)-ELISA is the preferred In wild R. fruticosus, there are faint to severe ringspots, method of detection and identification. Instructions to per- mottling and mosaic, yellow line patterns, leaf distortion form an ELISA test are provided in EPPO Standard PM 7/

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 135–145 PM 7/2 (2) Tobacco ringspot virus 137

125 ELISA tests for viruses (EPPO, 2015a) and further available for any of these tests, the following tests were information is provided in Appendix 2. selected on the basis of their relative performance under described conditions: 3.3.2 Molecular tests • Conventional RT-PCR test from Jossey & Babadoost For detection, the RT-PCR tests with generic primers for (2006) described in Appendix 4 Nepovirus subgroup A (Wei & Clover, 2008; Appendix 3) • Real-time RT-PCR test from Yang et al. (2007), and TRSV-specific primers as described under identification described in Appendix 5. can be used. Digiaro et al. (2007) described primers for Furthermore, a conventional RT-PCR test developed and conventional RT-PCR for generic detection of successfully used by M. Zeidan (PPIS, NL) is described in belonging to different subgroups and specific detection of Appendix 6. TRSV. However, since these tests are not widely used and experience with them is limited in the EPPO region, they 4.2.2 Sequencing are not described in this protocol. TRSV can be identified by sequence analysis of the ampli- con obtained by the generic RT-PCR from Wei & Clover 3.3.3 Bioassay (2008), as described in Appendix 3, following the guideli- Mechanical inoculation to herbaceous test plants is a simple nes described in Appendices 7 and 8 of the EPPO Standard method for virus detection. It does not lead to specific iden- 7/129, DNA barcoding as an identification tool for a tification of TRSV when used alone, as the symptoms pro- number of regulated pests and according to the species duced on test plants are generally the same for all demarcation criteria of the International Committee on Tax- nepoviruses. However, test-plant inoculations can be used onomy of Viruses (Sanfacßon et al., 2012). Note that in for virus detection and subsequent identification by other specific situations an additional test might be needed for methods. The bioassay is described in Appendix 1. confirmation of the identity.

3.3.4 Other tests 4.3 Other tests Electron microscopy (EM), in combination with symptoms, can give an indication of the presence of TRSV, when iso- Immunoelectron microscopy (IEM) is a quick technique metric particles with a diameter of approximately 28 nm and is described in EPPO Standard PM 7/126 (EPPO, are observed. Instructions to perform EM are provided in 2015b). Note that the source of antibodies is critical because EPPO Standard PM 7/126 Electron microscopy in diagnosis cross-reactions between nepoviruses can occur. Further of plant viruses (EPPO, 2015b). details are described in EPPO Standard PM 7/126 Electron microscopy in diagnosis of plant viruses (EPPO, 2015b). Antisera are described in Appendix 2. 4. Identification DAS-ELISA and molecular tests are recommended for 5. Reference material identification of TRSV. Reference isolates of TRSV are available from: American type culture collection (ATCC): http://www. 4.1 DAS-ELISA lgcstandards-atcc.org/en/Support/LGC_Contact_Us.aspx For TRSV several commercial antisera are available, and Leibniz-Institut DSMZ-Deutsche Sammlung von therefore ELISA can be used to detect and identify the Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße virus in herbaceous hosts, grapevine and blueberry using 7B, 38124, Braunschweig, Germany (https://www.d appropriate extraction buffers. In general, it is recom- smz.de/catalogues/catalogue-plant-viruses-and-antisera.html) mended to follow the protocol provided by the supplier of National Plant Protection Organization of the Netherlands the antiserum. Note that the source of antibodies is critical (NPPO-NL), PO Box 9102, 6700 HC Wageningen, the because cross-reactions with other nepoviruses can occur. Netherlands. Further details on DAS-ELISA can be found in EPPO Stan- Specifications and characteristics of available isolates can dard PM 7/125 (EPPO, 2015a) and Appendix 2. be found via Q-bank: http://www.q-bank.eu Wageningen University and Research (WUR), Postbus 16, 6700 AA Wageningen, the Netherlands. 4.2 Molecular methods 4.2.1 PCR tests 6. Reporting and documentation Several conventional and real-time RT-PCR tests have been described for identification of TRSV (Jossey & Babadoost, Guidelines on reporting and documentation are given in 2006; Yang et al., 2007; Martin et al., 2009; Fuchs et al., EPPO Standard PM 7/77 Documentation and reporting on 2010). As currently complete validation data are not a diagnosis.

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 135–145 138 Diagnostic

This protocol was reviewed by the Panel on Diagnostics 7. Performance criteria in Virology and Phytoplasmology. When performance criteria are available, these are provided with the description of the test. Validation data are also 12. References available in the EPPO Database on Diagnostic Expertise (http://dc.eppo.int), and it is recommended to consult this Abdalla OA, Bruton BD, Fish WW & Ali A (2012) First Confirmed database as additional information may be available there Report of Tobacco ringspot virus in Cucurbits Crops in Oklahoma. (e.g. more detailed information on analytical specificity, full Plant Disease, 96, 1705. Coneva E, Murphy JF, Boozer R & Velasquez N (2008) First report of validation reports, etc.). Tobacco ringspot virus in Blackberry (Rubus sp.) in Alabama. Plant Disease 92, 1708. 8. Further information Digiaro M, Elbeaino T & Martelli GP (2007) Development of degenerate and species-specific primers for the differential and simultaneous RT- Further information on the different tests for this PCR detection of grapevine-infecting nepoviruses of subgroups A, B can be obtained from: and C. Journal of Virological Methods 141,34–40. Fera, Plant Health Diagnostics and Advice, Sand Hutton, EPPO (2015a) PM 7/125 (1) ELISA tests for viruses. EPPO Bulletin 45, 445–449. York YO41 1LZ, United Kingdom (for DAS-ELISA) EPPO (2015b) PM 7/126 (1) Electron microscopy in diagnosis of plant French Agency for Food, Environmental and Occupational viruses. EPPO Bulletin 45, 450–453. Health and Safety (ANSES), Plant Health Laboratory, EPPO/CABI (1997). Tobacco ringspot nepovirus. In: Quarantine Pests Virology Team, 7 rue Jean Dixmeras, 49044 Angers, Cedex for Europe (Eds. Smith IM, McNamara DG, Scott PR & Holderness 01, France (for Bioassay and PCR detection) M), 2nd edn, pp. 1357–1362. CAB International, Wallingford (GB). Julius Kuhn-Institut,€ Messeweg 11-12, 38104 Braun- Fuchs M, Abawi GS, Marsella-Herrick P, Cox R, Cox KD, Carroll JE et al. (2010) Occurrence of Tomato ringspot virus and Tobacco schweig, Germany (for IEM and DAS-ELISA) ringspot virus in highbush blueberry in New York State. Journal of Naktuinbouw Laboratories, Sotaweg 25, PO Box 135, 2370 Plant Pathology 922, 451–459. AC Roelofarendsveen, the Netherlands (for Bioassay and Jossey S & Babadoost M (2006) First report of Tobacco ringspot virus DAS-ELISA) in pumpkin ( pepo) in Illinois. Plant Disease 90, 1361. National Plant Protection Organization, National Reference Kundu JK, Gadiou S, Schlesingerova G, DziakovaM&Cerm ak V Centre, PO Box 9102, 6700 HC Wageningen, the Nether- (2015) Emergence of quarantine Tobacco ringspot virus in Impatiens – lands (for Bioassay and DAS-ELISA). walleriana in the Czech Republic. Plant Protection Science 51, 115 122. Li JL, Cornman RS, Evans JD, Pettis JS, Zhao Y, Murphy C et al. 9. Feedback on this diagnostic protocol (2014) Systemic spread and propagation of a plant-pathogenic virus in European honeybees, Apis mellifera. MBio 5, e00898–13. If you have any feedback concerning this diagnostic proto- Martin RR, Pinkertona JN & Kraus J (2009) The use of collagenase to col, or any of the tests included, or if you can provide addi- improve the detection of plant viruses in vector by RT- tional validation data for tests included in this protocol that PCR. Journal of Virological Methods 155,91–95. you wish to share please contact [email protected]. Menzel M, Jelkmann W & Maiss E (2002) Detection of four viruses by multiplex RT-PCR assays with co-amplification of plant mRNA as internal control. Journal of Virological Methods 99,81–92. 10. Protocol revision Nemeth M, Ed. (1986) Virus diseases of stone fruit trees. In: ‘Virus, mycoplasma, and diseases of fruit trees’. Martinus Nijhoff An annual review process is in place to identify the need Publishers, Dordrecht (NL), pp. 256–545. for revision of diagnostic protocols. Protocols identified as NVWA (2006) report Tobacco Ringspot Virus (TRSV) on needing revision are marked as such on the EPPO website. Hemerocallis spp. and Iris spp. ornamental plants. file:///R:/Mijn% 20Documenten/Downloads/Risicobeoordeling%20PRATobacco% When errata and corrigenda are in press, this will also be 20Ringspot%20Virus%20(TRSV).pdf marked on the website. NVWA (2010) Tobacco ringspot virus (TRSV) on indoor grown Phlox subulata perennials. https://www.nvwa.nl/actueel/bestanden/bestand/ 11. Acknowledgements 2001031/pest-report-finding-trsv-in-phlox-subulata [accessed on 5 April 2016] This diagnostic protocol was originally drafted by: D. M. Ontario CropIPM (2009) Ministry of Food & Rural Affairs, Wright, Central Science Laboratory, Sand Hutton, York Canada. http://www.omafra.gov.on.ca/IPM/english/tomatoes/diseases- and-disorders/viruses.html [accessed on 31 December 2015) YO41 1LZ (GB) and H. J. Vetten, Institut fur€ Pflanzenvi- Roenhorst JW, Botermans M & Verhoeven JTJ (2013) Quality control rologie, Mikrobiologie und Biologische Sicherheit, Biolo- in bioassays used in screening for plant viruses. EPPO Bulletin 43, gische Bundesanstalt fur€ Land- und Forstwirtschaft, 244–249. Messeweg 11-12, 38104 Braunschweig (DE). It was revised Sanfacßon H, Iwanami T, Karasev AV, van der Vlugt R, Wellink J, by J. W. Roenhorst, National Plant Protection Organisation, Wetzel T et al. (2012) Family Secoviridae. In: Virus : PO Box 9102, 6700 HC Wageningen (NL). Ninth Report of the International Committee on Taxonomy of Viruses

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 135–145 PM 7/2 (2) Tobacco ringspot virus 139

(Eds King AMQ, Adams MJ, Carstens EB & Lefkowitz EJ), pp. spots and/or dieback; systemic infections are not always 881–899. Elsevier Academic Press, San Diego, CA (US). induced. Sneideris D, Zitikaite I, Zizyte M, Grigaliunaite B & Staniulis J (2012) Nicotiana benthamiana, , Nicotia- Identification of nepoviruses in tomato (Lycopersicon esculentum na occidentalis-P1 and : necrotic local Mill.). Zemdirbyste Agriculture 99, 173–178. Stace-Smith R (1985) Tobacco ringspot virus. CMI/AAB Descriptions lesions may develop into rings or ringspots; systemically of Plant Viruses No. 309 (no 17 revised). AAB, Wellesbourne (GB). infected leaves usually show ring or line patterns. Stace-Smith R (1987) Tobacco ringspot virus in Rubus. In: Viruses : necrotic spots on inoculated leaves; Diseases of Small Fruits, Agriculture Handbook Number no. 631 (Ed. systemically infected leaves may show spots and rings. The Converse RH ), pp. 227–228. USDA/ARS, Washington, DC (US). 0 growing tip may become necrotic. Wei T & Clover GRG (2008) Use of primers with 5 non- Cucumis sativus: chlorotic or necrotic local lesions; sys- complementary sequences in RT-PCR for the detection of nepovirus subgroups A and B. Journal of Virological Methods 153,16–21. temic mottling and dwarfing with apical distortion. Yang W, Yun Z, Chen Z, Zhang G & Wu S (2007) Detection of Tobacco ringspot virus by RT-real-time PCR. Acta Phytopathologica Appendix 2 – Data on antisera for DAS- Sinica 34, 157–160. ELISA and IEM Instructions to perform an ELISA test and IEM are pro- Appendix 1 – Biossay vided in EPPO Standards PM 7/125 ELISA tests for viruses Different types of plant material can be used for mechani- and PM 7/126 Electron microscopy in diagnosis of plant cal inoculation of test plants. Note that for some crops the viruses, respectively. success of inoculation may depend on the composition of The source of antibodies is critical. Several TRSV antis- the extraction buffer. era are available from various suppliers and may differ in reactivity. In general it is recommended to follow the proto- col provided by the supplier of the antiserum. In a compar- Extraction buffers to be used for test-plant inoculations ative test, commercial antisera from Agdia, DSMZ and The following buffers can be used to prepare inoculum Prime Diagnostics reacted with 15 different TRSV isolates from various plants for mechanical inoculation of test from different host plants and origins (E. Meekes, 2008, plants: unpublished data). • For most plant species, the infected material may be The antiserum of Prime Diagnostics has been recently val- ground in 0.02 M Na/K phosphate buffer, pH 7.0, con- idated for testing leaf material of N. occidentalis-P1 by the taining 2% (w/v) polyvinylpyrrolidone (PVP) (MW NPPO in the Netherlands. Following the instructions of the 10 000 to 40 000); supplier, the test could detect all four included TRSV iso- • For extraction from grapevine or blueberry, an aqueous lates from different host plants and origins up to a relative solution of nicotine may be used at the following concen- infection rate of 0.1%. Cross-reaction was observed with the trations: for grapevine 2.5% nicotine and for blueberry closely related black ringspot virus. No cross-reaction 2.0% nicotine or, alternatively, 0.5 M Tris-HCl buffer, occurred with other nepoviruses, i.e. Arabis , pH 8.2, containing 2% PVP MW 10 000 to 40 000; Arracacha virus B (tentative member), Beet ringspot virus, • For Pelargonium grinding in 0.06 M phosphate buffer, , , Tomato pH 7.6, containing 4% polyethylene glycol (PEG) (MW black ring virus, Tomato ringspot virus and the as-yet unas- 6000) is recommended. signed latent ringspot virus. No background reac- Celite is added to the inoculum as an abrasive. Alterna- tion was obtained for leaf material of N. occidentalis-P1. tively, carborundum can be used to dust leaves prior to In 2012, Naktuinbouw also validated the Prime Diagnos- inoculation. The abrasive should be washed off after inocu- tic antiserum for use in different ornamental crops. TRSV- lation to avoid damage to the inoculated leaves, which will infected leaf material of N. benthamiana was spiked at a mask symptoms. Prior to inoculation, plants may be main- ratio of 1:10 to healthy leaf material of different plant spe- tained for 12 h in the dark. Inoculated plants should be kept cies, i.e. Celosia sp., Hemerocallis fulva, Hosta sp., Iris sp., at 18–22°C in a glasshouse or growth chamber. Further Lobelia sp., N. benthamiana, Pentas sp., Petunia hybrida details on quality control for mechanical inoculation of test and Phlox paniculata. In these spiked samples, TRSV could plants are described by Roenhorst et al. (2013). be detected up to a relative infection rate of approximately 0.1%. No matrix effects were observed for any of these plant species. For Pelargonium sp. and Verbena sp., how- Recommended test plants and their symptoms2 ever, matrix effects (background) could occur. With regard amaranticolor and Chenopodium : to the specificity, no cross-reactions occurred with the fol- necrotic local lesions; systemically chlorotic and/or necrotic lowing viruses Angelonia flower break virus, Alfalfa mosaic virus, , Broad bean wilt virus, 2Note that symptoms may vary with respect to virus isolate, test-plant Calibrachoa mottle virus, mosaic virus, accession and climatic conditions. Chrysanthemum virus B, Hosta virus X, Impatiens necrotic

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 135–145 140 Diagnostic spot virus, mosaic virus, Pelargonium flower break virus, Pelargonium line pattern virus, Potato virus Y, Working Volume per Final Scrophularia mottle virus, Nemesia ring necrosis virus, Reagent concentration reaction (lL) concentration Tomato aspermy virus, Tomato black ring virus, Tomato Molecular-grade water* N.A. 6.1 N.A. bushy stunt virus, Tobacco mosaic virus, Tobacco necrosis RT-PCR buffer 29 12.5 19 virus, Tomato mosaic virus, Tomato ringspot virus, (Invitrogen)

Tobacco rattle virus and Tomato spotted wilt virus. In addi- MgSO4 50 mM 0.4 0.8 mM tion, in earlier studies no cross-reaction was observed for Forward primer: 10 lM 1.75 0.7 lM (E. Meekes, 2008, unpublished NepoA-F–Flap l l data). Repeatability and reproducibility were 100%. Reverse primer: 10 M 1.75 0.7 M NepoA-R RT/Taq Platinum 0.5 Appendix 3 – One step RT PCR (adapted polymerase from Wei & Clover, 2008) (Invitrogen) Subtotal 23.0 RNA extract 2.0 Total 25.0 1. General information *Molecular-grade water should be used preferably, or prepared purified 1.1 Two tests are used for generic detection of nepo- (deionized or distilled), sterile (autoclaved or 0.45-lm filtered) and viruses of subgroups A and B. TRSV belongs to nuclease-free. subgroup A and therefore only this test will be described. The original two-step RT-PCR described 2.2.2 One step RT-PCR using Super Script III Plat- by Wei & Clover (2008) is adapted to a one-step inum One Step qRT-PCR System (Invitrogen). format for which the choice of the reagents 2.2.3 RT-PCR cycling parameters: reverse transcription appeared critical (P. Gentit, pers. comm.). step at 50°C for 30 min; denaturation at 94°C for 1.2 The test can be performed on fresh or freeze-dried 3 min; 35 cycles consisting of denaturation at leaves of various crop and test plant species (see 94°C for 30 s, annealing at 55°C for 30 s, elonga- Wei & Clover, 2008, Table 1) tion at 69°C for 30 s, elongation at 72°C for 1.3 For subgroup A, the target sequences are two con- 5 min. served motifs ‘TSEGYP’ and ‘LPCQVGI’ in the RNA-dependent RNA polymerase (RdRp) gene, producing an amplicon of approximately 340 bp. 3. Essential procedural information 1.4 Oligonucleotides 3.1 Controls For a reliable test result to be obtained, the following (ex- Primer Sequence* ternal) controls should be included for each series of nucleic acid extraction and amplification of the target NepoA-F–Flap 50-AATAAATCATAAACDTCWGARGGITAYCC-30 0 0 organism and target nucleic acid, respectively: NepoA-R 5 -RATDCCYACYTGRCWIGGCA-3 • Negative isolation control (NIC) to monitor contamina- *Degenerate nucleotide sequence according to IUPAC nucleic tion during nucleic acid extraction: nucleic acid extraction acid notation. The underlined section corresponds to the Flap and subsequent amplification preferably of a sample of extension (Wei & Clover, 2008). uninfected matrix, or if not available clean extraction buffer. 1.5 The test has been successfully performed on a PCR • Positive isolation control (PIC) to ensure that nucleic acid thermocycler Labcycler (Sensoquest). of sufficient quantity and quality is isolated: nucleic acid extraction and subsequent amplification of the target organism or a matrix sample that contains the target 2. Methods organism (e.g. naturally infected host tissue, host tissue 2.1 Nucleic acid extraction and purification spiked with the target organism, or lyophilized infected 2.1.1 100 mg of plant material is used for total plant material). RNA extraction using the RNeasy Plant • Negative amplification control (NAC): molecular-grade Mini Kit from Qiagen. Alternative procedures water that was used to prepare the reaction mix. may work as well. • Positive amplification control (PAC) to monitor the effi- 2.1.2 Extracted RNA should be stored refrigerated ciency of the amplification: amplification of nucleic acid for short-term storage (<8 h), at 20°C of the target organism. This can include nucleic acid (<1 month) or –80°C for longer periods. extracted from the target organism, total nucleic acid 2.2. One-step reverse transcription PCR extracted from infected host tissue or a synthetic control 2.2.1 Master mix

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 135–145 PM 7/2 (2) Tobacco ringspot virus 141

(e.g. cloned PCR product3). The PAC should preferably In addition, the following data on the analytical speci- be near to the limit of detection. ficity are available from the original two-step RT-PCR for- mat as described by Wei & Clover (2008). 3.2 Interpretation of results Subgroup A primers detected all ten tested isolates Verification of the controls belonging to subgroup A: ArMV (one isolate), GFLV • NIC and NAC should produce no amplicons (four), PBRSV (one), TRSV (three), RpRSV (one). • PIC, PAC should produce amplicons of approximately No cross-reactions were observed with seven isolates 340 bp belonging to subgroup B: (one iso- When these conditions are met: late), Cycas necrotic stunt virus (three) and TBRV • A test will be considered positive if amplicons of approx- (three), and four isolates of species belonging to sub- imately 340 bp are produced group C: Blackcurrant reversion virus, CLRV, PRMV • A test will be considered negative if it produces no band and ToRSV. or a band of a different size Furthermore, no background reaction was observed in • Tests should be repeated if any contradictory or unclear healthy plants of 15 different species: Dianthus results are obtained caryophyllus (carnation), (), (hop), (walnut), batatas (), Malus 9 domestica (apple), 4 Performance criteria available (pepino), Solanum tuberosum (potato), The generic one-step RT-PCR for detection of nepoviruses (cherry), Ribes nigrum (blackcurrant), of subgroup A has been validated at ANSES (FR). The per- Fragaria vesca (strawberry), Solanum lycopersicum formance characteristics are described below. Further (tomato), (nasturtium), Vitis riparia details can be found in the EPPO Database on Diagnostic (grapevine), (grapevine). Expertise, i.e. validation data for diagnostic tests. 4.3 Data on repeatability 4.1 Analytical sensitivity data Each test was repeated 3 times. The level of repeatability Subgroup A primers were able to detect 103–105 dilutions reached for the RT-PCR targeting subgroup A of the genus of RNA extracted from different virus isolates in RNA Nepovirus was 94.1%. from healthy plant material. 4.4 Data on reproducibility 4.2 Analytical specificity data Not available. Subgroup A primers detected all five tested isolates belong- ing to subgroup A: Arabis mosaic virus (ArMV), Grapevine fan leaf virus (GFLV), Potato black ringspot virus Appendix 4 – Two-step Conventional RT (PBRSV), Tobacco ringspot virus (TRSV) and Raspberry PCR (Jossey & Babadoost, 2006) ringspot virus (RpRSV). No cross-reactions were observed with two isolates belonging to subgroup B, Beet ringspot virus, Tobacco 1. General information black ring virus (TBRV), and five isolates of species 1.1 This test is used for specific detection and identifi- belonging to subgroup C, Apricot latent ringspot virus, cation of TRSV, using primers originally designed Cherry leaf roll virus (CLRV), Myrobolan latent ringspot by Jossey & Babadoost (2006). virus, rosette mosaic virus (PRMV) and Tomato 1.2 This test has been successfully used for testing ringspot virus (ToRSV). Cross-reaction was observed with sativum, Begonia, Capsicum annuum, an isolate of Artichoke yellow ringspot virus, belonging to Chrysanthemum, Cucumis sativus, Cucurbita pepo, subgroup C. , Fragaria 9 ananassa, Fuchsia, Gerbera, Furthermore, no background was observed in healthy Gloxinia, Hyacinthus, Hemerocallis, Impatiens, Iris, plants of six different species, Cucumis sativus (cucumber), Ixia polystachya, Lilium, Narcissus, Pelargonium, Prunus avium (cherry), Prunus persicae (peach), Solanum Petunia, Primula, Ruellia, Solanum lycopersicum, lycopersicum (tomato), Solanum tuberosum (potato), Vitis Solanum melongena, Solanum tuberosum, vinifera (grapevine), and no cross-reactions with Plum pox Streptocarpus, , Tulipa, Viola odorata. virus and Strawberry latent ringspot virus on peach, and 1.3 The primers anneal to the conserved region in the Pepino mosaic virus, Tomato infectious chlorosis virus and coat gene, producing an amplicon of Tomato chlorosis virus (PepMV) on tomato. approximately 348 bp. The host sequence and exact location of the amplicon are not known. 1.4 Oligonucleotides 3Laboratories should take additional care to prevent risks of cross- contamination when using cloned PCR products.

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 135–145 142 Diagnostic

Table (continued) Primer Sequence Volume TRSV-F 50-CTTGCGGCCCAAATCTATAA-30 Working per reaction Final TRSV-R 50-ACTTGTGCCCAGGAGAGCTA-30 Reagent concentration (lL) concentration

1.5 The test has been successfully performed on a Bio- Subtotal 20.0 cDNA derived from 5.0 Rad C1000 TouchTM Thermal Cycler and an Eppen- the RT-step dorf Mastercycler Personal Thermal Cycler. Total 25.0

*Molecular-grade water should be used preferably, or prepared purified 2. Methods (deionized or distilled), sterile (autoclaved or 0.45-lm filtered) and 2.1 Nucleic acid extraction and purification nuclease-free. 2.1.1 One gram of symptomatic plant material is 2.3.2 PCR using a hot-start Taq DNA polymerase used for total RNA extraction using the (Dialat Ltd, Russia). RNeasy Mini Kit (Qiagen). 2.3.3 PCR cycling parameters: denaturation at 96°C 2.1.2 Extracted RNA should be stored refrigerated for 5 min; 35 cycles consisting of denaturation for short-term storage (<8 h) at –20°C at 96°C for 30 s, annealing at 60°Cfor30s, (<1 month) or at –80°C for longer periods. elongation at 72°C for 30 s; terminal elongation 2.2 Reverse transcription (RT; to produce cDNA from at 72°Cfor5 min. RNA) 2.2.1 Master mix 3. Essential procedural information Working Volume per Final Reagent concentration reaction (lL) concentration 3.1 Controls For a reliable test result to be obtained, the following (ex- * Molecular-grade water N.A. 3.0 N.A. ternal) controls should be included for each series of RT buffer (Evrogen) 59 4.0 19 nucleic acid extraction and amplification of the target dNTPs (each) 10 mM 2.0 1.0 mM Dithiothreitol (DTT) 20 mM 2.0 2.0 mM organism and target nucleic acid, respectively. Random decamer primers 20 lM 3.0 3.0 lM • Negative isolation control (NIC) to monitor contamination Reverse transcriptase 100 U/lL 1.0 5 U during nucleic acid extraction: nucleic acid extraction and (Evrogen; MMLV) subsequent amplification preferably of a sample of unin- Subtotal 15.0 fected matrix or if not available clean extraction buffer. RNA 5.0 • Positive isolation control (PIC) to ensure that nucleic acid Total 20.0 of sufficient quantity and quality is isolated: nucleic acid *Molecular-grade water should be used preferably, or prepared purified extraction and subsequent amplification of the target (deionized or distilled), sterile (autoclaved or 0.45-lm filtered) and organism or a matrix sample that contains the target nuclease-free. organism (e.g. naturally infected host tissue, host tissue spiked with the target organism, or lyophilized infected 2.2.2 RT step using MMLV reverse transcriptase plant material). (Evrogen, Russia). • Negative amplification control (NAC): molecular-grade ° 2.2.3 RT conditions: incubation at 40 C for 40 min water that was used to prepare the reaction mix. ° followed by a denaturation step at 70 C for • Positive amplification control (PAC) to monitor the effi- 10 min. ciency of the amplification: amplification of nucleic acid 2.3 Conventional PCR of the target organism. This can include nucleic acid 2.3.1 Master mix extracted from the target organism, total nucleic acid extracted from infected host tissue, or a synthetic control Volume (e.g. cloned PCR product4). The PAC should preferably Working per reaction Final Reagent concentration (lL) concentration be near to the limit of detection.

Molecular-grade water* N.A. 11.0 N.A. 3.2 Interpretation of results Mas TaqMix CF (Dialat 59 5.0 19 Verification of the controls Ltd) • NIC and NAC should produce no amplicons Forward primer: TRSV-F 10 lM 2.0 0.8 lM Reverse primer: TRSV-R 10 lM 2.0 0.8 lM

4 (continued) Laboratories should take additional care to prevent risks of cross-con- tamination when using cloned PCR products.

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 135–145 PM 7/2 (2) Tobacco ringspot virus 143

• PIC, PAC should produce amplicons of approximately 2.2 One-step real-time reverse transcription polymerase 348 bp chain reaction When these conditions are met: 2.2.1 Master mix • A test will be considered positive if amplicons of approx- imately 348 bp are produced Volume • A test will be considered negative, if it produces no band Working per reaction Final Reagent concentration (lL) concentration or a band of a different size • Tests should be repeated if any contradictory or unclear Molecular-grade N.A. 1.08 N.A. results are obtained water* Real-time RT PCR 29 6.0 19 master mix 4 Performance criteria available PCR forward primer: 10 lM 0.6 0.5 lM TRSV-FP No validation data is available. PCR reverse primer: 10 lM 0.6 0.5 lM TRSV-RP Probe: TaqMan-MGB 5 lM 0.24 0.1 lM Appendix 5 – One-step real-time RT-PCR TRSV FAM (adapted from Yang et al., 2007) RT enzyme 259 0.48 19 Subtotal 9.0 RNA 3.0 1. General information Total 12.0

1.1 This test is used for specific detection and identification *Molecular-grade water should be used preferably, or prepared purified of TRSV, using primers and probe described by Yang (deionized or distilled), sterile (autoclaved or 0.45-lm filtered) and et al. (2007). The protocol has been successfully nuclease-free. applied and slightly adaptedbyIVIA,Valencia(ES). 1.2 This test has been successfully used for testing 2.2.2 AgPath-ID one-step RT-PCR reagents Prunus avium, Rubus fruticosus, Vaccinium spp. (Applied Biosystems; refs: 4387424-500 reac- and Vitis vinifera. tions or 4387391-1000 reactions). 1.3 The primers and probe target the coat protein gene, pro- 2.2.3 RT-PCR cycling parameters: reverse transcrip- ° ° ducing an amplicon of approximately 91 bp. Primers tion at 45 C for 10 min; denaturation at 95 C and probe sequences based on CP sequences of NCBI for 10 min; 45 cycles consisting of denatura- ° GenBank accession numbers AF461163.1, AF461164.1, tion at 95 C for 15 s, annealing and elongation ° L09205.1, AY363727.1, covering nucleotides 3202– at 60 C for 60 s. 3220, 3236–3252, 3273–3292, in TRSV RNA 2 com- plete sequence, NCBI Sequence AY363727.1. 3. Essential procedural information 1.4 Oligonucleotides 3.1 Controls Primer/Probe Sequence For a reliable test result to be obtained, the following (ex-

0 0 ternal) controls should be included for each series of TRSV-FP 5 -GGGGTGCTTACTGGCAAGG-3 nucleic acid extraction and amplification of the target TRSV-RP 50-GCACCAGCGTAAGAACCCAA-30 0 0 organism and target nucleic acid, respectively. TaqMan-MGB 5 -FAM-TGATTTGCGGCGTACTG-MGB-3 • TRSV FAM Probe Negative isolation control (NIC) to monitor contamina- tion during nucleic acid extraction: nucleic acid extrac- tion and subsequent amplification preferably of a sample 1.5 The test has been successfully performed on a StepO- of uninfected matrix or if not available clean extraction nePlus real-time PCR system (Applied Biosystems). buffer. • Positive isolation control (PIC) to ensure that nucleic acid 2. Methods of sufficient quantity and quality is isolated: nucleic acid extraction and subsequent amplification of the target 2.1 Nucleic acid extraction and purification organism or a matrix sample that contains the target 2.1.1 Up to 50 mg leaf material is used for RNA organism (e.g. naturally infected host tissue, host tissue extraction by using the PowerPlant RNA isola- spiked with the target organism, or lyophilized infected tion kit (MOBIO), or alternative RNA extraction plant material). procedures. • Negative amplification control (NAC): molecular grade 2.1.2 Extracted RNA should be stored refrigerated for water that was used to prepare the reaction mix. short-term storage (<8 h) at –20°C (<1 month) or • Positive amplification control (PAC) to monitor the at –80°C for longer periods. efficiency of the amplification: amplification of

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 135–145 144 Diagnostic

nucleic acid of the target organism. This can include nucleic acid extracted from the target organism, total Primer Sequence

nucleic acid extracted from infected host tissue, or a 0 0 synthetic control (e.g. cloned PCR product5). The TRSV-F-Zeidan 5 -GCCTCTAAGGACGCAACTGTGACGCTC-3 TRSV-R-Zeidan 50-CACAAGCAGCTGACA GACAGACATTC-30 PAC should preferably be near to the limit of Nad5-F-Menzel* 50-GATGCTTCTTGGGGCTTCTTGTT-30 detection. Nad5-R-Menzel* 50-CTCCAGTCACCAACATTGGCATAA-30

* 3.2 Interpretation of results From Menzel et al., 2002. Verification of the controls 1.5 The test has been successfully performed on a PTC- • The PIC and PAC amplification curves should be expo- 200 DNA Engine thermal cycler (BioRad). nential. • NIC and NAC should give no amplification When these conditions are met: 2. Methods • A test will be considered positive if it produces an expo- 2.1 Nucleic acid extraction and purification nential amplification curve. 2.1.1 One gram of plant material is homogenised in • A test will be considered negative, if it does not produce 10 mL homogenization buffer (Bioreba gen- an amplification curve or if it produces a curve which is eral extraction solution) plus 0.2 g sodium not exponential. meta-bisulfite (Sigma 255556) in disposable • Tests should be repeated if any contradictory or unclear extraction bags (Agdia, Bioreba). results are obtained. 2.1.2 Two millilitres of the homogenate is trans- ferred into a microfuge tube and centrifuged 4 Performance criteria available for 1 min at 13 000 rpm. 2.1.3 RNA is extracted from 0.2 mL of the homo- No validation data is available. genate after centrifugation by using the AccuPrep Viral RNA Extraction Kit (Bio- Appendix 6 – One step conventional RT-PCR neer), RNeasy Mini Kit (Qiagen), or other kit. (Zeidan, unpublished) After addition of 0.4 mL of VB binding buf- fer or RLT buffer, respectively, the mixture is incubated for 10 min at room temperature, 1. General information and further steps are carried out following the manufacturer’s instructions. 1.1 This test is used for specific detection and identi- 2.1.4 Total RNA is eluted in 50 lL of storage buf- fication of TRSV, using primers designed by M. fer. It should be used immediately or stored Zeidan (PPIS, Israel). The test has been success- refrigerated for short-term storage (<8 h) or fully applied on flower bulbs, leaf material of at –80°C for longer periods. ornamentals and vegetables and green scrapings 2.1.5 Homogenized plant material (2 mL) can be of twigs of fruit trees. The protocol has been in stored at –80°C for RNA extraction at a later use since 2005. date. Extracted RNA should be stored refrig- 1.2 This test has been successfully used for testing erated for short-term storage (<8 h) at –20°C bulbs and leaves of Anemone sp., Gladiolus sp., (<1 month), or at –80°C for longer periods. Hyacinthus sp., Iris sp., Lilium sp., Narcissus 2.2 One-step reverse transcription PCR spp., Tulipa sp., leaf material of Cucurbitaceae, Dianthus sp., Glycine sp., Mentha longifolia, 2.2.1 Master mix (simplex reaction) Nicotiana sp., Pelargonium sp., and green scrap- ings of twigs of Malus sp., Prunus cerasus, Volume Vaccinium sp. and Vitis vinifera. Working per reaction Final 1.3 The primers are located in the CP gene on RNA Reagent concentration (lL) concentration 2, and produce an amplicon of approximately * 650 bp, covering nucleotides 3036–3683 in TRSV Molecular-grade water N.A. 15.0 N.A. RT-PCR premix kit 109 N.A. 19 RNA 2 complete sequence, NCBI reference (pellet) sequence NC_005096.1. PCR forward primer: 20 lM 0.5 0.5 lM 1.4 Oligonucleotides TRSV-F-Zeidan PCR reverse primer: 20 lM 0.5 0.5 lM TRSV-R-Zeidan

5Laboratories should take additional care to prevent risks of cross-con- tamination when using cloned PCR products. (continued)

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Table (continued) 95°C for 5 min; 40 cycles consisting of denaturation at 95°C for 30 s, annealing at Volume 56°C for 1 min, elongation at 72°C for Working per reaction Final 1 min; terminal elongation at 72°C for 5 min. Reagent concentration (lL) concentration

Subtotal 16.0 3. Essential procedural information Genomic RNA extract 4.0 Total 20.0 3.1 Controls For a reliable test result to be obtained, the following (ex- * Molecular-grade water should be used preferably, or prepared purified ternal) controls should be included for each series of l (deionized or distilled), sterile (autoclaved or 0.45- m filtered) and nucleic acid extraction and amplification of the target nuclease-free. organism and target nucleic acid, respectively. • Negative isolation control (NIC): Bioreba homogenization buffer. 2.2.2 Master mix (duplex reaction) • Positive isolation control (PIC): dried tissue of TRSV- Working Volume per Final infected plants, commercially available from DSMZ. Reagent concentration reaction (lL) concentration • Negative amplification control (NAC): molecular-grade water that was used to prepare the reaction mix. * Molecular-grade water N.A. 14.0 N.A. • Positive amplification control (PAC): for example, in- RT-PCR premix kit 109 N.A. 19 vitro transcribed RNA from a cloned TRSV fragment.1 (pellet) • PCR forward primer : 20 lM 0.5 0.5 lM Internal control (IC): Nad5 can be used as internal con- TRSV-F-Zeidan trol, in a separate reaction as well as in duplex format. PCR reverse primer : 20 lM 0.5 0.5 lM TRSV-R-Zeidan 3.2 Interpretation of results PCR forward primer: 20 lM 0.5 0.5 lM Verification of the controls nad5-F-Menzel • NIC and NAC should produce no amplicons PCR forward primer: 20 lM 0.5 0.5 lM • PIC, PAC should produce amplicons of approximately nad5-R-Menzel Subtotal 16.0 650 bp Genomic RNA extract 4.0 • IC should produce amplicon of approximately 180 bp (if Total 20.0 applicable) When these conditions are met: *Molecular-grade water should be used preferably, or prepared purified • A test will be considered positive if amplicons of approx- (deionized or distilled), sterile (autoclaved or 0.45-lm filtered) and nuclease-free. imately 650 bp are produced • A test will be considered negative if it does not produce 2.2.2 One-step RT-PCR using Maxime RT-PCR a band of 650 bp. Tests should be repeated if any contra- premix kit (INtRON Biotechnology) contain- dictory or unclear results are obtained ing a pellet of 109 RT-PCR buffer, dNTPs, OptiScript RT-system and Hotstart i-star Taq 4 Performance criteria available DNA polymerase. 2.2.3 RT-PCR cycling parameters: reverse tran- No specific validation data is available. scription at 45°C for 45 min; denaturation at

ª 2017 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 47, 135–145