According to ^{Dombrovsky and Smith (2017}), world seed trade has contributed to the transmission and dissemination of outbreaks of new diseases, particularly from viruses belonging to the genus Tobamovirus, including the ToBRFV. Tobacco, tomato and chili pepper (Solanaceae family) crops are severely affected by the virus of the genus Tobamovirus: Tobacco mosaic virus (TMV), Tobacco mild green mosaic virus (TMGMV), Tomato mosaic virus (ToMV) and Pepper mild mottle virus (PMMoV) (Dombrovsky and Smith, 2017).

The species of the Tobamovirus genus have rod-shaped particles, and a positive-sense single-stranded RNA genome of ~6.4 kb with four Open Reading Frames (ORFs). ORF1 and ORF2 are separated by a stop codon and they codify two proteins related to the replication of the virus with a weight of 126 and 183 kDa, respectively. ORF3 codifies the movement protein of 30 kDa, while ORF4 codifies the capsid of 17.5 kDa (^{Luria et al., 2017}).

The tobamoviruses are easily transmitted mechanically and via seeds (infectious particles in the testa) that infect sprouts when they germinate. The virus may remain in plant residues and soils contaminated with the virus for several months, and in infected seeds for several years (^{Dombrovsky and Smith, 2017}).

Recently, in Israel (^{Luria et al., 2017}) and Jordan (^{Salem et al., 2015}), there were reports of tomatoes infected by a new tobamovirus called Tomato brown rugose fruit virus (ToBRFV), which causes a reduction in the amount of flowers and fruits, necrosis in the peduncle of the fruit and leaves of the calyx. The fruits of infected plants presented yellow or brown areas and rough areas on their surface (Salem et al., 2015).

In 2018, tomato and chili farmers of the region of Yurecuaro and Tanhuato, Michoacan, have reported symptoms on fruits regarding yellow colors, green spots and deformations, green grooves and irregular brown spots (Fig. 1a), and on leaves, mosaic symptoms, spots and yellowing. The aim of the present work was to determine if ToBRFV is present in Yurecuaro and Tanhuato, Michoacan, affecting tomato and chili crops.

The present study was carried out in the facilities of the National Plant Health Reference Center. In September of 2018, in the municipal areas of Yurecuaro and Tanhuato, Michoacan, leaf tissue, flowers and fruits were taken from tomato and chili plants with symptoms of the disease: fruits showing yellowing, spots and green grooves, irregular brown spots and deformation, while the foliage presented chlorotic spots and mosaic. No symptoms were found in flowers, according to reports by other authors. In Yurecuaro, two samples were taken of tomato plants in different production units, and in Tanhuato, six samples were taken (five of tomato and one of chili). The samples were properly labeled and taken to the laboratory, where they were maintained at 4 ˚C.

In each sample, midribs of the leaves were separated and cut transversally to divide the sample into two parts: the first was used for the preparation of gridsfor the transmission electron microscope, and the second, for the extraction of total RNA.

Central midribs of infected leaves (0.1 g) were grinded in 500 µL of bi-distilled water using a sterile plastic mortar; the macerate was centrifuged at 9000 g for three minutes to eliminate any remaining plant tissue. Copper grids of 300 mesh (Electron Microscopy Sciences®) covered with a formvar/carbon® membrane, were covered with 10 µL of supernatant and 10 µL of phosphotungstic acid at 1 % (Fluka analytical), both were mixed with a pipette and left them for 30 seconds and the excess was eliminated with a piece of filter paper. Grids were observed under a transmission electron microscope and the images were photographed.

Total RNA was extracted from 100 mg of central midribsby the SV Total RNA Isolation System Start-Up^® (Promega™) kit, according to the indications of the manufacturer. The purity and concentration of the RNA extracted was quantified by spectrophotometry (Nano Drop 2000^®, Thermo Scientific™).

Retrotranscription was carried out using random primers (Random Hexamer, Invitrogen™), according to the manufacturer instructions. Conditions of amplification were: 1 cycle at 42 ˚C for 30 min, 1 cycle at 99 ˚C for 5 min, and finally, 12 ˚C for 5 min. PCR was performed with primers F-3666 (5´-ATGGTACGAACGGCGGCAG-3´) and R-4718 (5´-CAATCCTTGATGTG TTTAGCAC-3´) which amplified a fragment of 1052 pb of ORF2 (^{Luria et al., 2017}). The reaction mixture consisted of 12.5 µL of Platinum™ SuperFi™ PCR Master Mix (2X) (Invitrogen), 5 µL of Super FiTM GC Enhancer (5X) (Invitrogen), 3 µL of molecular biology degree water (Invitrogen), 1.25 µL of primer F-3666 (10 µM), 1.25 µL of primer R-4718 (10 µM) and 2 µL of cDNA template, in a final volume of 25 µL. The amplification was performed under the following conditions: 1 cycle at 98 ˚C for 5 min, 25 cycles at 98 ˚C for 1 min, 62 ˚C for 3 min and 72 ˚C for 3 min with a final extension of 72 ˚C for 10 min. The amplified products were observed by electrophoresis in an agarose gel at 1.5 %.

RT-PCR products were sequenced in the National Plant Health Reference Center Molecular Biology Lab. (Applied Biosystems, model 3130); sequences were assembled and edited to obtain sizes of 900 bp, and compared in the GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Local alignment was carried out with MEGA v7.0.26 and the algorithm MUSCLE. A matrix was built with Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV) and Tomato mottle mosaic virus (ToMMV) sequences to carry out a phylogenetic analysis. As an external group, we used two sequences from Pepper mild mottle virus (PMMoV) (NC_003630.1) and Tobacco mild green mosaic virus (TMGMV) (AB078435.1). The Maximum Likelihood (ML) model was used, based on the model by Tamura-Nei, with 1000 bootstrap iterations. The initial trees for the heuristic analysis were obtained automatically by applying the Neighbor-Join and BioNJ algorithms to a distance by estimated pairs matrix, using the Maximum Composite Likelihood (MCL) approach and selecting the topology with a higher log verosimilitude value with MEGA v7.0.26.

Symptoms in tomato fruits and plants observed in Yurecuaro and Tanhuato were similar to those reported by ^{Luria et al. (2017}) and ^{Salem et al. (2015}) (Figure 1a). The samples analyzed amplified the expected product of 1052 bp. Sequences obtained from samples M1 (MK273183), M2 (MK273184), M5 (MK273187) and M7 (MK273189) had a similarity of 100 % with those from Israel (KX619418.1) and Jordan (KT383474.1), while samples M3 (MK273185), M4 (MK273186), M6 (MK273188) and M8 (MK273190) had a similarity of 99 % for both isolates (Figure 1b).

The phylogenetic tree with the highest probability of registering (-2680.43) shows that the sequences of samples M1-M8 were grouped within the node that contains the Israeli (KX619418.1) and Jordanian sequences (KT383474.1) (Figure 1c). All groups of tobamoviruses have percentages of inference of 90 - 100 % from 1000 bootstrap iterations. In grids prepared with tomato leaf extracts, rod-shaped viral particles of approximately 300 nm in length, similar to those reported by ^{Luria et al. (2017}) were observed (Figure 1d).

These results indicate the presence of Tomato brown rugose fruit virus associated to tomato and chili pepper plants collected in Yurecuaro and Tanhuato, and suggest its introduction by commercial seeds produced in Israel and Jordan. The ToBRFV has only been reported in Asia and the Middle East, and there are reports that is easily mechanically transmitted and disseminated by seed (^{Dombrovsky and Smith, 2017}). Based on the results obtained, it is necessary to carry out pathogenicity tests in the future.

Conclusions

Based on the results obtained in the present study, it is established that Tomato brown rugose fruit virus is associated to commercial tomato and chili pepper crops from Yurecuaro and Tanhuato, Michoacan. These plants presented the symptoms described by ^{Luria et al. (2017}) and ^{Salem et al. (2015}).

Figure 1 Symptoms observed on tomato samples positive for ToBRFV. Fruits with yellow colourations (a), green spots and deformation (b), green striate (c) and fruits with brown spots (d). Products of 1052 bp obtained by RT-PCR using the oligonucleotides for the Tobamovirus genus (e). Maximum-likelihood tree of sequences of fragment ORF2 from samples ToBRFV with others Tobamovirus (TMV, ToMMV and ToMV) (f). Viral rods particle present in tomato samples characteristic of Tobamovirus (g).