In Mexico, the tomato (Solanum lycopersicum) is one of the most important horticultural crops, with a production of 3.7 million tons in 2024. Currently, 66% of the country’s tomato production takes place under covered cultivation, the rest is produced in open fields and approximately 5% is exported to the United States of America (SIAP, 2025). Viruses are an important limitation for this crop, since it directly affects fruit production and quality (Rojas et al., 2005a). The main viruses that affect tomato belong to the Bromoviridae, Potyviridae, Virgaviridae, Alphaflexiviridae, Secoviridae, Closteroviridae, Bunyaviridae and Geminiviridae families (Afouda et al., 2013; Hanssen et al., 2010; Massumi et al., 2009). Within the latter one, viruses from the Begomovirus genus are widely distributed in tropical and subtropical areas (Rojas et al., 2005b) and they are persistently transmitted by the whitefly (Bemisia tabaci) (Polston et al., 2014). The begomoviruses are composed of one or two circular single-strand DNA molecules of approximately 2.6 kb each, surrounded by a twinned icosahedral capsid, approximately 18 nanometers in diameter (Brown et al., 2015). The begomoviruses detected thus far in tomato crops in Mexico are the Begomovirus coheni (tomato yellow leaf curl virus), B. solanumflavi (the tomato yellow leaf curl China virus), B. capsicumhuastecoense (pepper huasteco yellow vein virus), B. solanumaureivariati (tomato golden mottle virus) and the

B. solanumseveri (tomato severe leaf curl virus) (Brown and Idris, 2006; González-Pérez et al., 2024; Idris et al., 2007; Zuñiga-Romano et al., 2019).

On the other hand, to date there are at least seven reported viroids of the Pospiviroidae family that infect tomato naturally: Pospiviroid exocortiscitri (citrus exocortis viroid), P. latenscolumneae (columnea latent viroid), P. parvicapsici (pepper chat fruit viroid), P. fusituberis (potato spindle tuber viroid), P. apicimpeditum (tomato apical stunt viroid), P. chloronani (tomato chlorotic dwarf viroid) and P. machoplantae (tomato planta macho viroid) (Zhang and Groth, 2024).

It has been proven that the coinfection of different pathogens in the same host does exist, such as of phytoplasmas and begomoviruses in eggplant (Solanum melongena) (Venkataravanappa et al., 2018) and the simultaneous infection of viruses such as Potyvirus yituberosi (potato virus Y), Crinivirus flavisolani (potato yellow vein virus) y Potexvirus ecspotati (potato virus X) in potato (García et al., 2019). Mixed infections between viruses and viroids have also been reported in grapevine (Vitis vinifera), such as Foveavirus rupestris (grapevine stem pitting-associated virus), Marafivirus syrahense (grapevine Syrah 1 virus) and the viroids Hostuviroid impedihumuli (hop-stunt viroid) and Apscaviroid alphaflavivitis (grapevine yellow speckle viroid 1) (Al Rwahnih et al., 2009; Giampetruzzi et al., 2012; Jo et al., 2015; Seguin et al., 2014). Up to 12 different viruses and viroids have been identified in tomato (Pospiviroid parvicapsici and P. latenscolumneae) (Choi et al., 2020). Co-infections can cause greater severity, which can lead to a strong reduction in production, with a significant effect on the crop yields (Hameed et al., 2014).

In Mexico, mixed infections have been found, caused by two or more species of begomoviruses or with viruses of another genus in the chili pepper crop (Anaya et al., 2003; Reveles et al., 2012). In the tomato crop under greenhouse conditions, the co-infection of two viroids has been reported: tomato planta macho viroid (Pospiviroid machoplantae), for some time erroneously known as the Mexican papita viroid, and the tomato chlorotic dwarf virus (P. chloronani) (Ling and Zhang, 2009). Lugo et al. (2011) found the co-infection of tomato yellow leaf curl virus (Begomovirus coheni) and squash leaf curl virus (B. cucurbitapeponis), whereas González-Pérez et al. (2024) reported the co-infection of tomato mosaic virus (Tobamovirus tomatotessellati), tomato golden mosaic virus (Begomovirus solanumaureimusivi) and Pepper huasteco yellow vein virus (B. capsicumhuastecoense).

In a high-technology tomato production greenhouse located in Atlacomulco, State of Mexico, an incidence was observed of up to 90% of plants with mosaic, inter-venial chlorosis, deformation, necrosis, blistering and leaf roughness in different stages of crop development, which affect fruit yield and quality. This situation has appeared in different crop cycles, therefore the aim of this investigation was to identify viruses or viroids associated with these symptoms, as well as the species of whitefly in question.

Collect of plant material and whiteflies. Every month for two years, leaf tissue was collected from symptomatic and asymptomatic tomato plants showing the alterations mentioned earlier in a high-technology greenhouse located in Atlacomulco, State of Mexico. The sampled plants were of different ages, and the number of collected plants every month varied, depending on the frequency with which the symptoms appeared. Likewise, in each sampling, leaves were taken from plants that showed no symptoms as a reference. The samples were wrapped in absorbent paper and placed in plastic bags to be transported to the laboratory, where they were stored at 4 °C until processing. In the base of the plants with symptoms, Moëricke traps were placed, containing water + liquid soap (1000:1 v/v), to collect insects in 15 mL Falcon tubes with 96% ethanol every week, from which whitefly adults were taken for their molecular identification by PCR and sequencing.

Detection of viruses and viroids. The samples were analyzed using ImmunoStrips® (Agdia Inc.) for impatiens necrotic spot virus (Orthotospovirus impatiensnecromaculae), tomato spotted wilt virus (O. tomatomaculae), tobacco mosaic virus (Tobamovirus tabaci), tomato mosaic virus (T. tomatotessellati), pepino mosaic virus (Potexvirus pepini) and cucumber mosaic virus (Cucumovirus CMV), following the manufacturer´s instructions. Additionally, total nucleic acids were extracted from 0.1 g of leaf tissue from plants with and without symptoms, following the protocol by Dellaporta et al., (1983) from all the samples analyzed in the study period. In the case of whitefly, the method used was CTAB 2%, from four samples, each composed of five adult individuals, following the methodology described by Zuñiga-Romano et al., (2019). RT-PCR and PCR (GoTaq® Flexi DNA Polymerase, Promega®) were carried out with universal oligonucleotides Pospiviroid For and Pospiviroid Rev for viroids of the Pospiviroid genus (Bostan et al., 2004). For retrotranscription, the M-MLV RT (Promega®) enzyme was used for 1 h at 42 °C, and subsequently, at 95 °C for 3 min to denature the enzime; PCR was carried out with 30 cycles of 30 s at 92 °C, 30 s at 62 °C and 90 s at 72 °C with a final extension of 10 min at 72 °C; for viruses of the Begomovirus genus, primers AV494 and AC1048 were used (Wyatt and Brown,1996) with the following amplification conditions: 35 cycles of 1 min each at 92 °C, 20 s at 60 °C and 30 s at 72 °C. The amplified products were separated and visualized by 1% agarose gel electrophoresis with GelRed® Nucleic Acid Stain 10,000X, Biotium, as an intercalating agent.

Identification of whitefly. In order to identify the whitefly species, a PCR (GoTaq® Flexi DNA Polymerase, Promega®) was carried out with oligonucleotides LCO1490 and HCO2198 (Folmer,1994), which amplify a ⁓710 bp fragment of the mitochondrial gene of subunit I of cytochrome C oxidase (mtCOI). Likewise, in order to find out whether the whiteflies were begomovirus carriers, the four samples composed of five adults were analyzed using PCR under the conditions mentioned earlier for these viruses. The amplified fragments were purified using Wizard® SV Gel and PCR Clean-Up System (Promega®) following the manufacturer’s instructions and nine pospiviroid and eight begomovirus samples were sequenced in Macrogen®. The obtained sequences were compared with those in GenBank® and incorporated into this database.

Phylogenetic analysis. For the Begomovirus genus, the phylogenetic tree was created in MEGAX, using the Neighbor-Joining method and the Tajima-Nei model, with 1,000 Bootstrap replications. As an external group, the alfalfa leaf curl virus (Capulavirus medicagonis) was used. For the case of the Pospiviroid genus, the phylogenetic tree was created using the Tamura 3-parameter model, and Coleus blumei viroid 1 (Coleviroid alphacolei) was used as an external group.

The symptoms of mosaic, intervenial chlorosis, leaf deformities and necrosis, blistering and roughness in tomato plants were associated to Pospiviroid machoplantae, Begomovirus solanumseveri or the coinfection of both pathogens. No symptom was found to be associated with any of the pathogens found or with their co-infection. Bemisia tabaci is the species of whitefly found in tomato crops as a possible carrier of the begomovirus. The asymptomatic condition observed solely in the infection of the planta macho viroid must be studied in order to understand the implications it can have in its spreading, distribution and management within the greenhouse. This is the first report of the natural co-infection of tomato planta macho viroid with tomato severe leaf curl virus in tomato.

Conflict of interest. The authors declare to have no conflict of interest regarding the research, authorship and/or publication of this Article.

Acknowledgements. To the Consejo Nacional de Ciencia y Tecnología (National Science and Technology Council - CONACYT) for the scholarship granted to the main author. To the Colegio de Postgraduados’ Graduate Program in Phytosanitary Sciences-Plant Pathology, for the opportunity to pursue graduate studies.