Occurrence and Distribution of Tomato Brown Rugose Fruit Virus Infecting Tomato Crop in Saudi Arabia.

During the growing season of 2021-2022, a total of 145 symptomatic tomato leaf and fruit samples were collected from different locations in Riyadh Region, Saudi Arabia, showing a moderate-to-severe mosaic with dark green wrinkling, blistering, narrowing, and deformation with necrosis spot on tomato leaves, while irregular brown necrotic lesions, deformation, and yellowing spots rendering the fruits non-marketable were observed on tomato fruits. These samples were tested serologically against important tomato viruses using enzyme-linked immunosorbent assay (ELISA), and the obtained results showed that 52.4% of symptomatic tomato samples were found positive for Tomato brown rugose fruit virus (ToBRFV), wherein 12 out of 76 samples were singly infected; however, 64 out of 145 had mixed infection. A sample with a single infection of ToBRFV was used for mechanical inoculation into a range of different host plants; symptoms were observed weekly, and the presence of the ToBRFV was confirmed by ELISA and reverse transcription-polymerase chain reaction (RT-PCR). A total RNA was extracted from selected ELISA-positive samples, and RT-PCR was carried out using specific primers F-3666 and R-4718, which amplified a fragment of 1052 bp. RT-PCR products were sequenced in both directions, and partial genome nucleotide sequences were submitted to GenBank under the following accession numbers: MZ130501, MZ130502, and MZ130503. BLAST analysis of Saudi isolates of ToBRFV showed that the sequence shared nucleotide identities (99-99.5%) among them and 99-100% identity with ToBRFV isolates in different countries. A ToBRFV isolate (MZ130503) was selected for mechanical inoculation and to evaluate symptom severity responses of 13 commonly grown tomato cultivars in Saudi Arabia. All of the tomato cultivars showed a wide range of symptoms. The disease severity index of the tested cultivars ranged between 52% and 96%. The importance ToBRFV disease severity and its expanding host range due to its resistance breaking ability was discussed.

Interaction estimation of pathogenicity determinant protein ßC1 encoded by Cotton leaf curl Multan Betasatellite with Nicotiana benthamiana Nuclear Transport Factor 2.

Background: Begomovirus is one of the most devastating pathogens that can cause more than 90% yield loss in various crop plants. The pathogenicity determinant ßC1, located on the betasatellite associated with monopartite begomoviruses, alters the host signaling mechanism to enhance the viral disease phenotype by undermining the host immunity. The understanding of its interacting proteins in host plants to develop disease symptoms such as curly leaves, enations, vein swelling, and chlorosis is crucial to enhance the disease resistance in crop plants. The current study was designed to reveal the contribution of ßC1 in disease pathogenicity and to unveil potential interacting partners of ßC1 protein in the model plant Nicotiana benthamiana. Methods: The ßC1 gene was cloned in pGKBT7 and used as bait against the cDNA library of N. benthamiana and its pathogenesis was tested against the healthy plant and the plants infiltrated with empty vectors. The yeast two-hybrid-based screening was performed to find the interacting factors. Successful interacting proteins were screened and evaluated in various steps and confirmed by sequence analysis. The three-dimensional structure of the Nuclear Transport Factor 2 (NTF2) protein was predicted, and in-silico protein-protein interaction was evaluated. Furthermore, protein sequence alignment and molecular phylogenetic analysis were carried out to identify its homologues in other related families. In-silico analyses were performed to validate the binding affinity of ßC1 protein with NTF2. The 3D model was predicted by using I-TASSER and then analyzed by SWISS MODEL-Workspace, RAMPAGE, and Verify 3D. The interacting amino acid residues of ßC1 protein with NTF2 were identified by using PyMOL and Chimera. Results: The agroinfiltrated leaf samples developed severe phenotypic symptoms of virus infection. The yeast-two-hybrid study identified the NTF2 as a strong interacting partner of the ßC1. The NTF2 in Solanaceae and Nicotiana was found to be evolved from the Brassica and Gossypium species. The in-silico interaction studies showed a strong binding affinity with releasing energy value of -730.6 KJ/mol, and the involvement of 10 amino acids from the middle portion towards the C-terminus and five amino acid residues from the middle portion of ßC1 to interact with six amino acids of NTF2. The study not only provided an insight into the molecular mechanism of pathogenicity but also put the foundation stone to develop the resistance genotypes for commercial purposes and food security.

Identification of Tomato black ring virus from tomato plants grown in greenhouses in Saudi Arabia.

A survey was conducted in Al-Kharj governorate, Riyadh region to identify viruses causing variety of virus-like symptoms on tomato plants. A total of 135 samples were collected from symptomatic tomato plants. Symptoms included mottling, deformation, necrosis of leaves and fruits. Eighteen viruses were tested by DAS-ELISA. Tomato black ring virus (TBRV) was the virus of concern as it was not detected in Saudi Arabia before and was detected in 52.6% of the collected samples in this study. RT-PCR was used to confirm detection of TBRV and to sequence the amplified products to determine molecular characteristics of this virus. In the host range test study that was performed using a purified isolate of TBRV, sixteen out of the twenty two tested plants showed symptoms. Brassica oleracea was not infected by this virus. Gel electrophoreses (2% agarose) yielded fragments of 978 bp of coat protein gene of TBRV. Nucleotide sequences of purified RT-PCR products for three TBRV Saudi isolates were deposited in the GenBank with the following accession numbers MT274656, MT274657, and MT274658. These isolates of TBRV indicated a close Phylogenetic relationship of (99-100%) among themselves and with five isolates from Poland (95-98%) but a distant relationship of 85% with isolates from England and Lithuania deposited in the GenBank. This is the first report for detection and molecular characterization of TBRV infecting tomato plants in Saudi Arabia.