Genetic Diversity of Tomato Black Ring Virus Satellite RNAs and Their Impact on Virus Replication.

Viral satellite RNAs (satRNAs) are small subviral particles that are associated with the genomic RNA of a helper virus (HV). Their replication, encapsidation, and movement depend on the HV. In this paper, we performed a global analysis of the satRNAs associated with different isolates of tomato black ring virus (TBRV). We checked the presence of satRNAs in 42 samples infected with TBRV, performed recombination and genetic diversity analyses, and examined the selective pressure affecting the satRNAs population. We identified 18 satRNAs in total that differed in length and the presence of point mutations. Moreover, we observed a strong effect of selection operating upon the satRNA population. We also constructed infectious cDNA clones of satRNA and examined the viral load of different TBRV isolates in the presence and absence of satRNAs, as well as the accumulation of satRNA molecules on infected plants. Our data provide evidence that the presence of satRNAs significantly affects viral load; however, the magnitude of this effect differs among viral isolates and plant hosts. We also showed a positive correlation between the number of viral genomic RNAs (gRNAs) and satRNAs for two analysed TBRV isolates.

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.

LNA probe-based assay for the detection of Tomato black ring virus isolates.

Tomato black ring virus (TBRV) infects a wide range of economically important plant species worldwide. In the present study we developed a locked nucleic acid (LNA) real-time RT-PCR assay for accurate detection of genetically diverse TBRV isolates collected from different hosts. The assay based on the LNA probe has a wide detection range, high sensitivity, stability and amplification efficiency. The assay amplified all tested TBRV isolates, but no signal was observed for the RNA from other nepoviruses and healthy plant species. Under optimum reaction conditions, the detection limit was estimated around 17 copies of the TBRV target region in total RNA. Real-time RT-PCR with the LNA probe described in this paper will serve as a valuable tool for robust, sensitive and reliable detection of TBRV isolates.

Development of Stable Infectious cDNA Clones of Tomato Black Ring Virus Tagged with Green Fluorescent Protein.

Tomato black ring virus (TBRV) is a member of the Nepovirus genus in the Secoviridae family, which infects a wide range of important crop species worldwide. In this work, we constructed four cDNA infectious clones of the TBRV tagged with the green fluorescent protein (TBRV-GFP), which varied in (i) the length of the sequences flanking the GFP insert, (ii) the position of the GFP insert within the RNA2 polyprotein, and (iii) the addition of a self-cutting 2A protein. The presence of the GFP coding sequence in infected plants was verified by RT-PCR, while the infectivity and stability of the constructs were verified by mechanical inoculation of the host plants. The systemic spread of TBRV-GFP within plants was observed under UV light at a macroscopic level, monitoring GFP-derived fluorescence in leaves, and at a microscopic level using confocal microscopy. The obtained clones are a valuable tool for future studies of TBRV-host interactions, virus biology, and the long-term monitoring of its distribution in infected plants.

Construction of Agrobacterium tumefaciens-mediated tomato black ring virus infectious cDNA clones.

Tomato black ring virus (TBRV, genus Nepovirus) infects a wide range of economically important plants such as tomato, potato, tobacco and cucumber. Here, a successful construction of infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2) is reported for the first time. The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS). The symptoms induced on plants agroinoculated with both constructs were indistinguishable from those caused by the wild-type virus. The infectivity of obtained clones was verified by reinoculation to Nicotiana tabacum cv. Xanthi, Chenopodium quinoa and Cucumis sativus. The presence of viral particles and RNA was confirmed by electron microscopy and reverse transcription polymerase chain reaction, respectively. Constructed full-length infectious cDNA clones will serve as an excellent tool to study virus-host-vector interactions.