First report of Tomato black ring virus infecting raspberry and blackberry in Poland

Raspberry (Rubus idaeus L.) and blackberry (Rubus fruticosus L.) are infected by at least 29 viruses, including the Tomato black ring virus (TBRV) (Martin et al. 2013). TBRV belongs to the genus Nepovirus (subgroup B) of the family Secoviridae and is listed as a plant pathogen in over 40 countries. TBRV infects a wide range of herbaceous and woody plants. In Poland, TBRV has been described on the plants of the following species: Tagetes patula, T. erecta, Cucumis sativus, Cucurbita pepo, Lactuca sativa, Solanum tuberosum, S. lycopersicum, Sambucus nigra, and Robinia pseudoacacia (Jończyk et al. 2004, Hasiów-Jaroszewska et al. 2015). To this date, there is no information on the incidence of TBRV in raspberry and blackberry in Poland. In the spring of 2019, 52 blackberry leaf samples and 408 raspberry leaf samples were collected from 4 plantations located in central Poland. None of the raspberry plants (cvs. Glen Ample, Polka, Sokolica), nor the blackberry plants (cvs. Thornfree, Polar, Gaj, Kotata) exhibited viral symptoms. Enzyme-linked immunosorbent assay (ELISA) was carried out for extracts from the 460 collected leaf samples to detect TBRV using commercial antisera (Loewe Biochemica GmbH, Germany). The results indicated that 9 samples (4 blackberry, 5 raspberry) were infected with TBRV. The isolates of the virus were transferred by sap inoculation and maintained in Nicotiana tabacum cv. Xanthi. Systemic ringspot, necrosis and patterned lines were observed on tobacco leaves. The presence of the virus in tobacco leaf samples was confirmed by reverse transcription PCR (RT-PCR). Total RNA was extracted from all 9 samples using the silica capture (SC) method described originally by Boom et al. (1990) and adapted to the detection of plant viruses by Malinowski (1997). Part of the CP gene was amplified with the CPF (5’-GCCTGTCTCTCTCGCAATG-3’) and CPR (5’-AAGGAGCCAAACTGAAATGT-3’) primer pair (Hasiów-Jaroszewska et al. 2015). Amplicons of the expected size (763 bp) were obtained for each sample. The amplified products were purified, sequenced in both directions, deposited in GenBank and assigned accession numbers: MT507387 to MT507390 and MT507394 for the isolates from Rubus idaeus and MT507391 to MT507393 and MN954654 for the isolates from Rubus fruticosus, respectively. The 9 newly obtained TBRV CP gene sequences, together with the 25 isolates deposited in GenBank, were aligned by ClustalW. The isolates obtained in this study showed a 99.0-100% nucleotides (nt) and a 98.7-100% amino acids (aa) identity in the part of the CP, respectively. Comparison of the part of the CP of the 4 blackberry and the 5 raspberry TBRV isolates with 25 TBRV isolates available in GenBank showed a 80.6-97.8% nt and a 87.9-99.5% aa identity, respectively. The results of the phylogenetic analysis have revealed that the TBRV isolates obtained in this study are closely related to 3 Polish isolates (AY157994, KR139941, KR139951) and 1 Bioreba ctrl Switzerland isolate (KT923164). These findings are of epidemiological significance due to the fact that TBRV was detected on symptomless Rubus plants, which therefore represent a reservoir of the virus and a threat in case of a symptomatic infection of sensitive cultivars. Accordingly, the results will assist in using appropriate strategies for reducing TBRV incidence in Rubus-growing areas. Moreover, this is, to the best of our knowledge, the first report of TBRV in raspberry and blackberry in Poland.

First Report of Tomato black ring virus (TBRV) on Tomato (Solanum lycopersicum) in Saudi Arabia

Tomato is a popular vegetable crop that is cultivated worldwide. It is also one of the most important crops in Saudi Arabia. In 2017, the area in which tomato was grown in Saudi Arabia was estimated to be 13317 ha and produced 306389 tons. Al Kharj Governorate in Riyadh region contributes the highest production of greenhouse tomatoes in Saudi Arabia (Ministry of Env. WTR & AGRI., 2017). In fall 2015, striking virus-like symptoms (mottling, leaf rolling, yellowing, and deformation, black strip on the stem, cracking on fruits, deformation, mottling, and mummification with severe yield losses) were observed on greenhouse tomato plants in several farms in Al Kharj Governorate. Samples were collected within the period of fall 2015 and the summer of 2017. The collected samples were tested serologically using enzyme linked immunosorbent assay (ELISA) for identification of the causal agent(s) using kits and protocols from AC Diagnostics Inc (Fayetteville, Arkansas, UAS). Out of 18 common tomato viruses tested, 14 viruses were detected in tomato plants in the region. The greatest concern was the presence of Tomato black ring virus (TBRV) as this was the first detection in Saudi Arabia and displayed the highest frequency of detection among all other detected viruses. Seventy-one out of the 135 tested samples were positive for TBRV. To confirm the presence of TBRV in the infected tomato samples, total RNA was extracted from positive samples and tested by RT-PCR with the newly designed primer pair F-TBRV (5′-GCAAACCAACGCTCTATGTTGT-3′)/R-TBRV (5'-AGAGCCAAACTGGAATGGTAGG-3') that is specific to the CP gene of TBRV. RT-PCR products of 978 bp in length were successfully obtained from the naturally infected tomato plants. One of the detected isolates was used to inoculate Chenopodium amaranticolor with the aim of obtaining a pure isolate from single local lesions that could be later used for propagation and maintenance in Nicotiana tabacum. A host range experiment was conducted using mechanical inoculation with the single-lesion isolate of TBRV on four replicates of 14 different plant species in parallel with healthy controls (Brunt et. al. 1996). Three weeks post-inoculation, varying reactions and symptoms ranging from local lesion to plant death, depending on host species, were observed on the tested plants (Supplementary Table 1). Host range results were largely similar to those reported in previous studies (Sneideris et al. 2012, and Rymelska et al. 2013). The presence of TBRV was confirmed both by ELISA and RT-PCR. Nucleotide sequences obtained from PCR products of selected samples were submitted to the GenBank and assigned the following accession numbers: MT274656, MT274657, and MT274658. Saudi isolates of TBRV were found to share 99-100% of their nucleotide sequences. They had the highest similarity of 98% with the Polish isolates (MG458221 and KX977561) and the lowest similarity of 85% with isolates from Lithuania (KF678369, and KF678370). To the best of our knowledge, this is the first report of occurrence of TBRV in Saudi Arabia. Since this virus is transmitted by seeds, it may have entered through imported seeds and spread in greenhouses through mechanical means. A survey of the different agricultural regions is encouraged to determine the incidence, distribution, and damage induced by this virus in Saudi Arabia.

Fundamental and practical bases for creating a blackcurrant field repository in Russia

Creating a virus-free repository of blackcurrant will allow an assessment of productivity, other qualitative characteristics and the use of pollen from healthy plants for breeding. The aim of the research was to reveal the most promising varieties and hybrids of blackcurrant, test plants for viruses and obtain nuclear stock plants for laying the repository. The studies were carried out in 2018-2020 using generally accepted methods of variety study, virus diagnosis and plants reproduction. Valuable blackcurrant varieties and hybrids of the All-Russian Horticultural Institute for Breeding, Agrotechnology and Nursery breeding were studied; the most productive clones were selected and tested by ELISA and PCR. The species composition of harmful viruses in the fruiting stands of blackcurrant under ex situ conditions is diverse. The total prevalence of Arabis mosaic virus (ArMV), Raspberry ringspot virus (RpRSV), Strawberry latent ringspot virus (SLRSV), Tomato black ring virus (TBRV) on blackcurrant plants was 31.5 %. The prevalence of SLRSV and RpRSV was about 15 %, TBRV – 14 %, ArMV – 7 %. Blackcurrant reversion virus (BRV) was absent. As a result of the monitoring carried out in 2018, blackcurrant plants of 8 varieties free from harmful viruses were identified and, after PCR, will be used to establish the repository and base mother nursery.

Effect of defective interfering RNAs on the vertical transmission of Tomato black ring virus

Viruses are thought to be the ultimate parasites, using host resources for multiplication. Interestingly, many viruses also have their own 'parasites', such as defective interfering RNAs (DI RNAs). One of the plant viruses whose infection can be accompanied by subviral RNAs is the Tomato black ring virus (TBRV). DI RNAs associated with the TBRV genome were generated de novo as a result of prolonged passages in one host. DI RNAs modulate the TBRV accumulation and the severity of the symptoms induced on the infected plants. In this study, we have addressed the question of whether DI RNAs can also affect TBRV vertical transmission through seeds. The experiments were conducted using the TBRV-Pi isolate and Chenopodium quinoa plants. C. quinoa plants were infected with TBRV-Pi with and without DI RNAs. Overall, 4 003 seeds were tested, and the analysis showed that the presence of DI RNAs made the TBRV-Pi seed transmission 44.76% more efficient. Moreover, for the first time, we showed that DI RNAs are being transferred from generation to generation.

An assessment of the transmission rate of Tomato black ring virus through tomato seeds

Tomato black ring virus (TBRV) infects a wide range of economically important plants, and is distributed worldwide. TBRV is transmitted by soil-inhabiting nematodes. However, a long-distance dispersion is possible via seeds and pollen. In this study, we provided evidence that virus can be efficiently transmitted through tomato seeds. Three tomato varieties (Beta Lux, Grace and Money Maker) and four genetically diverse TBRV isolates collected originally from different hosts were used in the experiments. The seedlings were grown in an insect-proof glasshouse and the presence of TBRV was verified by immunoassay (ELISA). The seed transmission was significantly dependent on tomato cultivar and virus isolate ranging from 1.69% up to 14.57%. Bioassays using Chenopodium quinoa plants confirmed the presence of infectious virus in the seeds.

Quantitative and qualitative impact of Tomato Black Ring Virus (TBRV) on Merlot and Cabernet franc

Aim: Fifteen nepoviruses are able to induce fanleaf degeneration in grapes, which is the viral disease with the highest economic impact. Grapevine FanLeaf Virus (GFLV) is the main causal agent of this disease worldwide and in Europe, followed by Arabis Mosaic Virus (ArMV). A third nepovirus has been described in France: Tomato Black Ring Virus (TBRV), detected in vines for the first time in France on a multi-varietal plot in 2009. The aim of this work is to evaluate the effect of TBRV on grape yield and berry quality potential in two grapevine varieties.Methods and results: Quantitative and qualitative impact of TBRV was investigated in 2010 and 2011 in the vine plot where the virus was first detected in France. Over 200 vines were analyzed by ELISA tests in order to determine their status regarding viral diseases. Vines were distributed in four groups: TBRV positive versus virus free for Merlot and Cabernet franc respectively. For each vine, the presence of eleven other viruses was investigated. In 2010 and 2011 shoot length was measured. Grape composition was also analyzed to determine technological maturity and phenol content in grapes of each vine in relation to its virus status. Total pruning weight was reduced in TBRV infected vines, while lateral shoot number and length were increased. All yield components were affected by the presence of the virus. Vines positive to TBRV produced less bunches and berries and smaller berries compared to healthy vines. Yield loss was greater on Merlot compared to Cabernet franc. Grape quality parameters seemed to be not negatively affected by the presence of TBRV.Conclusions: The major impact of TBRV was a significant yield decrease. The enhanced development of lateral shoots may be considered as a second negative impact that could increase production cost.Significance and impact of the study: These results provide essential information on the impact of TBRV in vineyards. Consequences for management of the viral disease in the vineyard are discussed.