Orchids are one of the most diverse families, with an estimated 25 to 30 thousand species, and several thousands of hybrids, both natural and artificial (^{Dressler, 1981}; Beutelspacher, 2012). In Mexico there are around 1260 known species of the family Orchidaceae (^{Hágsater et al., 2005}) and around 40% are endemic. Diseases caused by viruses are in the second place in terms of phytosanitary problems (^{Arauz, 1998}), and since orchids are long-lived herbaceous plants, this makes them more vulnerable to viral infections. Since orchids can be costly, and occasionally rare specimens, which leads to, even plants infected by viruses not being discarded, increasing the risk of infection to the others. Currently, at least 30 viruses species have been known and recorded to infect orchids, some of which can be transmitted by vectors such as the Tomato spotted wilt virus (TSWV) (^{Lawson, 2002}), but the most common ones, transmitted mechanically and via propagative material, are the Cymbidium ringspot virus (Tombusvirus), Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CymMV) (^{Freitas-Astúa, 2003}). CymMV and ORSV are the most economically important, since they reduce growth and flowering (Lawson, 2002) and in greenhouses in Mexico, they have been reported as infecting some genera (^{López-Hernández et al., 2014}). Nowadays, in the Miguel Ángel Soto Arenas (MAS) orchidarium, in the School of Science of the UNAM, plants of diverse orchid genera have been observed to present characteristic symptoms of viral infections. Many of these plants have historic, horticultural, scientific of biological value, and therefore cannot be sacrificed, but they must rather be diagnosed for their adequate handling. Therefore, the aim of this study was to determine the virus or viruses related to these symptoms via ELISA, RT-PCR and differential plants test, to describe them in the genera of orchids that have not been reported as hosts in Mexico, and using a phylogenetic analysis, determine if the infection comes from native genera.

Between April 22 and 24 of 2018, in the MAS orchidarium, samples were taken of tissues (leaves, roots and flowers) from plants with symptoms of variegation, chlorosis and foliar necrotic spots; these plants were marked and photographed. A total of 47 samples were taken from different plants. The samples were labeled and transported in a cooler to the virus lab of the Colegio de Postgraduados for processing. Out of these, we chose 12, which came from plants with more evident symptoms, and the extraction of total RNA was carried out, using CTAB (^{Sambrook and Rusell, 2001}) starting at 100 mg of plant tissue. The integrity of the extractions was verified by electrophoresis and the concentration was quantified using the Nanodrop^®. The retrotranscriptions were carried out using general primers to detect some of the most common phytopathogenic genera of viruses. For Potexvirus we used primers Potex1RC, Potex5 and Potex2RC described by ^{Van der Vlugt and Berendesen (2002}); for Tospovirus, primers BR60 and BR65 described by ^{Eiras et al. (2001}); for Potyvirus, primers NIb2F and NIb3R described by ^{Zheng et al. (2010}); and for Tomabovirus, primers TobRTup1 and TobRTdo2 described by ^{Dovas et al. (2004}). The general conditions for amplification were followed, along with the concentration of reactants recommended for each descriptor of the general primers. The products expected from the PCR were purified using the Wizard^® commercial kit, and were sequenced in Macrogen. The sequences were edited using the program BioEdit^® and compared with those reported in the GenBank. The consensus sequences were recorded in the GenBank and used to carry out the phylogenetic analyses using the maximum likelihood method (ML), based on the Kimura-2 parameter model, with a discreet Gamma distribution with 5 categories, using the free software MEGA7. For the comparison, we chose different sequences of the CymMV and ORSV from Costa Rica, Taiwan, Japan, France, Korea and New Zealand, available in the NCBI database.

The ELISA was performed using specific positive and negative ORSV and CymMV polyclonal antibodies (Agdia^®) following instructions from the manufacturer. We macerated 300 mg of plant tissue in 3 mL of extraction buffer. Each one of the 47 samples was analyzed in duplicates in order to ensure the reliability of the diagnosis. The optical density for the samples was obtained at 405 nm (OD405) in an ELISA slide reader. For the analysis with indicator plants, plant tissue positive to CymMV and ORSV was macerated individually in phosphate buffered saline, in order to mechanically inoculate Nicotiana tabacum var. xhanti, N. glutinosa, N. clevelandii, N. benthamiana, N. occidentalis, N. virginiana, N. rustica, Chenopodium quinoa., C. amaranthicolor and Datura stramonium plants. A total of three plants were inoculated for each species per virus, and as a control, a plant from each species was rubbed with water.

During the tour in the MAS orchidarium, plants were found with slight mosaic, with purple spots, or with generalized chlorosis, along with necrotic spots (Figure 1). Only in a few species of the genus Barkeria was found also variegated flowers.

These symptoms match those reported for viruses in orchids (mosaic, chlorotic rings, deformities, variegated and necrotic spots) (^{Albouy and Devergne, 2000}; ^{López-Hernández et al. 2014}), although their expression is highly variable and their severity will depend on the isolation of the virus, the host and the environmental conditions (Agrios, 2005). High temperatures and luminosity, prevalent in the greenhouses, generally favor the multiplication of the viruses and the mixture of infection by CymMV and ORSV will increase the severity (^{Yamane et al., 2008}). In plants positive for ORSV, no chlorotic rings were observed, although it is known that their appearance depends on how long the plants have been infected for, as well as on the environmental conditions (Yamane et al., 2008).

From the retrotranscriptions, amplicons were obtained from the expected fragment with the primers for Potexvirus (584 pb) and Tobamovirus (568 pb). The analyses for Tospovirus and Potyvirus were negative, and only the control samples were amplified (RNA of plants infected with TSWV and RNA of plants infected with the Papaya ringspot virus) (images not shown). The sequencing showed that the 568 pb amplicon had a similarity of 99% with Odontoglossum ringspot virus (KF85954.1), and it was found in eight of the 12 samples analyzed (Figure 2, left). The sequencing of the 584 pb fragment had a similarity of 98% with the Cymbidium mosaic virus (AJ270986.1) and it was found in only two samples (Figure 2, right).

The consensus sequence, isolation M48 (access number MK908224), from CymMV was grouped with a CymMV isolation from the Netherlands (AJ270986.1); it was separated from isolations from Costa Rica, France and Hawaii, and it was related with the isolations from Japan and Korea (Figure 3, right). Two consensus sequences of isolations M10 and M4 (access numbers MK902741 and MK902742) from ORSV were grouped with ORSV isolations from Korea and Taiwan (Figure 3, left). To date there are no cases of natural infections from viruses in wild orchids, and therefore the origin of these viruses is uncertain (^{Kull et al., 2009}). The group of isolations of CymMV and ORSV of this study with those reported in Korea, Japan and Taiwan, and far from those found in Costa Rica, suggests that the infection Could be due to the exchange between common plant material in orchid tourism (^{Pickering and Ballantyne, 2013}), and not to the existence of these viruses in Mexican ecosystems.

Figure 1 Symptoms observed during the sampling. Variegated flowers and a purple color in Barkeria scandens (A) and Barkeria whartoniana (B). Mosaic and necrotic lesions in Cattleya lobata (C). Chlorosis in Dendrobium nobile-type hybrid (D). Necrotic lesions coalescent in Sobralia macrantha (E). Purple coloring in Leochilus crocodiliceps (F), Chlorosis in Masdevalia floribunda (G). Chlorotic Stripes and necrotic stains in Rossioglossum Random Chester (H), Necrotic spots in Epidendrum jamiesonis (I), Necrotic spots in Oesrtedella sec. Epidendrum macdougallii (J). Mosaic in Guarianthe ×guatemalensis (K). Chlorotic stripes and necrotic spots Lycaste virginalis (L). 

Figure 2 Electrophoresis in 1% agarose gel of products of RT-PCR to detect the Odontoglossum ringspot virus (left) y al Cymbidium mosaic virus (right) in plants. 1 Barkeria whartoniana, 2 Lycaste híbrido, 3 B. skinneri, 4 B. scandens, 5 Bardendrum tanzanita, 6 Laelia anceps, 7 Laelia lobata, 8 Lycaste virginalis, 9 Masdevallia barlaeana, 10 Laelia anceps Mendehell, 11 Sobralia macrantha, 12 Encyclia spp. 1Kb: molecular marker of 1 kilobase; (-): water, free of nucleases. (+): plant infected with the Tobacco mosaic virus.  

With an analysis using DAS-ELISA, ORSV was found in 29 plants, CymMV in only four, both viruses were found in nine, and no virus was found in five (Table 1). In Mexico, ORSV had not been found until now in species of the genera Barkeria, Lycaste, Rossioglossum, Masdevallia, Leochilus, Stanhopea, Maxillaria and Oerstedella (Epidendrum); and CymMV in genera Dendrobium, Sobralia and Cuitlauzina. A greater incidence of ORSV was found than for CymMV in sampled plants, and despite all samples coming from plants with symptoms, some were negative, possibly due to the viral concentration of the sample taken was not high enough for any of the viruses to be found by ELISA. In general, CymMV is more stable than ORSV, and it is more prevalent (^{Khentry et al., 2006}); however, similar investigations with Phalaenopsis also found a higher proportion of ORSV than of CymMV (^{Yamane et al., 2008}). This can be explained by the origin of the plants, since it is known that the CymMV is not transmitted by seeds, unlike the ORSV (^{Pradhan et al., 2016}); but, whether the plants sampled in this investigation were generated from seeds or not, is unknown.

Figure 3 Representations of the phylogenetic relationships between the sequences of the fragments obtained with primers Potex5/Potex2cr para Cymbidium mosaic virus (rigt), and Tob Rtup1/TobRTdo2 for Odontoglossum ringspot virus (left), using the ML (Maximum Likelihood) molecular method, base on Kimura 2-parameters model and a 1000 boostrap. 

Local necrotic lesions were observed in Nicotiana tabacum var. xhanti and local chlorotic lesions in N. glutinosa, C. quinoa, C. amaranthicolor and Datura stramonium six days after inoculation (dai) with both viruses (Figure 4A-C). In Datura stramonium and N. occidentalis leaves inoculated with positive plant material only for CymMV, chlorotic rings were observed, whereas in N. benthamiana, it caused consistent systemic infection in leaf deformation and necrosis of nervations (Figure 4D-F). No symptoms were observed in control plants.

The most widely used indicator plants to diagnose CymMV are N. benthamiana, Cucumis sativus, D. stramonium, Gomphrena globosa and C. amarticolor (^{Brunt et al., 1996}), and based on the results of this study, it is also possible to use N. glutinosa and N. occidentalis. It has been corroborated that C. quinoa and C. amaranticolor are better hosts for ORSV and Datura stramonium for CymMV, although there is no selectivity (^{Cánovas et al., 2016}). However, this investigation found differences in the symptoms caused by CymMV and ORSV in D. stramonium, since the first caused smaller chlorotic lesions. The isolation of CymMV caused a systemic infection in N. benthamiana and it is known that there are isolations of this viruses capable of systemically infecting this plant, or not (^{Hsiang-Chia et al., 2009}).

This investigation reports, for the first time in Mexico, the presence of ORSV and CymMV in diverse genera of orchids with Mesoamerican species. Some species are under threat, according to the NOM-ECOL-059-2010, such as Barkeria scandens, B. skinneri, B. whartoniana, C. pendula, E. adenocaula, L. dawsonii, L. gouldiana, L. virginalis (= L. skinneri), S. tigrina and V. planifolia; and others are rare species, or under a high rate of illegal picking, and may soon be included in list of species in extinction, such as B. spectabilis, E. magnificum, L. autumnalis, L. furfuracea, O. reicheinheimii, O. Oliganthum, among others (^{Damon, 2017}).

Figure 4 Plants inoculated with ORSV 6 days after inoculation (dai): local necrotic lesions in N. tabacum var. xhanti (A); local chlorotic lesions in C. quinoa (B) and C. amaranthicolor (C). Plants inoculated with CymMV 17 dai: chlorotic rings in D. stramonium (D) and N. occidentalis (E), systemic infection in N. benthamiana (F). 

Thanks to S. in M. Patricia Olguín and undergraduate student Cekouat León for their assistance in the maintenance of the orchid collection. To the DGAPA UNAM for the funding (PAPIIT IN227319).