Tomato brown rugose fruit virus

Current scientific name: Tomato brown rugose fruit virus Class: Alsuviricetes Order: Martellivirales Family: Virgaviridae Genus: Tobamovirus Species: Tomato brown rugose fruit virus Synonyms: none EPPO Code:  TOBRFV  Common name: virus du fruit rugueux brun de la tomate Taxonomic rank: species

Tomato brown rugose fruit virus (ToBRFV) is a monopartite, linear, positive-sense single-stranded RNA (+ssRNA) non-enveloped virus. The genomic RNA has been sequenced for several ToBRFV isolates and sequences are publicly available in the  GenBank  database. The currently accepted reference sequence has the NC_028478.1 accession number (Salem et al., 2016).

Tomato brown rugose fruit virus is not currently listed as Union quarantine pest under  Regulation (EU) 2019/2072 , however, the pest is regulated by emergency measures in  Commission Implementing Regulation (EU) 2020/1191 . These emergency measures state that ToBRFV should not be introduced into or moved within the EU territory (Article 2). Capsicum spp. and S. lycopersicum and its hybrids, including their seeds and fruits, are listed as the major host plants of ToBRFV (Article 1). Member States have to conduct annual surveys on the specified plants for planting, seeds and fruits of the previously mentioned hosts. These surveys should include sampling and testing in accordance to the Annex and should be based on the assessed risk of introduction and spread of ToBRFV in the MS and on sound scientific and technical principles (Article 5). Eradication measures have to be implemented if the presence of ToBRFV is confirmed.

Plants for planting and seeds of Capsicum spp. and S. lycopersicum within the EU must be accompanied by a plant passport when moved within the EU and fulfil the conditions in Articles 6 and 7 of Commission Implementing Regulation (EU) 2020/1191, respectively.

Plants for planting and seeds of Capsicum spp. and S. lycopersicum must be accompanied by a phytosanitary certificate, when introduced into the EU, in line with the requirements listed in Articles 8 and 9 of Commission Implementing Regulation (EU) 2020/1191, respectively. Border control inspections shall be carried out o consignments of seeds and plants for planting, in accordance to Article 10 of Commission Implementing Regulation (EU) 2020/1191.

Resistant Capsicum spp. varieties are exempted from the requirements for testing.

Prohibitions and requirements for the movement of the major host commodities for ToBRFV are laid in Commission Implementing Regulation (EU) 2019/2072, however these are not specific for ToBRFV. Commission Implementing Regulation (EU) 2019/2072 sets import prohibitions and their related conditions for:

• Plants for planting of Solanum or their hybrids (Annex VI, 16).
• Plants for planting of Solanaceae (Annex VI, Point 18).

Commission Implementing Regulation (EU) 2019/2072 sets special import requirements for:

• Imports of seeds of Capsicum spp. and S. lycopersicum (Annex XI Part A Point 8).
• Imports of plants for planting of C. annuum, (Annex VII, Point 22) and S. lycopersicum (Annex VII, Point 22 and 26).
• Imports of parts of plants, other than fruits and seeds, of S. lycopersicum and S. melongena (Annex XI, Point 3) and for S. lycopersicum (Annex VII Point 23).
• Imports of fruits as per Annex VII; S. lycopersicum points 68, 69, Capsicum points 62, 72 and C. annuum point 68.

Special requirements for the movement of host plants commodities within the European Union territory are also listed in Commission Implementing Regulation (EU) 2019/2072:

• Seeds of S. lycopersicum and C. annuum (Annex XIII, Point 6).
• Plants for planting of stolon, or tuber-forming species of Solanum, or their hybrids (Annex VIII, Point 3).
• Plants for planting with roots, of Capsicum spp. and S. lycopersicum and plants for planting of C. annuum, S. lycopersicum (Annex VIII, points 12 and 13).

Movement of fruits of the main hosts within the EU are not subjected to any measures.

The general requirements for survey of quarantine pests in the EU territory are laid down in  Regulation (EU) 2016/2031  and  Commission Implementing Regulation (EU) 2020/1231 .

ToBRFV was first observed in 2014 and 2015 on tomato plants in Israel and Jordan respectively. Since these initial findings, the virus has been reported in America (Mexico, USA and Canada), Europe and Asia and has been found infecting both tomato and pepper crops (van de Vossenberg et al., 2020). ToBRFV has been reported in Asia (China, Iran, Israel, Jordan, Lebanon, Saudi Arabia and Uzbekistan) and is considered widespread in Syria. ToBRFV has been reported in various European locations (Albania, Norway, Switzerland and Turkey) and in 17 EU MS (table below). It was declared as eradicated in the UK ( EPPO, 2022a ), but recently found again ( EPPO, 2022b ).

Note: the information included in this section is aligned with the EPPO map updated on 07-06-2022

ToBRFV is a tobamovirus that systemically infects its host plants. Cell-to-cell movement occurs via plasmodesmata and is supported by the viral movement protein, while long distance movement within the plant occurs via the phloem (Dombrovsky and Smith, 2017). The ToBRFV is a recently discovered virus and, although aspects of its biology remain uncertain, these can be inferred through similarity to other tobamoviruses that have been extensively studied.

Tobamoviruses can survive outside the host plants either on inert or biological surfaces, while maintaining their infectivity (Li et al., 2016; Smith and and Dombrovsky, 2019). Tobamovirus particles were also found in soil (Lovelok et al., 2022) and run-off water (Bačnik et al., 2020). Tobamovirus particles are very stable and can persist on seeds for several years (Dombrovsky and Smith, 2017). Tobamoviruses can be found externally on the seed and also in the peripheral tissues of the seed (testa) and are generally transmitted at a low rate to seedlings during germination (Mink, 1993). Such viral particles may infect the embryo through small wounds happening during gemination (Dombrovsky and Smith, 2017).

ToBRFV mainly spreads mechanically. The persistence of tobamovirus particles outside its hosts allows for efficient mechanical transmission when a healthy host plant comes into contact with an infected: plant, soil, nutrient solutions (including circulating water, i.e. hydroponic culture or aerial and drip-irrigation), and surfaces, such as tools and materials used during agricultural practices (cutting tools, shovels, trays, pots); which cause wounds and microlesions to the plant tissue (Broadbent, 1976; Reingold et al., 2016; Dombrovsky and Smith, 2017). In tomato and peper plants, mechanical transmission of ToBRFV has been demonstrated experimentally (Panno et al., 2019b).

ToBRFV has been found on the seed coat and sometimes in the endosperm, but never in the embryo (Davino et al., 2020; Salem et al., 2022). Contaminated seeds may therefore lead to infected plantlets through mechanical transmission during germination (Salem et al., 2022). The transmission rate has been estimated at 1.8% of seedlings when testing the third true leaf (Davino et al., 2020), while a lower transmission rate of 0.08% has also been reported (Salem et al., 2022). The transmission rate is likely to vary depending on the viral load of the infected seeds.

To date, no natural vector has been identified for ToBRFV, however, transmission assisted by bumblebees Bombus terrestris (Hymenoptera: Apidae) has been demonstrated for this virus (Levitzky et al., 2019). It has been hypothesised that ToBRFV particles, adhering to pollen grains or present in sap on insect bodies and mandibles, can be transmitted through the wounds and lesions caused by bumblebees during pollination (Levitzky et al., 2019; Velthuis and van Doorn, 2006). Pollen transmission in the absence of insects is possible in theory, but considered unlikely ( EPPO, 2020 ).

This section provides the information needed to characterise the population of host plants to target in a survey, as described in the  ‘General guidelines for statistically sound and risk-based surveys of plant pests’  (EFSA et al., 2020). This includes the pest’s host range and main hosts in the EU ( Section 2.1 ), the suitability of EU environments to the pest’s establishment ( Section 2.2 ), the ability of the pest to spread ( Section 2.3 ), and the identification of risk factors associated with an increased probability of presence ( Section 2.4 ).

Once the above parameters have been defined, the target population can be structured in multiple levels. At level 1 is the survey area, which corresponds to the entirety or part of the Member State. At levels 2 and 3 are the epidemiological units that can be distinguished within the survey area. Epidemiological units can be chosen as administrative regions (e.g. EU NUTS areas or Member State-level regions) if they are homogeneous, or further subdivided into the environments where host plants are present using a land-use categorisation (e.g. urban, agricultural and natural areas, nurseries). At level 4, if risk factors are identified, the risk areas are defined around the risk locations. At level 5 are the inspection units, the elementary subdivisions of the target population that are inspected for the detection of the pest (e.g. host plants), depending on the pest detection method ( Section 3 ). For the definitions of the target population, epidemiological units and inspection units, see also the  glossary  of terms available at the end of this document.

The hierarchical structure of the target population should be tailored to the situation in each Member State. A possible structure of the target population for surveys of Tomato brown rugose fruit virus within the EU is proposed in  Section 2.5 .

To date, natural infections of ToBRFV have been reported only in tomato (S. lycopersicum), bell pepper (C. annuum) and habanero chilli pepper (C. chinense; Magaña-Álvarez et al., 2021). As some of the reports only mention chilli peppers as the host (Cambrón-Crisantos et al., 2019), without a clear indication of the species, there is some uncertainty on whether other Capsicum species might be natural hosts. In addition, uncertainty on the natural host status for S. melongena remains, due to diverging reports, as summarised in the table (below) on the experimental host plant species.

Resistance genes to tobamoviruses have been identified and described either in tomato or in pepper (De Ronde et al., 2014). However, in tomato, ToBRFV has been demonstrated to be able to overcome all three tobamovirus resistance genes (Tm1, Tm2 and Tm2 ² ) (Salem et al., 2016; Luria et al., 2017). Genetically modified ToBRFV-resistant tomato varieties were recently developed using the CRISPR/Cas9 technique (Ishikawa et al., 2022).

In pepper, ToBRFV has been reported to overcome L1 and L2, but not L3 and L4 resistance pepper genes. However, the resistance conferred by these genes seem to be thermosensitive and breaks when temperatures rise above 32°C (Fidan et al., 2021) or 30°C (Luria et al., 2017). Therefore, uncertainties remain on the resistance of Capsicum varieties depending on resistance gene(s) present and on the environmental conditions.

Large numbers of experimental hosts have been identified in a wide range of species belonging to Solanaceae and a few species belonging to the Amaranthaceae, Apocynaceae and Asteraceae families. Uncertainties remain on the natural host status of the experimental hosts (ANSES, 2020). A complete list of natural and experimental host plant species along with their associated references is reported in the tables below (last updated on 11 March 2022) ( EPPO, 2022b ). The species that are present in the EU and that could therefore be included for surveillance, are also indicated. Detection surveys should focus on the natural hosts present in the EU; S. lycopersicum and non-resistant C. annuum (unless under a high-temperature regime, in which resistance breaks down and therefore could also be included in detection surveys). Experimental hosts could be included for delimiting surveys if present in the survey area.

The main susceptible host crops of ToBRFV are either cultivated in open fields (in particular in southern EU countries) or under protected cultivation such as greenhouses throughout the EU countries. Consequently, wherever host plants are present, ToBRFV would be able to become established. Only the natural hosts, S. lycopersicum and susceptible C. annuum should be included in the host plant population targeted by EU detection surveys ( Section 2.1 ). For delimiting surveys some of the identified experimental hosts that are widely distributed in the EU as crops (such as potato, tobacco, eggplant), ornamentals (such as Petunia) or weeds (European black nightshade) could be included, if present around the infected area.

Natural spread

The main mechanism for ToBRFV transmission is by mechanical transmission. Natural dispersion of the virus may be mediated by contaminated seeds and pollinators e.g. bumblebees (Levitzky et al., 2019; Davino et al., 2020; Salem et al., 2022). Under experimental conditions, it has been demonstrated that the presence of a few infected plants in a greenhouse is sufficient to initiate an epidemy leading in a short time to spread to the entire crop, reaching almost 100% infection. In the experiment, transmission occurred by direct contact between contaminated and adjacent healthy tomato plants and probably by the action of bumblebees (Panno et al., 2020a). Pollinator movement between greenhouses and the outside commonly occurs, and susceptible species both in and around greenhouses could potentially become reservoirs of the virus inoculum (Oladokun et al., 2019). Another consideration and a possible driver of infection is the persistence of the virus from one cropping cycle to the next (via plant debris, soil).

Human-assisted spread

ToBRFV may spread to new areas through the trade and imports of infected seeds, plants for planting, plant tissue for vegetative propagation and fruits (Klap et al., 2020). Given the stability of the virus outside its hosts, ToBRFV can be mechanically transmitted through infected sap via agricultural practices, workers and the movement of contaminated tools and surfaces, including soil (ANSES, 2020) and water (Dombrovsky and Smith, 2017). However, the survival time of the virus outside its host remains uncertain.

Identification of risk factors and their relative risk estimation are essential for performing risk-based surveys. A risk factor is a biotic or abiotic factor that increases the probability of infestation by the pest in the area of interest. The risk factors that are relevant for surveillance need to be characterised by their relative risk (should have more than one level of risk for the target population) and the proportion of the overall target population on which they apply. The identification of risk factors needs to be tailored to the situation of each Member State. This section presents examples of risk factors for Tomato brown rugose fruit virus and is not necessarily exhaustive. 

For the identification of risk areas, it is first necessary to identify the activities that could contribute to introduction or spread of Tomato brown rugose fruit virus. These activities should then be connected to specific locations. Around these locations, risk areas can be defined, knowing that their size depends on the spread capacity of the target pest and the availability of host plants around these locations.  

The Member States can opt to utilise the information available on the EU Platforms of TRACES NT, EUROPHYT Interceptions and EUROPHYT Outbreaks. The information available, in particular, relating to the country of origin, type of commodity and hosts of intercepted or outbreak reports can be extracted from such platforms for specific harmful organisms. This information can allow Member States to identify potential pathways of introduction from previous historical findings. Thus, Member States might consider focusing their surveillance efforts around activities and locations related to previous interceptions and outbreaks.  

Such information should only be considered as indicative and given the possible dynamic changes, it should be reviewed and analysed periodically.

Example 1: importation and trade of seeds and plant for planting of host crops from infected areas

The main pathway for the entry of ToBRFV in a territory is via the introduction of infected host plant commodities such as seeds and plants for planting. Given that ToBRFV is already present in the EU, both the import and movement of seeds and plants for planting from third countries or areas within the EU where the virus is known to occur, are a potential pathway. Therefore, the imports into the EU and the movement and production within MS of these host plant commodities are considered the main activities associated with an increased risk of entry, establishment and spread of ToBRFV. Warehouses, nurseries and garden centres where these commodities are stored and distributed can be identified as risk locations, and the production sites that cultivate the imported and traded seeds and plants for planting, can be identified as risk areas. The pathway of entry of ToBRFV from seeds and plants for planting of the hosts is subject to special requirements as indicated in Section 1.2, to mitigate the risk of entry.

Example 2: re-use or sharing of equipment and packaging material related to host fruit production

Due to the persistence of the virus outside its hosts, the re-use and sharing of equipment related to the host fruit production, can be considered as a risk activity. An example for such a risk activity is a packing station handling fruit batches from diverse host production sites and, in particular, from outbreak sites, or host production sites that also pack fruits from other sites. In such activities, the movement and sharing of crates and equipment or even staff, could potentially create a pathway for spread of the virus (van der Gaag et al., 2021). Therefore, MS should evaluate the activities taking place in their territory and identify the potential risks involved. The sites in which the corresponding activities are carried out could be identified as risk locations. The fields and greenhouses surrounding such risk locations, and which (re)use equipment from such facilities, where ToBRFV host crops are grown, could be identified as the risk areas. The pathway of import of host fruits from third countries is regulated as indicated in  Section 1.2 , however the movement of host fruits within the EU is an open pathway. Imported and traded fruits can also be considered a pathway for the entry of ToBRFV. However, the transfer of the virus from the fruit intended for consumption to host plants in production, is considered unlikely and the risk is likely to be more associated with the sharing and re-use of equipment and transportation of the host fruit commodities.

Example 3: disposal of host waste

Composting or burial of contaminated plant debris in the soil at the end of the growing period, allows further potential spread of tobamoviruses via contaminated soil, as the virus can maintain its viability outside its hosts plants (Smith and Dombrovsky, 2019). This disposal of host debris following the harvesting season can therefore be considered as another risk activity and the areas where this occurs could be identified as risk locations (ANSES, 2020). Waste fruit disposal locations such as from tomato and pepper processing facilities could also be identified as risk locations. The fields and greenhouses surrounding the above-mentioned disposal areas and in which host plants are cultivated could therefore be considered as risk areas.

Example 4: history of previous outbreaks of ToBRFV on a production site

As the virus can potentially persist from one cropping season to the following, areas where previous outbreaks of ToBRFV have been confirmed could be identified as risk factors. The risk location is the production site with a history of previous outbreaks of ToBRFV, whereas the risk areas would include the production site itself as well as production sites in the vicinity, due to the potential movement of staff and the sharing of equipment between sites or companies.

The figure on the right panel gives examples of the components of a target population for Tomato brown rugose fruit virus and is not necessarily exhaustive.

ToBRFV induces a wide range of symptoms on S. lycopersicum and C. annuum. Some leaf and fruit symptoms elicited on tomato are however not specific and can be confused with those induced by other viruses (Alkowni et al., 2019). Sometimes infections may also be asymptomatic (Panno et al., 2020b;  EPPO, 2021 ; Abou Kubaa et al., 2022) and therefore the absence of symptoms does not necessarily indicate that ToBRFV is absent. Therefore, visual examination for the presence of symptoms is not sufficient for the detection and diagnosis of ToBRFV and its presence must be confirmed by sampling and molecular identification of the virus in the laboratory.

3.1.1. Visual examination

Symptoms

ToBRFV symptom expression on tomato may vary depending on the time of infection, the cultivar, the developmental stage of the plant and environmental conditions (CABI, 2022). Symptoms may be observed on leaves and on fruits. Leaf symptoms tend to appear first on young shoots at the top of the plant ( EPPO, 2021 ). Plants may show chlorosis, leaves with mosaic patterns and mottling, young leaves may be deformed and occasionally narrowed, and leaf surface can be blistered. Leaves may also wilt and become yellow, leading to plant death. Pedicels, petioles and sepals may develop brown necrotic lesions. ToBRFV causes fruit chlorotic or necrotic spots and marbling, deformations and uneven ripening of young fruits, brown rugose patterns ( EPPO, 2021 ). Viral infections may also reduce the number of fruits per branch (Salem et al., 2016; Dombrovsky and Smith, 2017; Cambrón Crisantos et al., 2019). Furthermore, mixed infections with other viruses may cause different symptoms, which can be worsened by physiological stresses.

Pepper plants carrying the L3 and L4 tobamovirus resistance genes are resistant to ToBRFV infection through a hypersensitive response. However, this resistance is thermosensitive such that at a 30–32°C hypersensitive-like necrosis develops, leading to systemic plant infection and development of symptoms (Luria et al., 2017; Fidan et al., 2021). Pepper plants infected with ToBRFV show blistering and corrugated fruit surfaces (Cambrón-Crisantos et al., 2019; CABI, 2022). Asymptomatic infection of ToBRFV has also been reported in C. annuum (Panno et al., 2020b). Other viruses may cause similar leaf and fruit symptoms in pepper plants and may consequently be confused with ToBRFV symptoms.

3.1.2. Sample collection

Viral concentration varies in plant tissues with an observed higher concentration in young leaves or sepals and a lower concentration in old leaves. Therefore, sampling of young leaves or sepals is recommended. Sampling of crops prior the development of fruits trusses should be conducted on young leaves from the top of the plant. Due to the erratic movement of the virus within mature plants following fruit setting, apart from sampling leaves from the top of the plant, additional sampling of sepals and/or fruits could also be considered (Skelton et al., 2021), however sampling of young leaves and sepals are preferred.

For symptomatic plants, sampling for laboratory testing is based on at least three symptomatic young leaflets collected from the top of the plants or shoots ( EPPO, 2021 ). Further information on the test sample requirements is available in the diagnostic protocol PM 7/146 (1) ( EPPO, 2021 ). Seeds and fruits may also be tested for the presence of ToBRFV. Symptomatic and asymptomatic samples should be collected, stored at cool temperature, and brought to the laboratory for testing.

Care should be taken when handling samples of tomato and pepper plants, since ToBRFV is a highly persistent virus and a high virus concentration may be present in infected plants, potentially leading to cross-contamination.

3.1.3. Timing of detection and identification

Tomato and pepper crops can be cultivated either in open field or in greenhouses, therefore availability of plant material to be collected for sampling will vary accordingly. To ensure early detection, sampling of young leaves from the top of the plant and sepals is recommended.

3.2.1. Laboratory testing

Various techniques are available for the detection of ToBRFV. If used, bioassays or serological assays need to be combined with molecular tests to unambiguously identify this virus. More information is available in the EPPO Diagnostic Protocol PM 7/14 (1) ( EPPO, 2021 ) and also in the soon available revised version of this protocol.

Biological assays

Assays rely on the inoculation of indicator seedlings and observation of symptom development. This can be obtained through mechanical inoculation of homogenates on the leaves of herbaceous indicator seedlings. In the literature, various herbaceous hosts have been used for ToBRFV (refer to  Section 2.1  and their references). Symptoms obtained on biological indicators are however not specific and are therefore not recommended. In addition, these tests should be complemented by laboratory assays for confirmation.

Serological assays

The enzyme-linked immunosorbent assay (ELISA) has been successfully adopted for the detection of tobamoviruses. Commercial kits targeting specifically the capsid protein are available, but these are generally not species specific (Dombrovsky and Smith, 2017). ToBRFV antisera were found to cross-react with other tobamoviruses and have a low diagnostic sensitivity for the detection of ToBRFV in seeds, when compared with real-time RT-PCR tests (Giesbers et al., 2021), therefore ELISA is not suitable for the detection of ToBRFV in seeds. ELISA may be used as a screening test on symptomatic plant material, however ELISA tests should be accompanied by a molecular test for confirmation of ToBRFV.

Molecular test

Several conventional and real-time reverse-transcriptase polymerase chain reaction (RT-PCR) protocols are available for ToBRFV detection (Alkowni et al., 2019; Panno et al., 2019b; Rodríguez-Mendoza et al., 2019; Bernabé-Orts et al., 2021; Menzel and Winter, 2021). Real-time RT-PCR protocols developed by ISF (2020), Menzel and Winter (2021) and Bernabé-Orts et al. (2021) were successfully applied for ToBRFV detection on seeds. These protocols, together with a commercial kit have been evaluated in a test performance study (TPS) under the European project VALITEST (Anthoine et al., 2020; Giesbers et al., 2021; Luigi et al., 2022). It was demonstrated that the conventional RT-PCR protocols described by Ling et al. (2019) and Panno et al. (2019a) cross-reacted with other tobamoviruses, but sequencing of the amplicon could allow unambiguous identification of the virus.

A sensitive loop-mediated isothermal amplification (LAMP) protocol for ToBRFV detection has been reported (Sarkes et al., 2020). However, when this protocol was applied for seeds by Giesbers et al. (2021), a lower diagnostic sensitivity (tomato: 79% and pepper: 7%) was achieved, in comparison with real-time PCR tests and therefore not recommended.

The diagnostic sensitivity of the applied method for identification of ToBRFV is one of the key survey parameters to be considered, as it directly impacts the sampling efforts required. The diagnostic sensitivities for the recommended protocols evaluated in the TPS in the frame of the European project VALITEST are shown in the table below (Luigi et al., 2022).

High-throughput sequencing

High-throughput sequencing techniques allow the detection of all viral species/strains present in a sample without a need for any preliminary knowledge (Olmos et al., 2018). While these techniques are not currently used as a routine detection method, such approaches have already been successfully applied for the detection and identification of ToBRFV (Luria et al., 2017; Rivarez et al., 2021).

Information on what, where, when and how to conduct survey activities for ToBRFV is summarised in the Table on the right panel. The identification of the target population needs to be tailored to the situation in each Member State (example shown below).

The figure below shows the next steps after the survey preparation for designing statistically sound and risk-based detection and delimiting surveys of ToBRFV. Guidance on selecting the type of survey, related survey preparation and design, is provided in the the  EFSA general guidelines for pest surveys  on the right panel (EFSA et al., 2020).

Scroll down the right panel to access the definitions included in the glossary.

 General guidelines for statistically sound and risk-based surveys of plant pests 

 Index of EFSA Plant Pest Survey Toolkit 

 Plant Pest Survey Cards Gallery 

 The statistical tool: RiBESS+ 

 The RiBESS+ manual 

 The RiBESS+ video tutorial