In Mexico, the tomato (Solanum lycopersicum) is the second most important crop, after the chili pepper, with a production of 3.2 million tons in 2017, and the diseases caused by begomoviruses can decrease its production. Begomoviruses are considered emerging pathogens from tropical and subtropical crops, they may have one or two circular single-stranded DNA molecules (designed as component A and B, respectively), of approximately 2.6 kb each, and are made up of two joined incomplete isometric particles. Component B depends on A for viral replication, both have a common region (CR) of approximately 200 bp, and they are necessary for cause infection. In Africa, Asia, Australasia and Europe, most begomoviruses have a monopartite genome, and some have a bipartite genome, whereas in America are mostly bipartite. In nature, begomoviruses are not transmitted mechanically, but rather do so eficiently by the complex of species of Bemisia tabaci in a persistent manner. The outbreaks of diseases caused by these viruses in tomato plants have been attributed to the introduction and spreading of diverse biotypes of B. tabaci that are polyphagous and easily colonize the crop (^{Brown et al., 2015}).

Most of the seed-transmitted viruses have an RNA genome. However, some begomoviruses have been reported which can also be transmitted in this way (^{Ortega et al., 2019}). High technology tomato-producing greenhouses allocate almost all of their entire production for export. In greenhouses located in Atlacomulco, Mexico State, the Impatiens necrotic spot virus (INSV), Tobacco mosaic virus (TMV) and Pepino mosaic virus (PepMV) have been found in plants showing mosaics and small necrotic lesions in the first case, as well as the Mexican papita viroid (MPVd) in plants with internodal shortening and reductions in the size of leaflets in the apical area. In recent months, incidences have been observed in 60 to 90% of plants, which included, not only mosaics in certain cases, but also leaf curling, spotting, yelowing, and, in severe situations, reduction in the size of the fruit, typical symptoms caused by begomoviruses, causing economic losses due to the reduction in the number of marketable fruits. Considering that plants with this new symptomatology have tested negative for INSV, TMV, PepMV and MPVd, the purpose of this investigation was to identify the related begomoviruses, to establish if they are seed-transmitted, to determine the species of whitefly present in the greenhouses, and confirm if this insect is a virus carrier.

Foliar tissue was collected from the top of tomato plants with the symptoms described above. At the base of the symptomatic plants, Moëricke traps containing water + liquid soap (1000:1 v/v) were placed in order to collect insects in 15 mL Falcon tubes containing 96% ethanol every week for one month. DNA was extracted from plants following the protocol by ^{Dellaporta et al. (1983}), from 0.1 g of the collected tissue; for whitefly DNA was extracted from 5 adult individuals with CTAB 2%. In the latter case, the insects were washed four times with distilled water and macerated in a 1.5 mL tube containing 500 µL of CTAB. The tubes were mixed in a vortex for 2 min, incubated at 65 °C in a thermoblock for 15 min, kept for 10 min at room temperature to add 500 µL of chloroform, and then mixed by inverting for 10 min. They were centrifuged at 13,000 rpm for 10 min and 400 µL of the supernatant were placed to a new 1.5 mL tube, then added 200 µL of isopropanol and incubated at -20 °C for 20 min. After being centrifuged at 13,000 rpm for 15 min at 4 °C, the supernatant was poured to add 1 mL of absolute ethanol and centrifuged again at 13,000 rpm for 10 min at 4 °C; the alcohol was poured, the pellet was dried at room temperature and resuspended in 50 µL of injectable water. In both cases, DNA concentration and purity was quantified in Nanodrop^® and its integrity was corroborated by gel electrophoresis in 1% agarose gel.

For plants, the universal primers for begomoviruses AV494 (5’-GCCYATRTA YAGRAAGCCMAG-3’)/AC1048 (5’-GGRTTD GARGCATGHGTACATG-3’) were used, which amplify a fragment of ~550 bp of the coat protein (^{Wyatt and Brown, 1996}).

To determine the species of whitefly, a PCR was carried out with primers LCO1490 and HCO2198 reported by ^{Vrijenhoek (1994}), which amplify a fragment of ~710 bp of the mitochondrial cytochrome C oxidase subunit I gene (COI). The fragments obtained for begomovirus and COI were sequenced and the sequences obtained were compared and registered in the GenBank^® database. In addition, to find out if the whiteflies collected were carriers of begomoviruses, the DNA obtained from these insects was analyzed by PCR with primers AV494/ AC1048. The amplified product was sequenced and compared with the GenBank^® database.

Seeds were obtained from mature fruits from plants tested positive for begomovirus, as well as from asymptomatic plants that were negative by PCR. For this, the fruit was cut in half, the seeds were extracted and placed in a container to wash them with tap water. They were later disinfested with 10% sodium hypochlorite for 3 min, washed five times with distilled water and dried on sterilized paper towels for 10 min.

DNA was extracted from 10 seeds taken from plants that tested positive, and another 10, from plants that tested negative for begomoviruses, respectively, following the protocol by ^{Dellaporta et al. (1983}). Afterwards, they were analyzed by PCR with universal primers for begomoviruses AV494/AC1048 (^{Wyatt and Brown, 1996}).

On the other hand, 50 seeds taken from fruits positive for begomovirus were planted in germination trays with sterile soil; as a control 50 seeds taken from fruits of plants that tested negative were used. In both cases, the trays were kept in a growth chamber, at 27 °C and a photoperiod of 16 h. True leaves were collected 14, 21 and 28 days after planting, and DNA was extracted (^{Dellaporta et al., 1983}) to carry out PCR.

The main symptoms observed in the plants collected were reduction in growth and leaf curling, as well as mosaics and chlorotic spots in some cases (Figures 1A-1C), which have been associated to begomovirus infection. Previously, plants were sporadically found with mosaic, distributed at random throughout the greenhouse; some tested positive for Impatiens necrotic spot virus, Tobacco mosaic virus or Pepino mosaic virus and negative for Cucumber mosaic virus and Tomato spotted wilt virus as well as a higher number (~30%) of plants with a shortening of internodes and small leaflets in the top, which tested positive for Mexican papita viroid (data not shown). The presence of a high percentage of plants with a severe reduction in growth and leaf curling suggested the presence of a virus or viroid different to those analyzed previously.

The adult whiteflies collected in Moëricke traps were morphologically similar, and were recovered practically intact (Figure 1D). The expected product for begomovirus in the tomato symptomatic plants was obtained (Figure 1E). The sequences of the products obtained (Accession number MK325267 and MK325268) had an identity of 99% with the Tomato severe leaf curl virus (ToSLCV). In Mexico, the ToSLCV was first reported in tomato crops in Baja California Sur (^{Holguín et al., 2003}), and then in Rioverde, San Luis Potosí and Xochitepec, Morelos. Only the begomoviruses Chino del tomate virus and Pepper Huasteco virus (currently Pepper huasteco yellow vein virus) had been reported in tomato grown under greenhouse conditions in Mexico.

The expected fragment of ~710 bp from whiteflies analyzed by PCR with primers of the COI gene was obtained (Figure 1F) and its sequence had an identity of 99% with Bemisia tabaci (access numbers MK290856, MK290857, MK290858 and MK290859). B. tabaci is the main vector species of begomoviruses wordlwide, and generally the symptoms of yellowing, leaf curling and growth reduction associated to the presence of this vector in tomato crops are considered an indication of the infection by these viruses.

Figure 1 Symptoms in tomato plants associated to begomovirus infection. A. Severe plant growth reduction and reduced size of terminal leaves. B. Leaf curling. C. Chlorotic spots. D. Adult whitefly collected in Moëricke traps. E) PCR products obtained with universal primers for begomoviruses AV494/AC1048 (~550 bp) in tomato plants collected in a greenhouse. Lane 1: Positive control (plant infected with Okra yellow mosaic Mexico virus); Lane 2: Negative control (water); Lanes 3-4: Symptomatic plants; Lane 5-6: Asymptomatic plants; Lane M: 100 bp DNA ladder (Promega®). F) PCR products obtained with primers LCO1490 and HCO2198 (~710 bp) from whiteflies DNA. Lane 1: Positive control (adult of Bemisia tabaci); Lane 2: Negative control (water); Lanes 3-6: Adults collected in traps near plants showing mosaic and leaf curling; Lane M: 100 bp DNA ladder (Promega®). 

In different studies, large populations of B. tabaci are related to infections of several species of begomoviruses in tomato crops in Mexico, which contrasts with observations in the present study, where the number of whiteflies was low.

The adult whitefly amplified the expected fragment for begomoviruses (~550 bp) (data not shown) and its sequence had an identity of 99% with ToSLCV (Accession numbers MK325269, MK325270 and MK325271). This correspondence between the virus found in tomato plants and associated whiteflies has been reported for Tomato golden mottle virus.

No begomoviruses were detected by PCR in seeds from tomato fruits obtained from symptomatic plants, nor in seedlings in the dates of evaluation. Certain begomoviruses which are not transmitted by seed of the crop, but rather in weeds, such as Okra yellow mosaic Mexico virus, which is not transmitted by roselle seed (Hibiscus sabdariffa L.), but it can transmit in Sida collina, S. acuta, S. haenkeana and Malacra fasciata seeds (^{Ortega et al., 2019}). These results, as well as the detection of ToSLCV in whiteflies and the high incidence of symptomatic plants, suggest that the population of B. tabaci found is efficient in the transmission of this begomovirus.

Based on the results obtained, it is the first report of Tomato severe leaf curl virus associated to tomato plants under greenhouse conditions, and its presence in adults of B. tabaci in Atlacomulco, Mexico State.