PM 7/146 (1) Tomato Brown Rugose Fruit Virus

Home , Pepper mottle virus, Tomato mosaic virus

Bulletin OEPP/EPPO Bulletin (2021) 51 (1), 178–197 ISSN 0250-8052. DOI: 10.1111/epp.12723

European and Mediterranean Plant Protection Organization Organisation Europe´enne et Me´diterrane´enne pour la Protection des Plantes PM 7/146 (1)

Diagnostics

PM 7/146 (1) Tomato brown rugose fruit virus

Diagnostic Speciﬁc approval and amendment

Approved in 2020-10. Speciﬁc scope

This Standard describes a diagnostic protocol for detection and identiﬁcation of tomato brown rugose fruit virus.1 This Standard should be used in conjunction with PM 7/ 76 Use of EPPO diagnostic protocols.

1. Introduction 2. Identity Tomato brown rugose fruit virus (ToBRFV genus Name: Tomato brown rugose fruit virus. Tobamovirus) was first observed in 2014 and 2015 on Synonyms: None. tomatoes in Israel and Jordan, and outbreaks have recently Acronym: ToBRFV. occurred in China, Mexico, the USA and several EPPO Taxonomic position: Virus, Riboviria, Virgaviridae, countries (EPPO, 2020). The virus is a major concern for Tobamovirus. growers of tomato and pepper as it reduces the vigour of EPPO Code: TOBRFV. the plant, causes yield losses and virus symptoms make the Phytosanitary categorization: EPPO Alert List, EU emer- fruits unmarketable. However, the virus may also be present gency measures. in asymptomatic foliage and fruit. Note Virus nomenclature in Diagnostic Protocols is based Tomato (Solanum lycopersicum) and pepper (Capsicum on the latest release of the official classification by the annuum) are the only confirmed natural cultivated hosts of International Committee on Taxonomy of Viruses (ICTV, ToBRFV (Salem et al., 2016, 2019; Luria et al., 2017; Release 2018b, https://talk.ictvonline.org/taxonomy/). NAPPO, 2018; Panno et al., 2020). ToBRFV was also Accepted species names are italicized when used in their detected in natural weed hosts (Chenopodium murale and taxonomic context, whereas virus names are not, corre- Solanum nigrum) in Israel (Dombrovsky, pers. comm., 2019). sponding to ICTV instructions. The integration of the genus The host status of Solanum melongena (aubergine) is not name within the name of the species is currently not consis- confirmed. tently adopted by ICTV working groups and therefore spe- Because ToBRFV is a relatively new described virus species names in diagnostic protocols do not include the genus cies, limited published scientific information is available name. Names of viruses not included in the official ICTV and evaluation of tests is underway. This Standard includes classification are based on first reports. tests which have been evaluated in laboratories from the EPPO region. 3. Detection Flow diagrams describing the diagnostic procedure for tomato brown rugose fruit virus in plant material and in 3.1. Disease symptoms seeds are presented in Figs 1 and 2. ToBRFV causes a wide range of symptoms. Symptoms 1Use of brand names of chemicals or equipment in these EPPO Stan- may range from very severe to mild, or plants can be dards implies no approval of them to the exclusion of others that may infected asymptomatically. Leaf symptoms often first also be suitable. appear in the young shoots at the top of the plant.

178 ª 2021 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 51, 178–197 PM 7/146 (1) Tomato brown rugose fruit virus 179

Plant material except seeds ELISA Symptomatic plant material only PM 7/125

Sample preparation Appendix 1 + -

Detection Sample preparation ToBRFV not detected One of the following tests Appendix 1 RT-PCR (Appendix 2 & 3) Real-time RT-PCR (Appendix 4 & 5)

- + Confirmation of detection and identification : One of the following tests (use a different test than for detection) RT-PCR (Appendix 2 & 3) OR Real-time RT-PCR (Appendix 4 & 5)

- +

Retesting or resampling ToBRFV identified

ToBRFV not detected

Fig. 1 Flow diagram describing the diagnostic procedure for tomato brown rugose fruit virus in plant material except seeds.

Seeds

Sample preparation Appendix 1

Detection One of the following tests Real-time RT-PCR (Appendix 4 & 5)

- +

Confirmation of detection and identification: One of the following tests (use a different test than for detection) Real-time RT-PCR (Appendix 4 & 5)

- +

ToBRFV not detected Retesting or resampling ToBRFV identified

Fig. 2 Flow diagram describing the diagnostic procedure for tomato brown rugose fruit virus in seeds.

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3.1.1. Symptoms on tomato - Blistering of the leaf surface (Fig. 4). The following virus symptoms may be observed on tomato - Wilting of leaves, followed by yellowing (Fig. 5) (Solanum lycopersicum) infected with ToBRFV (Salem and plant death. et al., 2016; Dombrovsky & Smith, 2017; Cambron-Crisan- tos et al., 2018; AHDB Horticulture, 2019): • Leaves or plants - Chlorosis, mosaic patterns (chlorotic/pale patches) (Fig. 3) and mottling often observed on young leaves at the top of the plant and on side-shoots.

Fig. 3 Mosaic symptoms caused by ToBRFV on tomato leaves. Courtesy: C. Picard (EPPO).

- Crumpling, puckering or deformation of young Fig. 5 Wilting of leaves, followed by yellowing and plant death caused leaves. by ToBRFV observed in Germany. Courtesy: H. Scholz-Dobelin€ (LWK - Narrowing of leaves (needle-like symptoms), occa- NRW). sionally observed (Fig. 4). • Pedicles (stems), calyx (sepals), and petioles - Brown necrotic lesions (Figs 6 and 7C illustrating symptoms caused by ToBRFV on fruits (see below) also show symptoms on stems and calices. • Fruits - Yellow (chlorotic) spots and marbling of fruits (Fig. 7A, B and C). - Dark-coloured (necrotic) spots on green fruits (Fig. 7B and D).

Fig. 6 Necrotic lesions caused by ToBRFV on the sepals of a young Fig. 4 Narrowing (needle-like symptoms) and blistering of the surface tomato fruit. Courtesy: Prof. S. Davino. of tomato leave caused by ToBRFV. Courtesy: C. Picard (EPPO).

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(B) Yellow and necrotic spots (a.b) and brown (A) Typical fruit symptoms with yellow (chlorotic) rugose (c) on tomato fruits spots. Courtesy: Dr. P. Keles Ozturk Courtesy: Dr A. Dombrovsky

(C) Yellow spots on tomato fruits and necrotic spots on stems and calix (D) Dark coloured (necrotic) spots on green Courtesy: Dr. P. Keles Ozturk fruits. Courtesy: D. Godínez

(E) Marbling of fruits and delay in ripening. Courtesy: Dr A. Dombrovsky

Fig. 7 Symptoms caused by ToBRFV in tomato fruits. (A) Typical fruit symptoms with yellow (chlorotic) spots. Courtesy: Dr A. Dombrovsky. (B) Yellow and necrotic spots (a, b) and brown rugose (c) on tomato fruits. Courtesy: Dr. P. Keles Ozturk. (C) Yellow spots on tomato fruits and necrotic spots on stems and calix. Courtesy: Dr. P. Keles Ozturk. (D) Dark-coloured (necrotic) spots on green fruits. Courtesy: D. Godınez. (E) Marbling of fruits and delay in ripening. Courtesy: Dr A. Dombrovsky.

- Deformation and uneven ripening of young fruits If any of the above symptoms are observed in a tomato (e.g. individual fruits can be red in some parts and variety harbouring tobamovirus resistance genes (e.g. Tm- showing green stripes, blotches or patches in other 22), there is a strong suspicion that ToBRFV is present as parts) (Fig. 7E). these tomato varieties are susceptible to ToBRFV (Luria - Orange fruits not turning red (variety Juanita in et al., 2017). However, similar symptoms might be caused Germany; Scholz-Dobelin,€ pers. comm., 2020). by other viruses and symptoms might be different in the - Brown rugose (wrinkled) patches (rarely observed). case of mixed infections. - Reduced number of fruits per branch.

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Confusion with other diseases of tomato 3.1.2. Symptoms on pepper Other viruses such as pepino mosaic virus (PepMV), Only pepper plants that do not harbour the L3 and L4 resistance physostegia chlorotic mottle virus (PhCMoV), tomato gene/alleles2 can be infected and therefore show symptoms mosaic virus (ToMV) and tobacco mosaic virus (TMV) may (Turina, pers. comm., 2019); plants harbouring those gene/alle- cause similar (nonspeciﬁc) leaf and fruit symptoms that may les react with a local hypersensitive response and are therefore be confused with ToBRFV symptoms (Alkowni et al., not systemically infected (Dombrowski, pers. comm., 2019). 2019). In some EPPO countries, tomato plants are inoculated Symptoms of a hypersensitive response are shown in Fig. 8. with mild strain(s) of PepMV in an early growth stage at the Based on pictures of symptoms on pepper from Mexico fruit production site to prevent infection by more severe available from Sader & Senasica (2019), pepper shows rela- strains (this technique is called cross-protection) (Aguero€ tively similar symptoms to those described for tomato, but et al., 2018; Pechinger et al., 2019). This can lead to mild more severe necrosis is seen on fruit. However, in pepper vari- virus symptoms early in the growing cycle, masking the eties that do not harbour the L resistance gene/alleles L3 and early ToBRFV symptoms and therefore delaying the detec- L4, ToBRFV often occurs in mixed infections with other tion of ToBRFV. The uneven ripening seen on fruit infected viruses (SADER & SENASICA, 2019). Therefore, the by ToBRFV is in general more severe than for PepMV observed symptoms are potentially due to a combination of (EPPO, 2020). Double infection of ToBRFV and another viruses. Symptoms on pepper from an outbreak in Sicily (IT) tobamovirus may result in accelerated virus accumulation in are described in Panno et al. (2020). The infected variety did the plants. This phenomenon has been observed in suscepti- not harbour the L resistance gene (Davino, pers. comm., 2020). ble wild-type tomato plants and in tomato plants harbouring Symptoms of pepper plants that do not harbour the gene/ the Tm-22 resistance gene infected by paprika mild mottle alleles L3 and L4 obtained after mechanical inoculation are virus (PaMMV) and ToBRFV (Luria et al., 2018). shown in Fig. 9.

Fig. 8 Symptoms of a hypersensitive response (HR) to infection by ToBRFV in pepper plants having L resistance genes (Luria et al., 2017). (A)–(D) Symptoms developed following mechanical inoculation of leaves: (A) necrotic lesions; (B) yellowing; (C, D) leaf necrosis. (E)–(G) HR symptoms developed following mechanical inoculation of roots, showing necrotic spots on stems and growth reduction.

2It is not known for certain whether differences in resistance are caused by allelic variants of the same gene, by the presence/absence of different genes or by variations in different homologous genes. In this protocol the different variants will be referred to as L resistance genes/allele.

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Fig. 9 Symptoms caused by ToBRFV on pepper not harbouring the gene/alleles L3 and L4 after mechanical inoculation (the picture on the top left side shows expanded lesions and necrosis after (A) 26 days or (B) 2 months post inoculation). Courtesy: CREA.

Confusion with other diseases of pepper mainly based on testing of plants for fruit production. In Other viruses may cause similar (nonspecific) leaf and the United Kingdom, for asymptomatic plants, sampling is fruit symptoms that may be confused with ToBRFV based on the collection of 200 leaftlets/leaves per site of symptoms. production and cultivar. This corresponds to the number of units that should be sampled to achieve a 99% confidence level to detect an infection level 9 efficacy of detection of 3.2. Test sample requirements approximately 2%. The virus concentration in different plant parts can vary Young leaflets/leaves should be collected from the top of significantly (high concentration in young leaves and fruits, different plants. and low concentration in old leaves). Since limited data is For molecular testing, leaflets can be pooled in subsam- available so far, only a preliminary recommendation can be ples. In the United Kingdom and France a maximum of 10 made on test sample requirements. leaflets are pooled for one test [A. Fox (pers. comm.) and P. Gentit (pers. comm.), respectively, corresponding to 20 3.2.1. Test sample requirements for leaves subsamples to be tested for one sample of 200 leaflets]. In Tables 1, 3 and 4 of ISPM 31 Methodologies for sampling of Italy and Netherlands pooling up to 50 and 60 leaflets (re- consignments (IPPC, 2008) provide information on the num- spectively) has been shown to be effective [L. Tomassoli ber of units to be sampled to detect varying level of infection (pers. comm.) and Naktuinbouw (unpub. data), respec- depending on the size of a lot and for different confidence tively]. As the number of pooled leaftlets/leaves per sub- levels, and are considered useful to determine sample sizes sample depends on the test, it should be validated. for both consignments and places of production. Sample preparation for molecular tests is described in Experimental data to recommend a specific sample size Appendix 1. Sample preparation for ELISA is described in is lacking. Experience in the EPPO region is currently PM7/125.

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For symptomatic plants, sampling for laboratory testing validated for real-time RT-PCR (ISF, 2020). A sample of is based on at least three symptomatic leaflets collected 3000 seeds allows an infection level of 0.1% to be from the top of the plants. detected with an efficacy of detection of 100% and a confidence level of 95% in lots of more than 200 000 seeds Tomato crops (IPPC, 2008). Guidance on sampling is provided in the EU Sampling requirement was assessed on five symptomatic Commission Implementing Regulation (EU) 2020/1191 and five asymptomatic tomato plants grown in a commer- (2020). Subsample size may also require adaptation if cial greenhouse during an outbreak (NVWA, NL, unpub. other tests need to be performed with smaller subsample data). Samples were taken from the oldest leaf, a leaf at size requirements. 2.5 m height, young leaves in the top of the plant and Seed treatments might influence the outcome of a test. sepals from the calyx (leaves on top of the fruit). For symp- Tests should be performed on seeds that are not primed, tomatic plants, the results indicated that the highest concen- pelleted and/or coated unless it has been either validated trations are found in the young leaves and calyx. Moreover, that these treatments have no significant effect on the per- testing of the older leaves of these infected plants did not formance of the test or internal controls are used to monitor always produce positive results. In the symptomless plants, possible inhibition. Sample preparation is described in the virus was detected in two of the five plants, i.e. in a Appendix 1. young leaf of one plant and in the calyx of another, while all other samples tested negative. 3.3. Screening tests Based on these preliminary data and the fact that virus concentrations are usually the highest in actively growing For the detection of ToBRFV the following tests are recom- tissue, it is recommended for both symptomatic and asymp- mended. tomatic plants to sample young leaflets in the top of the plant. Sampling of the calyx needs further confirmation. 3.3.1. Molecular methods Several specific molecular tests have been described for the Pepper crops detection of ToBRFV (Luria et al., 2017; Alkowni et al., No data is available for testing of different parts of pep- 2019; Ling et al., 2019; Rodrıguez-Mendoza et al., 2019; per plants and therefore based on general experience the Panno et al., 2019a, 2019b; ISF, 2020). sampling of young leaves is currently recommended. The Expert Working Group which drafted this protocol (see Acknowledgements section) discussed the different tests Propagation material to be recommended on different matrices and selected the No data is available on the testing of young plants grown following tests for inclusion in the protocol mainly based on in nurseries. It is not known at which growth stage of the their analytical specificity. For extraction, see Appendix 1. plants the virus can be detected reliably. Seed to seedling transmission in tomato has been found to be low (0.2% 3.3.1.1. Molecular tests for testing plant material (except transmission rate; Dombrovski, pers. comm., 2019). seeds). Molecular tests included in this protocol are those Because there is no data available on the limitations of test- which have been selected based on preliminary validation ing young plants, testing should ideally be carried out as studies by CREA (IT) before a Test Performance Study late as practically possible before the plants leave the nurs- (TPS) organized in the framework of the EU-funded project ery. It should be noted that the likelihood of spread from VALITEST, including a commercial kit (Anthoine et al., an initial infected plant to other plants is higher for grafted 2020). Outcomes of the TPS will be made available plants because of the handling of the plants during the through the EPPO database on diagnostic expertise. grafting process. - One-step conventional RT-PCR: using the primers from Alkowni et al. (2019) – Appendix 2. 3.2.2. Test sample requirements for fruits - One-step conventional RT-PCR: Loewe Biochemica ToBRFV has been successfully detected in symptomatic GmbH using the primers from Rodrıguez-Mendoza et al. tomato and pepper fruits. Further details on sampling strate- (2019) – Appendix 3. gies are not available. Testing of fruits is mainly performed - Real-time RT-PCR: using the CaTa28 and CSP1325 pri- on imported or marketed symptomatic fruits. Sample prepa- mers and probes from ISF (2020) – Appendix 4. ration is described in Appendix 1. - Real-time RT-PCR: using the primers and probe from For fruits with calix, the calix should preferably be tested. Menzel & Winter (in press) – Appendix 5. The tests described in Ling et al., (2019) and Panno 3.2.3. Test sample requirements for seeds et al. (2019a) are not included in this Standard due to poor For seed testing it is difficult to recommend sample sizes analytical specificity (cross-reactions) (Anthoine et al., and bulking rates. For seed lots of tomato and pepper, pro- 2020). The primers of Rodrıguez-Mendoza et al., 2019 are tocols using weighed samples of approximately 3000 used in the test described in Appendix 3 (Loewe Biochem- seeds, tested in three subsamples of 1000 seeds, have been ica GmbH kit). The test described in Luria et al. (2017) has

ª 2021 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 51, 178–197 PM 7/146 (1) Tomato brown rugose fruit virus 185 not been included as other tests have a better analytical in Appendix 1 of PM 7/2 (EPPO, 2017). Test plants recom- sensitivity as well as analytical specificity. mended are Nicotiana glutinosa or Nicotiana tabacum cv. The real-time PCR test using primers from Panno et al. Xanthi NN (ISF, 2020). Plants should be examined for local (2019b) is under evaluation and will be considered for a lesions (positive and negative controls should be included). revision of this protocol. 4. Identification 3.3.1.2. Molecular tests for testing seeds. Molecular tests included in this protocol are those recommended by ISF and 4.1. Molecular tests one based preliminary validation studies by CREA (IT). - Real-time RT-PCR using the CaTa28 and CSP1325 pri- The four molecular tests described in section 3.3.1.1 are mers and probes from ISF (2020) – Appendix 4. recommended for identification and confirmation. - Real-time RT-PCR using the primers and probe from The choice of the test will depend on the matrix, i.e. the Menzel & Winter (in press) - Appendix 5. expected virus concentration. For leaf material, both real- A TPS specific for seed testing will be organized in the time and conventional RT-PCR tests can be used, whereas framework of Euphresco and data from this TPS will be for seeds real-time RT-PCR tests are recommended. used to prepare a revision of the current protocol.

4.2. Other tests 3.3.2. Other screening tests 3.3.2.1. Other molecular tests. Generic tobamovirus RT- 4.2.1. Sequencing PCR tests such as Li et al. (2018), Letschert et al. (2002), Sanger sequencing or high-throughput sequencing analysis Levitzky et al. (2019) and Menzel et al. (2019) may also be can be used for further confirmation when the virus concen- used for screening of plant material (except seeds), but they also tration allows for its application. detect other species. Confirmation of positive tests with Sequence analysis of amplicons obtained from specific sequence analysis or a specific molecular test (see section RT-PCR (3.3.1.1) or generic RT-PCR (3.3.2.1) can be used 3.3.1.) is required. No validation data is available for these tests. for identification. Sequence analysis should follow the A LAMP test for the detection of ToBRFV has been devel- guidelines described in Appendices 7 and 8 of the EPPO oped in Canada (Sarkes et al., 2020). However, there is no Standard PM 7/129 DNA barcoding as an identification experience with this test in the EPPO region. Two LAMP tests tool for a number of regulated pests (EPPO, 2021 (in this are being evaluated at Fera Science Ltd (GB) (including an issue of the EPPO Bulletin)). adaptation of the test of Sarkes et al., 2020) and the inclusion The Panel on Diagnostics in Virology and Phytoplasmol- of these tests will be considered for a revision of this protocol. ogy noted that high-throughput sequencing technologies may be used for obtaining complete or near-complete gen- 3.3.2.2. Serological tests. ELISA may be used as a screen- ome sequences, which can be analysed for identification of ing test on symptomatic material. ToBRFV antisera currently a virus isolate. available were found to cross-react with other tobamoviruses; antisera raised to related tobamoviruses, e.g. ToMV, detect 5. Reference material ToBRFV at lower analytical sensitivity than the homologous virus. Instructions for performing an ELISA are provided in ToBRFV isolates for reference are available from: the EPPO Standard PM 7/125 ELISA tests for viruses (EPPO, DSMZ Leibniz-Institut DSMZ-Deutsche Sammlung von 2015). Confirmation of positive test with a molecular test Mikro- organismen und Zellkulturen GmbH Inhoffenstraße (see section 3.3.1.1.) is required because of cross-reactions 7 B 38124 Braunschweig (DE). with other tobamoviruses. Consiglio per la ricerca in agricoltura e l’analisi A comparison between serological and molecular tests, dell’economia agraria (CREA), Centro di Ricerca Difesa e particularly regarding the analytical sensitivity, would be Certificazione (CREA-DC), Via C.G. Bertero, 22, 00156 needed before the inclusion of serological tests for testing Rome, (IT) ([email protected]). of asymptomatic material or seeds can be considered for inclusion in a revision of this protocol. 6. Reporting and documentation 3.3.2.3. Bioassay. There is no available validation data on Guidelines on reporting and documentation are given in the use of bioassay for detection of ToBRFV and for the EPPO Standard PM 7/77 Documentation and reporting on confirmation of the presence of viable ToBRFV on plant a diagnosis. material and seeds. In general, analytical sensitivity of bioassay is known to be lower than for ELISA and molecu- 7. Performance characteristics lar tests. However, experience in laboratories in the region indicates that it may be used for detection from symptomatic When performance characteristics are available, these are material. Extraction buffers that can be used are described provided with the description of the test. Validation data is

ª 2021 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 51, 178–197 186 Diagnostic also available in the EPPO Database on Diagnostic Exper- Journal of Virological Methods. 187,43–50. https://doi.org/10.1016/j. tise (http://dc.eppo.int), and it is recommended to consult jviromet.2012.09.004. this database as additional information may be available Brittain Anthoine, Dreo Chabirand, Gueniau Faggioli, Luigi Harrison, Mehle Lukezic, Bavnikar Mezzalam Mouaziz, Spadaro Renvoise & there (e.g. more detailed information on analytical speci- Vucurovic Tomassoli (2020) List of tests for validation – Round – ficity, full validation reports, etc.) deliverable N° 1.3 – VALITEST project (Grant agreement N. 773139). https://www.valitest.eu/media/files/deliverables/WP6/D1.3_ List_of_tests_for_validation_-_Round_2.pdf [accessed 15 April 8. Further information 2020]. Further information on this organism can be obtained from: Cambron-Crisantos JM, Rodrıguez-mendoza J, Valencia-luna JB, Botermans M (NVWA, NL), Fox A (Fera, GB), Mehle N Alcasia Rangel S, Garcia-Avila CG, Lopez-Buenfil JA et al. (2018) First report of Tomato brown rugose fruit virus (ToBRFV) in (NIB, SI, EURL on Viruses, Viroids and Phytoplasmas), Michoacan, Mexico. Mexican Journal of Phytopathology, 37(1), 1–8. Oplaat AG (NVWA, NL), Roenhorst JW (NVWA, NL, http://www.rmf.smf.org.mx/Vol3712019/RMF1810-5.pdf [accessed EURL on Viruses, Viroids and Phytoplasmas), Tomassoli L on 15 April 2020]. (CREA, IT, EURL on Viruses, Viroids and Phytoplasmas) Dombrovsky A & Smith E. (2017) Seed Transmission of and Ziebell H (JKI, DE). Tobamoviruses: Aspects of Global Disease Distribution. In: Advances in Seed Biology, Chapter 12, pp. 233–260. INTECH. https://www. intechopen.com/books/advances-in-seed-biology/ [accessed on 15 9. Feedback on this diagnostic protocol April 2020]. EPPO (2015) PM 7/125 (1) ELISA tests for viruses. EPPO Bulletin 45, If you have any feedback concerning this Diagnostic Proto- 445–449. https://doi.org/10.1111/epp.12259 [accessed 15 April 2020]. col, or any of the tests included, or if you can provide addi- EPPO (2017) PM 7/2 (2) Tobacco ringspot virus. EPPO Bulletin 47, tional validation data for tests included in this protocol that 135–145. https://doi.org/10.1111/epp.12376. you wish to share please contact [email protected]. EPPO (2020) EPPO Global Database (available online). https://gd.eppo. int [accessed 15 April 2020]. EU (2020) Commission Implementing Regulation (EU) 2020/1191 of 10. Protocol revision 11 August 2020 establishing measures to prevent the introduction into and the spread within the Union of Tomato brown rugose fruit An annual review process is in place to identify the need virus (ToBRFV) and repealing Implementing Decision (EU) 2019/ for revision of diagnostic protocols. Protocols identified as 1615 C/2020/5453. needing revision are marked as such on the EPPO website. Gambino G, Perrone I & Gribaudo I (2008) A Rapid and effective method for RNA extraction from different tissues of grapevine and When errata and corrigenda are in press, this will also be other woody plants. Phytochemical AnalysisPhytochemical Analysis. marked on the website. 19(6), 520–525. https://doi.org/10.1002/pca.1078 IPPC (2008) ISPM 31 Methodologies for sampling of consignments, FAO, Rome, Italy. Acknowledgements ISF (2020) Detection of Infectious Tomato brown rugose fruit virus (ToBRFV) in Tomato and Pepper Seed. https://www.worldseed.org/ This protocol was prepared by an Expert Working Group wp-content/uploads/2020/03/Tomato-ToBRFV_2020.03.pdf [accessed composed of Fox A (Fera, GB lead author), Mehle N (NIB, 15 April 2020]. SI, EURL on Viruses, Viroids and Phytoplasmas), Roen- Letschert B, Adam G, Lesemann D-E, Willingmann P & Heinze C horst JW (NVWA, NL, EURL on Viruses, Viroids and (2002) Detection and differentiation of serologically cross-reacting Phytoplasmas), Oplaat AG (NVWA, NL), Tomassoli L tobamoviruses of economical importance by RT-PCR and RT-PCR- – (CREA, IT, EURL on Viruses, Viroids and Phytoplasmas), RFLP. Journal of Virological Methods 106,1 10. Levitzky Naama, Smith Elisheva, Lachman Oded, Luria Neta, Mizrahi Ziebell H (JKI, DE) it was reviewed by the Panel on Virol- Yaniv, Bakelman Helen et al. (2019) The bumblebee Bombus ogy and Phytoplasmology. terrestris carries a primary inoculum of Tomato brown rugose fruit virus contributing to disease spread in tomatoes. PLoS One 14(1), References e0210871. https://doi.org/10.1371/journal.pone.0210871. Li Y, Tan G, Lan P, Zhang A, Liu Y, Li R & et al. (2018) Detection Aguero€ J, Gomez-Aix C, Sempere RN, Garcıa-Villalba J, Garcıa-Nunez~ of tobamoviruses by RT-PCR using a novel pair of degenerate – J, Hernando Y et al. (2018) Stable and broad spectrum cross- primers. Journal of Virological Methods 259, 122 128. https://doi. protection against pepino mosaic virus attained by mixed infection. org/10.1016/j.jviromet.2018.06.012. Frontiers in Plant Science 9,1–12. https://doi.org/10.3389/fpls.2018. Ling KS, Tian T, Gurung S, Salati R & Gilliard A (2019) First report 01810. of Tomato brown rugose fruit virus infecting greenhouse tomato in AHDB Horticulture (2019) Tomato Brown Rugose Fruit Virus the United States. Plant Disease 103, 1439. (ToBRFV). AHDB Horticulture, pp. 1–2. Luria N, Smith E, Reingold V, Bekelman I, Lapidot M, Levin I et al. Alkowni R, Alabdallah O & Fadda Z (2019) Molecular identification of (2017) A new Israeli Tobamovirus isolate infects tomato plants 2 – tomato brown rugose fruit virus in tomato in Palestine. Journal of harboring Tm-2 resistance genes. PLoS One 1 19, https://doi.org/ Plant Pathology 101(3), 719–723. 10.1371/journal.pone.0170429. Botermans M, van de Vossenberg BT, Verhoeven JT, Roenhorst JW, Luria N, Smith E, Sela N, Lachman O, Bekelman I, Koren A et al. Hooftman M, Dekter R et al. (2013) Development and validation of (2018) A local strain of Paprika mild mottle virus breaks 3 a real-time RT-PCR assay for generic detection of pospiviroids. L resistance in peppers and is accelerated in Tomato brown rugose

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fruit virus-infected Tm-22-resistant tomatoes. Virus Genes 54, 280– control using real-time RT-PCR. Journal of Virological Methods 289. https://doi.org/10.1007/s11262-018-1539-2. 176,53–59. Menzel W, Jelkmann W & Maiss E (2002) Detection of four apple Pechinger K, Chooi KM, MacDiarmid RM, Harper SJ & Ziebell HA viruses by multiplex RT-PCR assays with coamplification of plant (2019) New Era for Mild Strain Cross-Protection. Viruses 11, 670. mRNA as internal control. Journal of Virological Methods. 99,81–92. https://doi.org/10.3390/v11070670. Menzel W, Knierim D, Winter S, Hamacher J & Heupel M (2019) Rodrıguez-Mendoza J, Garcia-Avila CJ, Lopez-Buenfil JA, Araujo-Ruiz First report of Tomato brown rugose fruit virus infecting tomato in K, Quezada A, Cambron-Crisantos JM et al. (2019) Identification of Germany. New Disease Reports 39, 1. https://doi.org/10.5197/j.2044- Tomato brown rugose fruit virus by RT-PCR from a coding region 0588.2019.039.001. or replicase. Mexican Journal of Phytopathology 37(2), 346–356. Menzel W & Winter S. (in press) Identification of novel and known SADER & SENASICA (2019) Tomato brown rugose fruit virus tobamoviruses in tomato and other solanaceous crops using a new (ToBRFV): caso Mexico. Descargue Aquı Las Presentaciones Del pair of generic primers and development of a specific RT-qPCR for SEMINARIO: Tomato Brown Rugose Fruit Virus (ToBRFV) ToBRFV. Acta Horticulturae. Realizado El 8 de Marzo de 2019 En Cd. Obregon, Sonora. SADER NAPPO (2018) Official Pest Reports. Tomato Brown Rugose Fruit Virus: & SENASICA. https://issuu.com/cesavesonora/docs/1_antecedente Detected in the Municipality of Yurecuaro, Michoacan. NAPPO. s_e_identificacion_tom https://www.pestalerts.org/oprDetail.cfm?oprID=765 [accessed 15 Salem NM, Cao MJ, Odeh S, Turina M & Tahzima R (2019) First April 2020]. report of tobacco mild green mosaic virus and tomato brown rugose Panno S, Caruso AG, Blanco G & Davino S (2020) First report of fruit virus infecting Capsicum annuum in Jordan. APS Publication 1– Tomato brown rugose fruit virus infecting sweet pepper in Italy. New 3. https://doi.org/10.1094/PDIS-06-19-1189-PDN. Disease Reports 41, 20. https://doi.org/10.5197/j.2044-0588.2020.041. Salem N, Mansour A, Ciuffo M, Falk BW & Turina M (2016) A new 020. tobamovirus infecting tomato crops in Jordan. Archives of Virology Panno S, Caruso AG & Davino S (2019a) First report of tomato brown 161(2), 503–506. https://doi.org/10.1007/s00705-015-2677-7. rugose fruit virus on tomato crops in Italy. Plant Disease 103, 1443. Sarkes A, Fu H, Feindel D, Harding MW & Feng J. (2020) Development Panno S, Ruiz-Ruiz S, Caruso AG, Alfaro-Fernandez A, Ambrosio FS and evaluation of a loop-mediated isothermal amplification (LAMP) & Davino S(2019b) Real-time reverse transcription polymerase chain assay for the detection of Tomato brown rugose fruit virus (ToBRFV). reaction development for rapid detection of Tomato brown rugose bioRxiv. https://doi.org/10.1101/2020.03.02.972885 fruit virus and comparison with other techniques. PeerJ 7, e7928. Weller SA, Elphinstone JG, Smith NC, Boonham N & Stead DE https://doi.org/10.7717/peerj.7928. (2000) Detection of Ralstonia solanacearum Strains with a Papayiannis LC, Harkou IS, Markou YM, Demetriou CN & Katis NI quantitative, multiplex, real-time, fluorogenic PCR (TaqMan). Assay. (2011) Rapid discrimination of Tomato chlorosis virus, Tomato Applied and Environmental Microbiology 66(7), 2853–2858. https:// infectious chlorosis virus and co-amplification of plant internal doi.org/10.1128/AEM.66.7.2853-2858.2000.

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GH+ buffer (see Table 1). Samples are incubated for Appendix 1 – Sample preparation and RNA 10 min at 65°C. After centrifugation at 12 000g for 2 min, extraction for molecular methods 500 µL of supernatant is loaded on the QIAshredder spin This appendix describes sample preparation and RNA column and centrifuged. Thereafter, the manufacturer’s extraction methods for different hosts and types of plant instructions in the RNeasy Plant Mini kit (Qiagen) should material. These initial steps are critical for the results of a be followed. test and are often more related to the matrix than the speci- For high-throughput RNA extraction, the Sbeadex maxi fic test. Therefore, they are described in this separate plant kit can be used in combination with a Kingfisher appendix. KF96 system. In this system 250 lL of the supernatant is A wide range of RNA extraction methods may be used, transferred to a binding plate containing 450 lL of binding from commercial kits to methods published in scientific buffer and 50 lL of particle suspension (both included in journals. RNA extraction using CTAB (Gambino et al., the kit) and RNA is extracted following the manufacturer’s 2008) is robust and is considered by the Panel on Diagnos- instructions. tics in Virology and Phytoplasmology as an appropriate method for RNA extraction for all types of tissues which 2. Seeds will not affect the performance of the molecular tests. The CTAB method is not further described in this appendix. Sample preparation, including grinding, and RNA extrac- Care should be taken when handling samples as high tion from seeds can be challenging. Two procedures using concentration of virus may be present. different homogenization buffers are described. Extracted RNA should be stored refrigerated for short- 2.1. Homogenization in phosphate buffer term storage (<8h)at20°C(<1 month) or at 80°C for For both tomato and pepper, 12 subsamples of 250 seeds longer periods. can be immersed in 10 mL of 0.1 M phosphate buffer

(Na2HPO4/KH2PO4, pH 7.2), incubated at 4 2°C over- night, and then ground with, for example, a FastPrep 1. Plant material (leaves or fruits) homogenizer at 5 m s1 for 40 s. After centrifugation at 1.1. Individual plants and/or small samples 10 000g at 4°C for 10 min, the supernatant can be used for For testing individual plants and/or small samples the RNA extraction using the RNeasy Plant Mini kit (Qiagen), RNeasy Plant Mini kit (Qiagen) can be used according to following the manufacturer’s instructions with some minor the manufacturer’s instructions. modifications. The homogenates can be processed sepa- 1.2. Pooled samples rately or combined to three subsamples of four homoge- Pooled samples of tomato leaflets or sepals, pepper nates. leaves, or fruits should consist of equal amounts of each Briefly, 600 µL of the supernatant is added to 600 µLof plant. For leaf material, this can be achieved by, for exam- RLT buffer (without b-mercaptoethanol). Two aliquots of ple, stacking leaves and preparing leaf discs using a dispos- 600 µL of this mix are loaded one after the other on the able 4-mm leaf punch or by cutting or tearing the top parts. same QIAshredder spin column and centrifuged. Subse- For fruits samples of tomato and pepper, a small piece of quently, the manufacturers protocol is followed until the the skin and adjacent tissue should be sampled from at least elution step. RNA is eluted from the RNeasy Mini Spin three different locations of each fruit. For pooled samples, columns by applying 50 µL of RNase-free warm water however, larger amounts of plant material maybe involved. (65°C) followed by centrifugation. To maximize RNA In this case other buffers can be used for homogenization, recovery, an additional elution step is performed using the such as ELISA sample buffer or GH+ buffer as specified in same procedure. Table 1. 2.2. Homogenization using GH+ buffer For both tomato and pepper three subsamples of 1000 Table 1. GH+ buffer seeds are transferred to a grinding bag (e.g. Interscience BagPage 100 mL) and 20 mL of GH+ buffer (Table 1) is Amount Final concentration added. The seeds are soaked at room temperature for 30– 60 min before homogenization (e.g. with an Interscience Guanidine hydrochloride 573.18 g 6 M BagMixer on position 4) for 90 s (tomato) or 4 min (pep- Sodium acetate (4 M, pH 5.2) 50 mL 0.2 M per). EDTA Na2 2H2O 9.3 g 25 mM PVP-10 25.0 g 2.5% w/v Alternatively, dry seeds are ground with a Genogrinder. Distilled water to 1.0 L Three subsamples of 1000 tomato or six subsamples of 500 pepper seeds are transferred to a 50-mL tube and a steel ball (14 mm) is added. Seeds are ground, tubes upside down, at 1700 rpm for 4 min for tomato and 7 min for Approximately 1 g of plant tissue is put in an extraction pepper seeds. After grinding GH+ buffer is added, 20 mL bag and homogenized in 3.5 mL (range 1:2–1:5 (w/v)) of for tomato or 10 mL for pepper samples. Tubes are shaken

ª 2021 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 51, 178–197 PM 7/146 (1) Tomato brown rugose fruit virus 189 by hand to obtain homogenous solutions. Duplicate pepper (Thermo Scientific, Fera Science Ltd) and One Taq One- homogenates are combined and mixed before further pro- Step RT-PCR Kit with Quick-Load buffer (NEB, JKI) (data cessing to make three subsamples. not shown). One millilitre of the seed homogenate is transferred into a 1.5-mL tube and 30 lL of dithiothreitol (DTT, 5 M) is added, 2. Methods followed by incubation in a thermoshaker at 850 rpm and 65°C for 15 min. After centrifugation at 16 000g for 10 min, 2.1. Nucleic acid extraction and purification 750 µL of supernatant is loaded on the QIAshredder spin col- See Appendix 1, alternative procedures may also be suit- umn and centrifuged. Thereafter the manufacturer’s instruc- able. tions for the RNeasy Plant Mini Kit (Qiagen) are followed. 2.2. One-step RT-PCR For high-throughput RNA extractions, the Sbeadex 2.2.1. Master Mix maxi plant kit can be used in combination with a Kingfisher KF96 system. In this system 250 lL of the supernatant is Working Volume per Final µ transferred to a binding plate containing 600 lL of binding Reagent concentration reaction ( L) concentration l buffer (kit) and 50 L of particle suspension (both included Molecular-grade NA 11.6 in the kit) and RNA is extracted following the manufac- water* turer’s instructions. Onestep RT-PCR 59 4.0 19 Note that when Bacopa chlorosis virus (BaCV) is used as Buffer (Qiagen) internal control, this virus is added to GH+ buffer (BaCV dNTPs 10 mM 0.8 0.4 mM ToBRFV-F 10 µM 0.4 0.2 µM infected leaf material homogenized, in a dilution of approx- µ µ 4 ToBRFV-R 10 M 0.4 0.2 M imately 10 -fold aiming for a Ct value of 28-32). Onestep RT-PCR NA 0.8 NA Enzyme mix Appendix 2 – One-step conventional RT-PCR (Qiagen) Subtotal 18.0 test for ToBRFV (using the primers from Sample RNA extract NA 2 NA Alkowni et al., 2019) Total 20.0 The test below is described as it will be carried out to gen- *Molecular grade water should be used preferably, or prepared purified erate the validation data in the framework of the EU project (deionized or distilled), sterile (autoclaved or 0.22 µm filtered) and VALITEST. Other equipment, kits or reagents may be used nuclease-free. provided that a verification (see PM 7/98) is carried out. 2.2.2. RT-PCR cycling conditions Reverse transcription at 50°C for 30 min; denaturation at 1. General Information 95°C for 15 min; 35 cycles consisting of denaturation at 1.1. This conventional RT-PCR is suitable for the detec- 94°C for 30 s, annealing at 58°C for 30 s, extension at tion and identification of ToBRFV in leaves, calyx and fruit 72°C for 30 s; final extension at 72°C for 10 min. of tomato and pepper. Note: If other enzymes are used reaction conditions may 1.2. The test uses the primers of Alkowni et al., 2019. be changed. The original protocol was adapted to a one-step format. 1.3. The ToBRFV-F/ToBRFV-R primers were designed 3. Essential procedural information to target the small replicase subunit region of isolate TBRFV-Ps, accession number MK165457 (primer posi- 3.1. Controls tions: forward 735-755, reverse 1271-1294). For a reliable test result to be obtained, the following 1.4. Oligonucleotides and average amplicon size: controls should be included for each series of nucleic acid extraction and amplification of the target organism and tar- Amplicon get nucleic acid, respectively. Primer Sequence size • Negative isolation control (NIC) to monitor contamination Forward primer ToBRFV-F 50-AAT GTC CAT 560 bp during nucleic acid extraction: nucleic acid extraction and GTT TGT TAC subsequent amplification preferably of a sample of unin- GCC-30 fected matrix or if not available clean extraction buffer. 0 Reverse primer ToBRFV-R 5 -CGA ATG TGA • Positive isolation control (PIC) to ensure that nucleic acid TTT AAA ACT of sufficient quantity and quality is isolated: nucleic acid GTG AAT-30 extraction and subsequent amplification of the target organism or a matrix sample that contains the target 1.5. The test has been successfully performed using the organism (e.g. naturally infected host tissue). OneStep RT-PCR Kit (Qiagen, ref 21021). Alternative • Negative amplification control (NAC) to rule out false reagents were used: Verso 1-Step RT-PCR Kit ReddyMix positives due to contamination during the preparation of

ª 2021 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 51, 178–197 190 Diagnostic

the reaction mix: amplification of molecular-grade water No cross-reactions occurred with isolates of any of the that was used to prepare the reaction mix. viruses or viroids listed below (Source: Fera unless other- • Positive amplification control (PAC) to monitor the effi- wise stated, further validation is in progress). ciency of the amplification of nucleic acid of the target Tobamoviruses: bell pepper mosaic virus (JKI), cucum- organism. This can include a known amount of nucleic ber green mottle mosaic virus (JKI), pepper mild mottle acid extracted from the target organism, total nucleic acid virus (Fera and JKI), tobacco mosaic virus (Fera and JKI), extracted from infected host tissue or a synthetic control tomato mosaic virus (Fera and JKI), tomato mottle mosaic (e.g. cloned PCR product, artificial RNA or DNA). The virus (JKI), ullucus tobamovirus-1. PAC should preferably be near to the limit of detection. Common tomato affecting viruses: pepino mosaic virus As an alternative (or in addition) to the external positive (All strains), southern tomato virus, tomato spotted wilt controls (PIC and PAC), internal positive controls (IPCs) virus (Fera and JKI), tomato black ring virus (JKI), tomato can be used to monitor each individual sample separately. bushy stunt virus (JKI), tomato ringspot virus (JKI). IPC can include an endogenous nucleic acid of the matrix Other viruses: barley stripe mosaic virus (JKI), pea early using conserved primers, preferably amplifying RNA tar- browning virus (JKI), potato mop-top virus (JKI), soil- gets, such as nad5 (Menzel et al., 2002). borne cereal mosaic virus (JKI). 3.2. Interpretation of results Pospiviroids: citrus exocortis viroid, columnea latent vir- Verification of the controls oid, pepper chat fruit viroid, potato spindle tuber viroid • NIC and NAC: no band is visualized. tomato apical stunt viroid, tomato planta macho viroid. • PIC and PAC: a band of the expected size (~560 bp) is Mixed infections: potato virus X with tomato mosaic visualized. virus, potato virus Y with tobacco mosaic virus. • IPC if used: a band of the expected size (e.g. ~181 bp for 4.3. Selectivity nad5) is visualized. Data from JKI was obtained using a two-step RT-PCR When these conditions are met protocol. • A test will be considered positive if a band of the The test does not give false positive reactions with leaf expected size (~560 bp) is visualized. material of Capsicum annuum (pepper) (Fera, JKI), • A test will be considered negative if no band or a band Chenopodium murale (JKI), Petunia 9 hybrida, variety of a different size than expected is visualized. Himmelsroschen€ (JKI), Solanum lycopersicum (tomato) • Tests should be repeated if any contradictory or unclear (Fera), Solanum melongena (aubergine) (JKI) and Ullucus results are obtained. tuberosus (Fera). It should be noted that, in general, for viruses and vir- 4.4. Repeatability and reproducibility oids, bands of different sizes may correspond to strains of Although the performance characteristics for repeatability the target organism and care should be taken when inter- and reproducibility have not been evaluated according to preting the results of conventional PCR, in particular the PM 7/98, the test has been repeated and reproduced on sev- sizes of bands. eral occasions with consistent results.

4. Performance criteria available Appendix 3 – One-step conventional RT-PCR (Loewe Biochemica GmbH, using primers Limited data was obtained from JKI and Fera Science Ltd from Rodrıguez-Mendoza et al., 2019) (GB). However, the test will be validated according to PM 7/98 Specific requirements for laboratories preparing The test below is described as it will be carried out to gen- accreditation for a plant pest diagnostic activity within EU erate the validation data in the framework of the EU project project VALITEST. As soon as validation data from VALITEST. Other equipment, kits or reagents may be used those two projects is available, it will be published at provided that a verification (see PM 7/98) is carried out. http://dc.eppo.int/validationlist.php. 4.1. Analytical sensitivity data 1. General information Using the one-step RT-PCR the virus can be detected at a dilution of 10-8 based on a dilution series prepared from 1.1. This test can be used for detection and identification of RNA extracts of infected plant material mixed with RNA tomato brown rugose fruit virus (ToBRFV) using primers extracted from non-infected plants (Source: JKI, further val- designed by Rodrıguez-Mendoza et al. (2019). The original idation is in progress). protocol was adapted to a one-step format and further opti- 4.2. Analytical specificity data mized for routine use by the Loewe Biochemica GmbH. • Inclusivity 1.2. This test has been successfully used for testing Evaluated with isolates from Germany and the United fresh, frozen or dried leaf and fruit material from tomato Kingdom (it should be noted that low isolate variation is and pepper. observed worldwide). 1.3. Primers ToBRFV-FMX and ToBRFV-RMX were • Exclusivity designed to target part of the putative RNA-dependent RNA

ª 2021 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 51, 178–197 PM 7/146 (1) Tomato brown rugose fruit virus 191 polymerase gene of isolate ToBRFV-IL, accession number can be used to monitor each individual sample separately. KX619418 (primer position: forward 2063–2086; reverse IPC can include an endogenous nucleic acid of the matrix 2514–2537). using conserved primers, preferably amplifying RNA tar- 1.4. Oligonucleotides gets, such as nad5 (Menzel et al., 2002). 3.2. Interpretation of results Amplicon Verification of the controls Primer Sequence size • NIC and NAC: no band is visualized. • ~ Forward ToBRFV-FMX 5’-AAC CAG AGT CTT ~475 bp PIC and PAC: a band of the expected size ( 475 bp) primer CCT ATA CTC GGA A-3’ should be visualized. Reverse ToBRFV-RMX 5’-CTC WCC ATC TCT • IPC if used: a band of the expected size (e.g. ~181 bp for primer TAA TAA TCT CCT-3’ nad5) is visualized. When these conditions are met • A test will be considered positive if a band of the expected size (~475 bp) is visualized. 2. Methods • A test will be considered negative if no band or a band 2.1. Nucleic acid extraction of a different size than expected is visualized. See Appendix 1. Alternative procedures may also be suit- • Tests should be repeated if any contradictory or unclear able. results are obtained. 2.2. One-step RT-PCR (Loewe Biochemica GmbH) It should be noted that, in general, for viruses and viroids 2.3.1. Prepare master mix according to manufacturer’s bands of different sizes may correspond to strains of the tar- instructions. get organism and care should be taken when interpreting the 2.3.2. Reverse transcription PCR cycling parameters results of conventional PCR, in particular the sizes of bands. according to manufacturer’s instructions. 4. Performance criteria available 3. Essential procedural information Validation data was generated according PM 7/98 (4) 3.1. Controls Specific requirements for laboratories preparing For a reliable test result to be obtained, the following accreditation for a plant pest diagnostic activity. Validation (external) controls should be included for each series of was performed at CREA-DC (Italy) in the framework of nucleic acid extraction and amplification of the target VALITEST project (Horizon 2020). Additional validation organism and target nucleic acid, respectively. data obtained in the framework of VALITEST will be pub- • Negative isolation control (NIC) to monitor contamina- lished when available at http://dc.eppo.int/validationlist.php. tion during nucleic acid extraction: nucleic acid extraction 4.1. Analytical sensitivity data and subsequent amplification preferably of a sample of The virus can be detected at a dilution of 105 dilution uninfected matrix or if not available clean extraction buf- of homogenate of infected tomato leaves diluted in homo- fer. genate of healthy tomato leaves and 103 of homogenate of • Positive isolation control (PIC) to ensure that nucleic acid infected-pepper leaves diluted in homogenate of healthy of sufficient quantity and quality is isolated: nucleic acid pepper leaves. extraction and subsequent amplification of the target 4.2. Analytical specificity data organism or a matrix sample that contains the target 4.2.1. Inclusivity organism (e.g. naturally infected host tissue). Evaluated with isolates of ToBRFV PV-1236 and PV- • Negative amplification control (NAC) to rule out false 1241 (from DSMZ, Leibniz Institute, Germany) and Italian positives due to contamination during the preparation of isolates of ToBRFV from Sicily (ToB-SIC01/19) and Pied- the reaction mix: amplification of molecular grade water mont (T1103). that was used to prepare the reaction mix. 4.2.2. Exclusivity • Positive amplification control (PAC) to monitor the effi- No cross-reactions were observed with isolates of bell ciency of the amplification: amplification of nucleic acid pepper mottle virus (BPeMV), tobacco mild green mottle of the target organism. This can include nucleic acid virus (TMGMV), tobacco mosaic virus (TMV), tomato extracted from the target organism, total nucleic acid mosaic virus (ToMV), obtained from DSMZ (isolates PV- extracted from infected host tissue, whole-genome ampli- 0170, PV-0124, PV-1252 and PV-0141, respectively) and fied DNA or a synthetic control (e.g. cloned PCR pro- pepper mild mottle virus (PMMV) isolate from Italy duct). The PAC should preferably be near to the limit of (PMMV-218/14 CREA-DC). detection. 4.3. Selectivity data As an alternative (or in addition) to the external positive The test does not give false positive reactions with controls (PIC and PAC), internal positive controls (IPCs) healthy tomato and pepper leaves and tomato fruits.

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4.4. Repeatability data Table (continued) 100% of repeatability was obtained testing samples of 5 3 tomato leaves (10 dilution) and pepper leaves (10 dilu- Amplicon tion) on different occasions. Primer/probe Sequence size

Forward primer* BaCV-F 5’-CGA TGG GAA TTC 87 Appendix 4 – Duplex real-time RT-PCR test Reverse primer* BaCV-R ACT TTC GT-3’ for ToBRFV (using CaTa28 and CSP1325 Probe* BaCV-P 5’-AAT CCA CAT CGC primers and probe from ISF, 2020) ACA CAA GA-3’ 5’-TxR - CAA TCC TCA CAT GAT GAG ATG CCG-BHQ2-3’ A. Test as in ISF-ISHI-Veg (2020) *When BaCV is used as IPC. The test below is described as in ISF-ISHI-Veg (2020). 1.4. The test has been validated using UltraPlexTM 1- Other equipment, kits or reagents may be used provided Step ToughMix (QuantaBio) using CFX 96 (Bio-Rad). that a veriﬁcation (see PM 7/98) is carried out.

2. Methods 1. General Information 2.1. Nucleic acid extraction and purification 1.1. This one-step triplex real-time RT-PCR protocol See Appendix 1. Alternative procedures may also be suit- was developed for the detection and identification of able. ToBRFV in tomato and pepper seeds but can also be used 2.2. One-step real-time RT-PCR for leaf material. 2.2.1. Master Mix 1.2. The test is based on CaTa28 primers and probe developed by Enza Zaden BV (NL) in combination with the CSP1325 primers and probe developed by CSP Labs Working Volume per Final (US). This test includes the BaCV primers and probe devel- Reagent concentration reaction (µL) concentration oped by Naktuinbouw (NL) as internal positive control. * The target sequence of the CaTa28 primers is located Molecular grade water NA Up to 20 UltraPlex 1-Step 49 6.25 19 within the movement protein gene; using the nucleotide ToughMix (QuantaBio) sequence of GenBank accession no NC_028478.1, the for- CaTa28 Fw 10 µM 0.75 0.3 µM ward and reverse primer start at position 5163 and 5283, CaTa28 Rv 10 µM 0.75 0.3 µM respectively, and the probe covers positions 5191–5211. CaTa28 Pr 10 µM 0.50 0.2 µM The target sequence of the CSP1325 primers is located at CSP1325 Fw 10 µM 0.75 0.3 µM the end of the coat protein gene, using the nucleotide CSP1325 Rv 10 µM 0.75 0.3 µM µ µ sequence of GenBank accession no NC_028478.1, the for- CSP1325 Pr 10 M 0.50 0.2 M BaCV-F** 10 µM 0.75 0.3 µM ward and reverse primers start at positions 6144 and 6223, BaCV-R** 10 µM 0.75 0.3 µM – respectively, and the probe covers positions 6169 6195. BaCV-P** 10 µM 0.50 0.2 µM 1.3 Oligonucleotides Subtotal 20.00 RNA 5.00 Amplicon Total 25.00 Primer/probe Sequence size *Molecular-grade water should be used preferably, or prepared purified Forward primer CaTa28 Fw 5’-GGT GGT GTC AGT 139 (deionized or distilled), sterile (autoclaved or 0.22 µm filtered) and Reverse primer CaTa28 Rv GTC TGT TT-3’ nuclease-free. Probe CaTa28 Pr 5’-GCG TCC TTG GTA **When BaCV is used as IPC. GTG ATG TT-3’ 5’-6FAM-AGA GAA 2.2.2. Real-time RT-PCR cycling conditions: reverse TGG AGA GAG CGG transcription at 50°C for 10 min; denaturation at 95°C ACG AGG-BHQ’1-3’ ° Forward primer CSP1325 Fw 5’-CAT TTG AAA GTG 100 for 3 min; 40 cycles of denaturation at 95 C for 10 s ° Reverse primer CSP1325 Rv CAT CCG GTT T-3’ and annealing and elongation at 60 C for 60 s. Probe CSP1325 Pr 5’-GTA CCA CGT GTG Note: If other enzymes are used reaction conditions may TTT GCA GAC A-3’ be changed. 5’-VIC-ATG GTC CTC TGC ACC TGC ATC TTG AGA-BHQ’1-3’

(continued)

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needs to be verified in each laboratory when implementing 3. Essential procedural information the test. 3.1. Controls For a reliable test result to be obtained, the following 4. Performance characteristics available (external) controls should be included for each series of nucleic acid extraction and amplification of the target A validation report has been prepared by ISF in March organism and target nucleic acid, respectively. 2020 (unpublished data) and has been provided to EPPO • Negative isolation control (NIC) to monitor contamina- (Ranganathan, pers. comm., 2020). Data is presented tion during nucleic acid extraction: nucleic acid extraction below. and subsequent amplification preferably of a sample of A Euphresco project is in progress regarding seed testing uninfected matrix or if not available clean extraction buf- for ToBRFV when available data will be published in the fer. EPPO database on diagnostic expertise. • Positive isolation control (PIC) to ensure that nucleic acid 4.1. Analytical sensitivity (relative) of sufficient quantity and quality is isolated: nucleic acid Two seed samples from different crop species (Solanum extraction and subsequent amplification of the target lycopersicum and Capsicum annuum) were used to prepare organism or a matrix sample that contains the target healthy seed extracts. The seed extracts were spiked with organism (e.g. naturally infected host tissue). ToBRFV-infected leaf material (5 mg of dried leaves) and • Negative amplification control (NAC) to rule out false used to prepare three individual 10-fold dilution series up positives due to contamination during the preparation of to six dilutions per seed sample. A pre-test was performed the reaction mix: amplification of molecular grade water to determine the starting concentration of the ToBRFV-in- that was used to prepare the reaction mix. fected leaf material used for spiking with the aim to reach • Positive amplification control (PAC) to monitor the effi- the analytical sensitivity (relative) of the ELISA at the sec- ciency of the amplification: amplification of nucleic acid ond or third dilution step (details available in the report). A of the target organism. This can include nucleic acid total of 42 spiked samples (2 seed samples 9 3 dilution extracted from the target organism, total nucleic acid series 9 7 concentrations) were tested in triplicate. extracted from infected host tissue, whole-genome ampli- Samples at a dilution of 108 were consistently detected fied DNA or a synthetic control (e.g. cloned PCR pro- with RNeasy Plant Mini Kit (Qiagen). Samples at a dilution duct). The PAC should preferably be near to the limit of of 107 were consistently detected with Sbeadex Plant detection. Maxi kit (LGC Genomics). As alternative (or in addition) to the external positive 4.2. Analytical specificity controls (PIC and PAC), internal positive controls (IPC) Multiple company data from leaf and/or seed samples can be used to monitor each individual sample separately. from various origins infected with the target virus ToBRFV In ISF-ISHI-Veg (2020), Bacopa chlorosis virus (BaCV) is or nontarget virus/viroid was compiled. used as an IPC. IPC can also include endogenous nucleic • Inclusivity: All 17 available ToBRFV isolates, originating acid of the matrix using conserved primers, preferably from eight different countries (Israel, Saudi Arabia, Mex- amplifying RNA targets such as nad5 (Botermans et al., ico, Germany, Egypt, UK, Jordan), were detected with 2013). However, for seed samples, nad5 might not perform both primer/probe sets. consistently. Alternatively, COX (e.g. Weller et al., 2000 or • Exclusivity: Exclusivity was evaluated on bell pepper Papayiannis et al., 2011) can also be used as IPC. mottle virus (BepMV), columnea latent viroid (CLVd), 3.2. Interpretation of results cucumber green mottle mosaic virus (CGMMV), cucum- Verification of the controls ber mosaic virus (CMV), dahlia latent viroid (DLVd), • NIC and NAC should be negative. kyuri green mottle mosaic virus (KGMMV), paprika mild • The PIC and PAC (as well as IPC, if applicable) should mottle virus (PaMMV, 2 strains), pepino mosaic virus be positive. (PepMV, 4 strains), pepper chat fruit viroid (PCFVd), When these conditions are met pepper mild mottle virus (PMMoV, 4 strains), tobacco • A test will be considered positive if it produces an expo- mild green mosaic virus (TMGMV), tobacco mosaic virus nential amplification curve. (TMV, 3 strains), tomato apical stunt viroid (TASVd), • A test will be considered negative if it does not produce tomato chlorotic dwarf viroid (TCDVd), tomato mosaic an amplification curve or if it produces a curve which is virus (ToMV, 7 strains), tomato mottle mosaic virus not exponential. (ToMMV, 2 strains), tomato planta macho viroid • Tests should be repeated if any contradictory or unclear (TPMVd), tropical soda apple mosaic virus (TSAMV). results are obtained. None of the non-ToBRFV strains reacted with either of It should be noted that a preliminary cut-off value at 32 the two sets. has been indicated in ISF-ISHI-Veg (2020). As a Ct cut-off 4.3. Repeatability value is equipment, material and chemistry dependent it 100%.

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For both S. lycopersicum and C. annuum ToBRFV could Table (continued) be detected in all three replicates up to a dilution of 108 when using the RNeasy Plant Mini Kit (Qiagen). Amplicon 4.4. Reproducibility Primer/probe Sequence size 100% at 107 with Sbeadex Plant Maxi kit (LGC Geno- Forward primer CSP1325 Fw 5’-CAT TTG AAA GTG 100 mics). 8 Reverse primer CSP1325 Rv CAT CCG GTT T-3’ 100% at 10 with RNeasy Plant Mini Kit (Qiagen). Probe CSP1325 Pr 5’-GTA CCA CGT GTG Evaluated in two laboratories. TTT GCA GAC A-3’ 4.5. Other information 5’-VIC-ATG GTC CTC When compared with ELISA the test was 10009 more TGC ACC TGC ATC sensitive using the RNeasy Plant Mini Kit (Qiagen) and TTG AGA-BHQ’1-3’ 1009 more sensitive with Sbeadex Plant Maxi kit (LGC Genomics) 1.4. The test has been validated using a TaqMan RNA-to-CtTM 1-Step Kit (Thermo Fisher Scientific) and an iTaqTM Universal Probes One-Step kit (Bio-Rad) using B. Adapted ISF-ISHI-Veg test for leaf testing (Master Mix CFX96 optical reaction module with C1000 Touch thermal and PCR conditions) cycler (Bio-Rad). The test below is described as it will be carried out to gen- erate the validation data on leaf material in the framework 2. Methods of EU project VALITEST. Other equipment, kits or reagents may be used provided that a verification (see PM 2.1. Nucleic acid extraction and purification 7/98) is carried out. See Appendix 1. Alternative procedures may also be suitable. 2.2. One-step real-time RT-PCR 1. General information 2.2.1. Master Mix 1.1. This one-step duplex real-time RT-PCR protocol based on ISF-ISHI-Veg (2020) was developed for the Working Volume per Final detection and identification of ToBRFV in leaf material. Reagent concentration reaction (µL) concentration Primers and probes concentrations are changed in compar- Molecular-grade NA 5.10 ison to the original protocol. water* 1.2. The test is based on CaTa28 primers and probe devel- TaqMan RNA-to- 29 10.00 19 oped by Enza Zaden BV (NL) in combination with the CtTM 1-Step master CSP1325 primers and probe developed by CSP Labs (US). mix (Thermo Fisher The target sequence of the CaTa28 primers is located Scientific) CaTa28 Fw 10 µM 0.30 0.15 µM within the movement protein gene; using the nucleotide CaTa28 Rv 10 µM 0.30 0.15 µM sequence of GenBank accession no NC_028478.1, the for- CaTa28 Pr 10 µM 0.20 0.10 µM ward and reverse primers start at positions 5163 and 5283, CSP1325 Fw 10 µM 0.30 0.15 µM respectively, and the probe covers positions 5191–5211. CSP1325 Rv 10 µM 0.30 0.15 µM The target sequence of the CSP1325 primers is located at CSP1325 Pr 10 µM 0.20 0.10 µM the end of the coat protein gene; using the nucleotide TaqMan RNA-to- 409 0.50 19 TM sequence of GenBank accession no NC_028478.1, the for- Ct 1-Step RT enzyme mix (Thermo ward and reverse primers start at positions 6144 and 6223, Fisher Scientific) – respectively, and the probe covers positions 6169 6195. Subtotal 18.00 1.3. Oligonucleotides RNA 2.00 Total 20.00 Amplicon Primer/probe Sequence size *Molecular-grade water should be used preferably, or prepared purified (deionized or distilled), sterile (autoclaved or 0.22 µm filtered) and Forward primer CaTa28 Fw 5’-GGT GGT GTC AGT 139 nuclease-free. Reverse primer CaTa28 Rv GTC TGT TT-3’ Probe CaTa28 Pr 5’-GCG TCC TTG GTA 2.2.2. Real-time RT-PCR cycling conditions: reverse GTG ATG TT-3’ transcription at 48°C for 15 min; denaturation at 95°C for 5’-6FAM- AGA GAA ° TGG AGA GAG CGG 10 min; 40 cycles of denaturation at 95 C for 15 s and ACG AGG-BHQ’1-3’ annealing and elongation at 60°C for 60 s. Note: If other enzymes are used reaction conditions may (continued) be changed.

ª 2021 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 51, 178–197 PM 7/146 (1) Tomato brown rugose fruit virus 195

VALITEST test performance study. This value will be eval- 3. Essential procedural information uated in the TPS. As a Ct cut-off value is equipment, mate- 3.1. Controls rial and chemistry dependent it needs to be verified in each For a reliable test result to be obtained, the following laboratory when implementing the test. (external) controls should be included for each series of nucleic acid extraction and amplification of the target organism and target nucleic acid, respectively. 4. Performance characteristics available • Negative isolation control (NIC) to monitor contamina- The test has been validated according to PM 7/98 Specific tion during nucleic acid extraction: nucleic acid extraction requirements for laboratories preparing accreditation for a and subsequent amplification preferably of a sample of plant pest diagnostic activity within EU project VALIT- uninfected matrix or if not available clean extraction buf- EST. As soon as validation data from the project is avail- fer. able, it will be published at http://dc.eppo.int/validationlist. • Positive isolation control (PIC) to ensure that nucleic acid php. 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 Appendix 5 – Real-time RT-PCR test for organism (e.g. naturally infected host tissue). ToBRFV (Menzel & Winter, in press) • Negative amplification control (NAC) to rule out false ‘The test below is described as it will be carried out to gen- positives due to contamination during the preparation of erate the validation data on leaf material in the framework the reaction mix: amplification of molecular grade water of EU project VALITEST. Other equipment, kits or that was used to prepare the reaction mix. • reagents may be used provided that a verification (see PM Positive amplification control (PAC) to monitor the effi- 7/98) is carried out. ciency of the amplification: amplification of nucleic acid of the target organism. This can include nucleic acid extracted from the target organism, total nucleic acid 1. General information extracted from infected host tissue, whole-genome ampli- 1.1. This one-step real-time RT-PCR protocol is suitable fied DNA or a synthetic control (e.g. cloned PCR pro- for the detection and identification of ToBRFV in leaf and duct). The PAC should preferably be near to the limit of seed material of tomato and pepper plants. detection. 1.2. The test is based on primers and probe published As alternative (or in addition) to the external positive by Menzel & Winter (in press). controls (PIC and PAC), internal positive controls (IPC) 1.3. Primers and probe target a fragment from the end can be used to monitor each individual sample separately. of the coat protein gene to the middle of 3-NTR (position IPC can include endogenous nucleic acid of the matrix 6133-6228 for Genbank accession no. NC_028478). using conserved primers preferably amplifying RNA targets 1.4. Oligonucleotides such as nad5 (Botermans et al., 2013). Alternatively, a BaCV spike prepared from a plant infected with bacopa Primer/probe Sequence chlorosis virus (BaCV) may be added during the RNA extraction, see Appendix 1. BaCV-specific primers and Forward primer ToBRFV qs1 5’-CAA TCA GAG CAC probes should be used (Naktuinbouw, the Netherlands; ISF, Reverse primer ToBRFV qas2 ATT TGA AAG TGC Probe ToBRFV p1 A-3’ 2020) see Part A. 5’-CAG ACA CAA TCT 3.2. Interpretation of results GTT ATT TAA GCA Verification of the controls TC-3’ • NIC and NAC should be negative. 5’-6FAM- ACA ATG • The PIC and PAC (as well as IPC, if applicable) should GTC CTC TGC ACC be positive. TG-BHQ1-3’ When these conditions are met • A test will be considered positive if it produces an expo- 1.5. The test has been successfully performed using the nential amplification curve. TaqMan RNA-to-CtTM 1-Step Kit (Thermo Fisher Scien- • A test will be considered negative if it does not produce tific), iTaqTM Universal Probes One-Step Kit (Bio-Rad), an amplification curve or if it produces a curve which is AgPath-ID One-Step RT-qPCR mix (Applied Biosystems) not exponential. or Superscript IV (Invitrogen) in a one-tube assay with Fer- • Tests should be repeated if any contradictory or unclear mentas Maxima Probe qPCR Mix (Thermo Fisher Scientific) results are obtained. and a range of different real-time PCR systems including It should be noted that a preliminary cut-off value at 35 Bio-Rad (CFX96 optical reaction module with C1000 Touch has been indicated in the test description established for the thermal cycler) and Eppendorf realplex 4.

ª 2021 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 51, 178–197 196 Diagnostic

the reaction mix: amplification of molecular grade water 2. Methods that was used to prepare the reaction mix. 2.1. Nucleic acid extraction and purification • Positive amplification control (PAC) to monitor the effi- See Appendix 1. Alternative procedures may also be suit- ciency of the amplification: amplification of nucleic acid able. of the target organism. This can include nucleic acid 2.2. One-step real-time RT-PCR extracted from the target organism, total nucleic acid 2.2.1. Master Mix extracted from infected host tissue, whole-genome ampli- fied DNA or a synthetic control (e.g. cloned PCR pro- Working Volume per Final duct). The PAC should preferably be near to the limit of µ Reagent concentration reaction ( L) concentration detection. Molecular-grade NA 5.8 NA As alternative or in addition to the external positive con- water* trols (PIC and PAC), internal positive controls (IPCs) can TaqMan RNA-to- 29 10.0 19 be used to monitor each individual sample separately. IPC CtTM 1-Step Kit, can include endogenous nucleic acid of the matrix using µ µ Forward Primer 10 M 0.6 0.3 M conserved primers preferably amplifying RNA targets such ToBRFV qs1 as nad5 (Botermans et al., 2013). However, for seed sam- Reverse Primer 10 µM 0.6 0.3 µM ToBRFV qas2 ples, nad5 might not perform consistently. In this case, Probe ToBRFV p1 10 µM 0.5 0.25 µM COX (e.g. Weller et al., 2000 or Papayiannis et al., 2011) TaqMan RT Enzyme 409 0.5 19 can be used as IPC. Alternatively, a BaCV spike prepared Mix (Thermo Fisher from a plant infected with bacopa chlorosis virus (BaCV) Scientific) may be added during the RNA extraction, see Appendix 1. Subtotal 18.0 BaCV-specific primers and probes should be used (Naktuin- RNA 2.0 bouw, the Netherlands; ISF, 2020) see Part Appendix 4 A. Total 20.0 A preliminary evaluation of the use of the BaCV in combi- NA. nation with the Menzel & Winter Primers has been made *Molecular-grade water should be used preferably, or prepared purified by Naktuinbouw (unpub. data, pers. comm., Koenraadt, µ (deionized or distilled), sterile (autoclaved or 0.22 m filtered) and 2020). nuclease-free. 3.2. Interpretation of results Verification of the controls 2.2.2. Real-time RT-PCR cycling conditions: reverse • NIC and NAC should be negative. transcription at 48°C for 15 min, denaturation at 95°C for • The PIC and PAC (as well as IPC, if applicable) should 10 min; 40 cycles of denaturation at 95°C for 15 s and be positive. annealing and elongation at 60°C for 1 min. When these conditions are met Note: If other enzymes are used reaction conditions may • A test will be considered positive if it produces an expo- be changed. nential amplification curve. • A test will be considered negative if it does not produce an amplification curve or if it produces a curve which is 3. Essential procedural information not exponential. 3.1. Controls • Tests should be repeated if any contradictory or unclear For a reliable test result to be obtained, the following results are obtained. (external) controls should be included for each series of It should be noted that a preliminary cut-off value at 35 nucleic acid extraction and amplification of the target has been indicated in the test description established for the organism and target nucleic acid, respectively. VALITEST test performance study. This value will be eval- • Negative isolation control (NIC) to monitor contamina- uated. As a Ct cut-off value is equipment, material and tion during nucleic acid extraction: nucleic acid extraction chemistry dependent it needs to be verified in each labora- and subsequent amplification preferably of a sample of tory when implementing the test. uninfected matrix or if not available clean extraction buffer. 4. Performance characteristics available • Positive isolation control (PIC) to ensure that nucleic acid of sufficient quantity and quality is isolated: nucleic acid Limited data on the analytical sensitivity and data on the extraction and subsequent amplification of the target analytical specificity were obtained from Menzel & Winter organism or a matrix sample that contains the target (in press), where the test has been carried out by using organism (e.g. naturally infected host tissue). Superscript IV (Invitrogen) in a one-tube assay with Fer- • Negative amplification control (NAC) to rule out false mentas Maxima Probe qPCR Mix (ThermoFisher Scien- positives due to contamination during the preparation of tific).

ª 2021 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 51, 178–197 PM 7/146 (1) Tomato brown rugose fruit virus 197

The test will be validated according to PM 7/98 Specific virus (PV-1176), odontoglossum ringspot virus (PV-1048, requirements for laboratories preparing accreditation for a PV-0625), paprika mild mottle virus (PV-0606), pea early plant pest diagnostic activity within EU project VALITEST browning virus (PV-0298), peanut clump virus (PV-0291), (on leaves) and within a Euphresco project (on seeds). As pepino mosaic virus (PV-1022), pepper mild mottle virus soon as validation data from those two projects is available, (PV-0166), piper chlorosis virus (PV-1126), ribgrass mosaic it will be published at http://dc.eppo.int/validationlist.php. virus (PV-0436), soil-borne cereal mosaic virus (PV-0552), 4.1. Analytical sensitivity data soil-borne wheat mosaic virus (PV-0748), streptocarpus ToBRFV was detected up to a 10-6 dilution in 10-fold flower break virus (PV-1058), sunn-hemp mosaic virus serial dilutions of extracted RNA from ToBRFV-infected (PV-0156), tobacco mild green mosaic virus (PV-0887), tomato leaf homogenates in healthy tomato leaf RNA tobacco mosaic virus (PV-0107), tobacco rattle virus (PV- extract. 0354), tomato aspermy virus (PV-0220), tomato black ring 4.2. Analytical specificity data virus (PV-0191), tomato bushy stunt virus (PV-0269), 4.3. Inclusivity tomato chlorosis virus (PV-1023), tomato leaf curl New Three out of three ToBRFV isolates gave positive results, Delhi virus (PV-1109), tomato mosaic virus (PV-0135), i.e. the following isolates from DSMZ collection: PV-1236, tomato ringspot virus (PV-0380), tomato spotted wilt virus PV-1241, PV-1244. (PV-0182), tomato yellow leaf curl virus (PV-0588), tomato 4.4. Exclusivity yellow ring virus (PV-0526), tropical soda apple mosaic No cross-reactions occurred with the following nontarget virus (PV-1223), turnip vein clearing virus (PV-0148), you- viruses (accession number of tested isolates from DSMZ cai mosaic virus (PV-0527). collection is given in parentheses): barley stripe mosaic 4.5. Selectivity virus (PV-0330), beet soil-borne virus (PV-0576), beet virus Not determined. Q (PV-0961), bell pepper mottle virus (PV-0170), cucum- 4.6. Repeatability and reproducibility ber green mottle mosaic virus (PV-0375), obuda pepper Not determined.

ª 2021 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 51, 178–197 DOI: 10.1111/epp.12761

ADDENDUM

PM 7/146 (1) Tomato brown rugose fruit virus

In the EPPO Diagnostic protocol PM 7/146 (1) Tomato brown rugose fruit virus, approved in October 2020 and published in the April 2021 issue of the Bulletin, a reference is made to the March 2020 version of the International Seed Federation (ISF) protocol. The ISF has informed the EPPO Secretariat that this ISF protocol was updated in November 2020 to clarify the seed sampling and subsample size. They note that this new version is available at https://www.worldseed.org/wp-content/ uploads/2020/11/Tomat o- ToBRFV_2020v1.5.pdf In addition, in Appendix 4, for the ‘Test as in ISF-ISHI-Veg (2020)’ in the section 4 ‘Performance Characteristics available’ it states that

A validation report has been prepared by ISF in March 2020 (unpublished data) and has been provided to EPPO. (Ranganathan, personal communication, 2020)

It is noted that this information is publicly available on the ISF website at the following link: https://www.worldseed. org/wp-content/uploads/2020/09/2020_Validation-Report-_ToBRFV_Tomato-Pepper_W.pdf The EPPO Secretariat thanks the ISF for this update and clarification.

REFERENCE EPPO. (2021). PM 7/146 (1) Tomato brown rugose fruit virus. EPPO Bulletin, 51, 178–197.