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.


Introduction
Tomato (Solanum lycopersicum L.; family: Solanaceae) is one of the most popular and economically important vegetable crops worldwide [1]. In Saudi Arabia, tomato is considered one of the most economically important vegetable crops grown in different regions. The total area under tomato cultivation is estimated to be 12,454.3 hectares, producing 351,212.4 tons of fresh tomato fruits [2]. Tomato plants are affected by many phytopathogens including several phytopathogenic fungi, bacteria, viruses, nematodes,

Visual Inspection and Detection of Tomato Viruses Using ELISA
The tomato plant symptoms induced by ToBRFV were wide and complex, wherein leaf symptoms showed moderate-to-severe mosaic with dark green wrinkling, blistering, narrowing, and deformation with necrosis spots on tomato leaves, while the fruit symptoms showed irregular brown necrotic lesions, deformation, and yellowing spots, rendering the fruits non-marketable, all observed in most of the tomato crops in many different locations in Riyadh Region (Figure 1).
All collected samples were tested with ELISA against ToCV, TSWV, TCSV, TAV, TBSV, TBRV, TRSV, ToMV, PepMV, TYLCV, and ToBRFV. The obtained results as indicated in Table 1 showed that 77.24% (112/145) of symptomatic tomato samples were found positive against at least one of the tested viruses by ELISA. The obtained results showed that 52.4% (76/145) of symptomatic tomato samples were found positive for ToBRFV, 12 out of 76 samples (6.9%) were infected with ToBRFV only, and 64 out of 145 (44%) had mixed infection between ToBRFV and at least one of the tested viruses.   Total  145  76  26  34  23  37  9  5  24  11  6  8  64  12 Percentage   All collected samples were tested with ELISA against ToCV, TSWV, TCSV, TAV,  TBSV, TBRV, TRSV, ToMV, PepMV, TYLCV, and ToBRFV. The obtained results as indicated in Table 1 showed that 77.24% (112/145) of symptomatic tomato samples were found positive against at least one of the tested viruses by ELISA. The obtained results showed that 52.4% (76/145) of symptomatic tomato samples were found positive for ToBRFV, 12 out of 76 samples (6.9%) were infected with ToBRFV only, and 64 out of 145 (44%) had mixed infection between ToBRFV and at least one of the tested viruses.

Mechanical Inoculation and Host Range Determination
The selected plant species for host range showed a wide range of symptoms compared with the non-inoculated control plants of the same species. Necrotic local lesions were developed on the inoculated leaves of N. tabacum and N. glutinosa plants. N. benthamiana and N. occidentalis showed yellowing, necrosis, and collapsed plants. On the other hand, S. lycopersicum plants showed leaf blistering, mosaic, narrowing, and leaf deformation, whereas C. annum plants showed symptoms of necrotic lesions in inoculated leaves and stems. Chlorotic local lesions were observed in the Chenpodium spp. (Figure 2). All these host plants showed positive reactions in DAS-ELISA and RT-PCR detection. S. melongena and S. tuberosum showed no symptoms and had a positive reaction when tested by doubleantibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and RT-PCR, while C. lanatus, C. melo, and C. sativus were asymptomatic but had a negative reaction when tested by the same techniques. All symptoms were recorded on inoculated (local symptoms) and non-inoculated leaves (systemic symptoms), as mentioned in Table 2.
mation, whereas C. annum plants showed symptoms of necrotic lesions in inoculated leaves and stems. Chlorotic local lesions were observed in the Chenpodium spp. (Figure 2). All these host plants showed positive reactions in DAS-ELISA and RT-PCR detection. S. melongena and S. tuberosum showed no symptoms and had a positive reaction when tested by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and RT-PCR, while C. lanatus, C. melo, and C. sativus were asymptomatic but had a negative reaction when tested by the same techniques. All symptoms were recorded on inoculated (local symptoms) and non-inoculated leaves (systemic symptoms), as mentioned in Table  2.

Detection of ToBRFV by RT-PCR
The obtained results using PCR products from cDNA synthesized in the selected samples from different locations during the survey revealed that ToBRFV was detected when running on agarose gel electrophoresis (1%), giving specific bands (1052 bp), and it was clearly visible in all tested samples and their comparison with 1 Kb DNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA), confirming the presence of ToBRFV in these samples ( Figure 3).

Detection of ToBRFV by RT-PCR
The obtained results using PCR products from cDNA synthesized in the selected samples from different locations during the survey revealed that ToBRFV was detected when running on agarose gel electrophoresis (1%), giving specific bands (1052 bp), and it was clearly visible in all tested samples and their comparison with 1 Kb DNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA), confirming the presence of ToBRFV in these samples ( Figure 3).

Partial Genome Nucleotide Sequencing and Phylogenetic Analysis
Sequences of the three selected isolates representing Riyadh Region (Al-Kharj, Az-Zulfi, and Al-Hareeq) were submitted to GenBank, named as ToBRFV-SA-F8, ToBRFV-SA-L10, and ToBRFV-SA-L24, respectively, and had the accession numbers MZ130501, MZ130502, and MZ130503, respectively. Sequences of three samples showed high similarity to all ToBRFV isolates that were registered in the NCBI. The data obtained from the phylogenetic tree revealed limited genetic variability among all Saudi isolates of ToBRFV and the sequences of other isolates available in the NCBI isolated from different host species and from different geographical countries. BLAST analysis of the three Saudi Arabian isolates of ToBRFV showed that the sequence shared nucleotide identities ranged between 99% and 99.5% among them and showed 98.9-99.9% identity with other GenBank isolates, including Palestine ( Table 3). A homology tree was designed to compare the phylogenetic relationship between virus isolates ( Figure 4).

Evaluation of Tomato Cultivars Responses to the Saudi Isolate of ToBRFV
This experiment was carried out to evaluate the responses of thirteen commercial tomato cultivars to mechanical inoculation with a Saudi Arabian isolate of ToBRFV (accession no. MZ130503). Symptoms started to show up on upper and systemic new leaves in all cultivars after 2-3 weeks after inoculation. All of the tomato cultivars showed a wide range of symptoms including mosaic, mottling, leaf deformations, leaf narrowing, leaf rolling, blistering, and shoestring ( Figure 5). The DSI ranged between 52% and 96% (Table  4). ToBRFV was detected in all thirteen tested commercial tomato cultivars by RT-PCR ( Figure 6).

Evaluation of Tomato Cultivars Responses to the Saudi Isolate of ToBRFV
This experiment was carried out to evaluate the responses of thirteen commercial tomato cultivars to mechanical inoculation with a Saudi Arabian isolate of ToBRFV (accession no. MZ130503). Symptoms started to show up on upper and systemic new leaves in all cultivars after 2-3 weeks after inoculation. All of the tomato cultivars showed a wide range of symptoms including mosaic, mottling, leaf deformations, leaf narrowing, leaf rolling, blistering, and shoestring ( Figure 5). The DSI ranged between 52% and 96% (Table 4). To-BRFV was detected in all thirteen tested commercial tomato cultivars by RT-PCR ( Figure 6).

Discussion
ToBRFV is an emerging viral pathogen that is highly virulent, very aggressive, and fast spreading. ToBRFV belongs to the genus Tobamovirus and causes significant yield losses to tomato crop and its fruit quality as well, and therefore early detection is important in orderto thwart its spread [15]. In this study, various disease symptoms on tomato plants caused by ToBRFV in Riyadh Region, Saudi Arabia, were reported such as moderate-to-severe mosaic with dark green wrinkling, blistering, narrowing, and deformation with necrosis spots on tomato leaves, while irregular brown necrotic lesions, deformation, and yellowing spots rendering the fruits non-marketable were observed on tomato fruits. Symptoms of ToBRFV in tomato vary among environmental conditions and

Discussion
ToBRFV is an emerging viral pathogen that is highly virulent, very aggressive, and fast spreading. ToBRFV belongs to the genus Tobamovirus and causes significant yield losses to tomato crop and its fruit quality as well, and therefore early detection is important in orderto thwart its spread [15]. In this study, various disease symptoms on tomato plants caused by ToBRFV in Riyadh Region, Saudi Arabia, were reported such as moderate-to-severe mosaic with dark green wrinkling, blistering, narrowing, and deformation with necrosis spots on tomato leaves, while irregular brown necrotic lesions, deformation, and yellowing spots rendering the fruits non-marketable were observed on tomato fruits. Symptoms of ToBRFV in tomato vary among environmental conditions and tomato cultivars [16]. In Jordan, ToBRFV causes brown rugose symptoms on tomato fruits, and hence the virus was named tomato brown rugose fruit virus [12]. Besides brown rugose, ToBRFV induced yellow spots on tomato fruits and brown rugose symptoms in some cultivars in Israel [16]. ToBRFV also caused yellow spots on fruits of tomato plants in Germany, Palestine, and China [19,21,35].
ELISA is considered as a valuable tool for virus detection and it is an easy test to deal with a large number of samples [36,37]. ELISA is a robust technique that enables the detection of viral capsid protein subunits of tobamoviruses. ELISA was adapted successfully for the detection of tobamoviruses, including ToBRFV. The ELISA test was conducted to screen the most common tomato viruses using kits that were available in our laboratory, including our survey, and the ELISA results clearly demonstrated that ToBRFV was present in commercial tomato crops collected from Riyadh Region, Saudi Arabia.
Various plants were proven to be experimental hosts to ToBRFV, and similar symptoms to those in our study were described by several investigators on C. quinoa, C. amaranticolor, C. album, N. benthamiana, N. glutinosa, N. tabacum, S. lycopersicum, C. annum, S. nigrum, and D. stramonium [16,21,35,38]. Petunia plants are symptomless hosts, while eggplants and potatoes are non-hosts for the virus [16]. On the other hand, ToBRFV causes latent infection on eggplants and potatoes [35]. Plants from the family Cucurbitaceae were found to be non-hosts for ToBRFV [35,38].
PCR and RT-PCR, which are specific and sensitive nucleic-acid-based methods for detection of plant viruses [39], were performed to diagnose viruses. All tissue samples (leaves or fruits) of the selected tomato plants tested positive for ToBRFV by RT-PCR, as the expected size of the 1052 bp fragment of the ORF encoding the RNA dependent RNA polymerase [16] was amplified, sequenced, and confirmed in terms of the presence of the virus in the study area. The main purpose of the molecular studies was to confirm the presence of ToBRFV in the study area and makeup of Saudi isolates of ToBRFV and their percentage identity and phylogenetic relationship with other isolates reported worldwide. The identity analyses and phylogenetic analysis of the Saudi isolates shared the highest nucleotide and amino acid identities with isolates from the Netherlands, Canada, the United Kingdom, and Jordan.
The Saudi isolate (MZ130503) infected all tested tomato cultivars and caused a wide range of symptoms including mosaic, mottling, leaf deformations, leaf narrowing, leaf rolling, blistering, and shoestring. DSI ranged between 52% and 96%. A similar study from China [35] reported that all the 50 tomato cultivars tested were highly sensitive to mechanical inoculation of ToBRFV, and systemic leaves of inoculated tomato plants showed symptoms of yellowing, curling, rolling, narrowing, blistering, and mosaic. In another study, all cultivated tomatoes and the great majority of wild tomatoes showed a wide range of symptoms including mosaic, leaf deformations, mottling, shoestring, and stunting in their reaction to ToBRFV inoculation [40].

Mechanical Inoculation and Host Range Determination
For ToBRFV biological purification, a single local lesion technique was carried out by mechanical inoculation using C. amaranticolor as a local lesion host, whereas N. tabacum was used as a propagative host for the following experiments [41,42]. For mechanical inoculation, the plant leaves were dusted with carborundum. The extracted sap was passed through a cheesecloth and used to inoculate the selected 19 plant species belonging to different botanical families, namely, S. lycopersicum, S. melongena, S. nigrum, S. tuberosum, C. annum, N. glutinosa, N. occidentalis, N. tabacum, N. benthamiana, C. amaranticolor, C. glaucum, C. quinoa, C. album, G. globosa, D. stramonium, P. hybrida, C. lanatus, C. melo, and C. sativus. All inoculated plants were kept in an insect-proof cage inside a greenhouse for 2−3 weeks, and symptom expressions on each species were described and recorded. DAS-ELISA and RT-PCR were used to confirm the presence of the ToBRFV.  [16]. The reaction mixture was performed in a 25 µL volume using a Thermo Scientific DreamTaq Green PCR Master Mix (2X). PCR was performed using the following parameters: 94 • C for 5 min as a heating step of Taq DNA polymerase, 94 • C for 30 s (denaturation), 56 • C for 30 s (annealing), and 72 • C for 1 min (extension) for 35 cycles, followed by final extension at 72 • C for 5 min. PCR products were analyzed by electrophoresis on a 1% agarose. A 1 Kb DNA Ladder (Thermo Scientific, USA) was used to determine the size of DNA-amplified cDNA products.

Partial Genome Nucleotides Sequencing and Phylogenetic Analysis
RT-PCR products of the three selected tomato samples were sent to Macrogen Inc., Seoul, South Korea, for Sanger sequencing on strands through its entirety. The sequence results were analyzed through BLASTn for comparison with published ToBRFV gene RdRp sequences retrieved from the National Center for Biotechnology Information (NCBI). The construction of the phylogenetic tree from the aligned sequences was conducted by using MEGA X [43], applying the maximum likelihood method (ML) algorithm. ToBRFV-SA-F8, ToBRFV-SA-L10, and ToBRFV-SA-L24 isolates were compared with 27 different isolates obtained from GenBank from different hosts and countries, a homology tree was designed to compare the phylogenetic relationship among virus isolates, and pairwise nucleotide sequence identity tables were made.

Evaluation of Tomato Cultivar Responses to the Saudi Isolate of ToBRFV
This experiment was carried out to evaluate the responses of thirteen commercially cultivated tomato cultivars (Newton, Quaresma, Dafnis, JV 15, Jamilah, Seraj, Mawal (Syngenta Basel Switzerland.), Baikonour, Mulla F1 (Westfrisian seeds Gorredijk, Netherlands), Dusmo F1, Meghina F1 (ISI SEMENTI Fidenza PR, Italy), Tone Gutar (Seminis vegetable seeds, Mumbai, India ), and Titanic (Huizer Zaden, Rilland, Netherlands) by mechanical inoculation using the Saudi isolate of ToBRFV (accession no. MZ130503). All inoculated plants were kept in an insect-proof cage inside a greenhouse as mentioned above. The severity of the symptoms was evaluated as per the following scale: mild (+), moderate (++), severe (+++), very severe (++++), and symptomless (−) [44]. The disease severity index (DSI) listed in Table 5 was calculated by the followed formula [40]: (1) DSI = disease severity index; e = class; Re = number of plants in class (e); N = total number of plants. RT-PCR was used to confirm the presence of the ToBRFV using the newly developed systemic leaves [35,40]. Mild mosaic or mottling, followed by recovery 2 Mild mosaic or mottling with leaf deformation 3 Moderate mosaic or mottling and leaf deformed followed by leaf rolling 4 Severe mosaic or mottling, and leaf deformation 5 Severe mosaic or mottling, leaf deformation, and shoestring

Conclusions
In summary, on the basis of the results obtained from this study, ToBRFV induced moderate-to-severe mosaic with dark green wrinkling, blistering, narrowing, and deformation with necrosis spots on tomato leaves, while irregular brown necrotic lesions, deformation, and yellowing spots rendering the fruits non-marketable were observed on tomato fruits. These symptoms were similar to those described in other studies (Salem et al. (2016) [12] and Luria et al. (2017) [16]). In 145 symptomatic tomato samples collected from different locations in Riyadh Region, 52.4% were found to be positive for ToBRFV, 6.85% were singly infected, and 44.13% had mixed infection between ToBRFV and with at least one of tested viruses. In the host range experiment, the plants S. lycopersicum, S. melongena, S. nigrum, S. tuberosum, C. annum, N. glutinosa, N. occidentalis, N. tabacum, N. benthamiana, C. amaranticolor, C. glaucum, C. quinoa, C. album, G. globose, D. stramonium, and P. hybrida were found to be susceptible to ToBRFV, while, plants of C. lanatus, C. melo, and C. sativus of the family Cucurbitaceae were found to be non-susceptible to ToBRFV. All the thirteen tested commercially tomato cultivars grown in Saudi Arabia showed a wide range of symptoms, including mosaic, mottling, leaf deformations, leaf narrowing, leaf rolling, blistering, and shoestring in response to mechanical inoculation with a Saudi isolate of ToBRFV (accession no. MZ130503). On the basis of the obtained results in this present study, it was established that Tomato brown rugose fruit virus (ToBRFV) is associated with the commercial tomato crop from Riyadh Region, Saudi Arabia.

Conflicts of Interest:
The authors declare no conflict of interest, and all authors approved the manuscript and agreed to submit it to Plants for publication.