Longidorus attenuates and L. elongates are the vectors of TBRV (Harrison et al, 1961).

Method	Instructions	NAPIS Survey Method
Tissue Sample	Take a random sample of host material.	3011 - General Tissue Sample

Many of the hosts of TBRV are asymptomatic (not showing symptoms) but still contain the virus. Green leaves of mature plants should be sampled randomly irrespective of whether they are showing symptoms.

No specific signs are present.

In strawberry: Symptoms in Fragaria vesca (strawberry) indicator plants include leaf blotching and necrotic ring spots. However, the symptoms often disappear after the first season, and some hosts were completely symptomless (Martin and Tzanetakis, 2006).

In raspberry: Susceptible cultivars react to infections with chlorotic mottling, vein yellowing, yellow speckling, yellow or chlorotic ringspotting, leaf curling, reduced vigor, stunting, and deformation of fruit (Martelli and Taylor, 1990).

In other hosts: Symptoms of numerous other infected hosts, including tomato, celery, artichoke, potato, cucumber, bean, pea, tobacco, and spinach, have been studied (Pospieszny et al. 2004; Šneideris et al., 2012; Šneideris and Staniulis 2014). Common symptoms include: chlorotic spotting, mosaic symptoms, necrotic lesions, ring spots, or pattern lines, and deformed fruit. A patchy distribution of diseased plants is characteristic of outbreaks where infection has been contracted from the soil (Harrison et al., 1961).

ID/Diagnostic: Multiple

Serological for screening and Molecular for confirmation.

Screening: A TBRV-specific ELISA kit is commercially available (Neogen Europe Ltd ). However, this ELISA was found to cross react with other nepoviruses and failed to identify one of six tested strains of TBRV (Harper et al., 2011).

Agdia offers testing services for TBRV in the United States via (DAS ELISA), but reagents do not appear to be available from Agdia. The ELISA reagents, however, are available from other companies, including Bioreba, which is represented by Eurofin Labs in the United States. AC Diagnostics, based out of Fayettville, AR, recently began to offer a DAS ELISA testing kit and reagents for TBRV.

Bioreba also offers a DAS ELISA testing kit for TBRV The product description mentions that "the virus shows quite some antigenic variation between strains", suggesting that the kit might not detect all isolates of TBRV. Bioreba"s kit uses a mix of antibodies to grapevine and potato virus isolates, and different extraction buffers are recommended depending on the host material.

Confirmation: Work instructions (WI-B-T-G-23 and WI-B-T-G-24) were developed by the CPHST Beltsville Lab. These work instructions were developed using a synthetic target (a mini gene) spiked into healthy plant RNA.

Note: These work instructions require additional validation by CPHST Beltsville using field collected plant samples before work instructions will be made available to the CAPS community.

Detection of Tomato black ring virus (TBRV) using a Multiplex Reverse-Transcription (RT) Conventional PCR (Work Instruction: WI-B-T-G-23)
TBRV RNA2 sequences available in National Center for Biotechnology Information (NCBI) nucleotide databases (NC-004440, AY157944 and X80831) were used to design the virus-specific forward primer PTBRV2f and reverse primer PTBRV486mr, a modified version of the published reverse primer TBRV-sp-a (Digiaro et al. 2007). The TBRV assay targets a segment of the capsid protein gene on RNA2 using TBRV-specific primers (PTBRV2f and PTBRV486mr) multiplexed with Nad5 plant gene-specific primers (Menzel et al., 2002) as an internal control in a two-step RT-PCR using a commercial kit.

Detection of Tomato black ring virus (TBRV) using a Multiplex RT quantitative PCR (qPCR) in a Cepheid SmartCycler (Work Instruction: WI-B-T-G-24)
The TBRV assay targets a segment of the capsid protein gene on RNA2 using TBRV-specific primers (TBRV2f and TBRV4r) and TaqMan FAM probe (TBRV125p), developed by the Beltsville Lab, multiplexed with plant gene (Nad5) specific primers and TaqMan TET probe (Nad5p1) as an internal control in a two-step RT qPCR using a commercial kit.

The number of viruses known to infect strawberry has grown significantly in the last decade (Tzanetakis and Martin, 2013). Strawberry necrotic shock virus is the predominant in the United States (Tzanetakis and Martin, 2013). Other nematode-transmitted viruses in strawberry include: Strawberry latent ringspot virus (SLRSV), Arabis mosaic virus (ArMV), Raspberry ringspot virus (RpRSV), Tomato black ring virus (TBRV), and Tomato ringspot virus (ToRSV) (Tzanetakis and Martin, 2013). In Europe, TBRV is often found in mixed infections with Raspberry ringspot virus (RpRSV). Both of these viruses are vectored by L. elongatus, and they exhibit similar symptoms in strawberry (Martin and Tzanetakis, 2006).

Digiaro et al., (2007) developed a one-step RT-PCR with primers specific to TBRV. This assay was tested by Harper et al. (2011) and found to be effective. Wei and Clover (2008) developed a nepovirus group-B specific one-step RT-PCR, which was successfully used by Šneideris et al., (2012) to identify TBRV in Lithuania. However, this protocol failed to detect one of six tested strains of TBRV when tested by Harper et al. (2011). In addition, degenerate primers RT-PCR developed by Wei and Clover (2008) could be used for identification of the suspect samples via RT-PCR and sequencing. Harper et al. (2011) developed a one-step real-time RT-PCR which can detect TBRV with high sensitivity and does not cross-react with other common nepoviruses.

Šneideris and Staniulis (2014) developed primers which amplify the full copy of the TBRV coat protein gene sequence in a two-step RT-PCR. These primers successfully identified twelve different strains of TBRV.

A TBRV specific reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) assay was also recently described by Hasiόw-Jaroszewska et al. (2015). This protocol was developed using TBRV isolates from 16 different hosts, most which came from Poland. The authors claim the RT-LAMP assay is 100 fold more sensitive than RT-PCR protocols in detecting TBRV.