Roselle (Hibiscus sabdariffa L.) is known to be affected by viruses in the genus Begomovirus (Geminiviridae), which possess a circular monopartite (DNA-A) or a bipartite DNA genome (DNA-A and DNA-B), infect a wide range of plant species and are transmitted by a complex of cryptic species of whiteflies (Bemisia tabaci) (^{Brown et al., 2015}). Among the begomovirus species that have been reported in association with roselle are: Cotton leaf curl Multan virus (CLCuV), Mesta yellow vein mosaic virus (MeYVMV), Hibiscus variegation virus (HiVV, provisional name) (^{Hernández-Zepeda et al., 2007}; ^{Chatterjee and Ghosh, 2008}; ^{Muhammad et al., 2017}) and Okra yellow mosaic Mexico virus (OYMMV). The latter was recently reported in the state of Guerrero, Mexico in association with yellowing of roselle at incidences of up to 90 to 100 % (^{Velázquez et al., 2016}). This virus was also detected in Abutilon permolle, Corchorus siliquosus and Sida acuta in Yucatán, Mexico (Hernández-Zepeda et al., 2007). In addition to infecting cultivated plants, begomoviruses can also infect weeds. Weeds are a potential reservoir of economically important viruses and different host weeds are crucial in the epidemiology of begomoviruses (^{Prajapat et al., 2014}); additionally, several of them can transmit viruses by seed (^{Dikova, 2005}; ^{Sharman et al., 2009}). Sweet potato leaf curl virus (SPLCV, a DNA virus) in sweet potato (Ipomoea batatas) (^{Kim et al., 2015}), Mung bean yellow mosaic virus (MYMV, a DNA virus) in Vigna mungo (^{Kothandaraman et al., 2016}) and of Tomato yellow leaf curl virus (TYLCV, a DNA virus) in tomato (Solanum lycopersicum) (^{Kil et al., 2016}) are transmitted by seed. In Guerrero, a high incidence of roselle plants showing yellowing or mosaic were infected with OYMMV (^{Velázquez et al., 2016}) and Whitefly-associated begomovirus 3, respectively, both with bipartite genome. Additionally, different weeds associated to the crop show yellowing but it is unknown if they are hosts of these begomoviruses or if they are transmitted by seed. The purpose of this study was to determine if begomoviruses detected in roselle plants are also found in weeds associated to the crop and if they are transmitted by seed. During August and November of 2016, roselle plants of three cultivars with yellowing or mosaic symptoms were collected from a commercial field in Guerrero, Mexico. Weeds showing yellowing and foliar distortion were also collected from inside and on the periphery of the field. Asymptomatic plants of both roselle and the weeds were collected as controls, and weeds were identified at the species level. Total nucleic acids were extracted from young roselle and weed leaves with Concert ™ Plant RNA Reagent (Cat. 12322-012, Invitrogen) according to the manufacturer’s instructions. Universal primers for begomoviruses Av494 (5’-GCCYATRTAYAGRAAGCCMAG-3’) and Ac1048 (5’-GGRTTDGARGCATGHG TACATG-3’) were used to amplify a 550 bp fragment of the protein coat (CP) gene (^{Wyatt and Brown 1996}). The reaction mixture consisted of 2 μL of reaction buffer (5X Green GoTaq^®), 0.6 μL of MgCl₂ (25 mM), 0.2 μL of dNTPs (10 mM), 0.55 μL (10 μM) of each begomovirus primer, 0.05 μL (10 μM) of each 18s rRNA gene primer that amplify an 844bp fragment as an indicator of PCR effectiveness (^{Gambino and Gribaudo 2006}), 4.9 μL ultra-pure water, 0.1 μL GoTaq^® DNA Polymerase (5U/μL PROMEGA) and 1 μL DNA (20 ng / μL) in a final volume of 10 μL. PCR was done with the following conditions: initial denaturation at 95 °C/4 min, followed by 30 cycles at 95 °C/1 min, 55 °C/2 min, 72 °C/1 min and a final extension of 72 °C/10 min. The PCR products obtained were sequenced (Macrogen Inc., Korea), the sequences were edited with DNA baser (http://www.dnabaser.com/index.html) and they were compared with those from the GenBank^® database. The samples of roselle and weeds positive to begomovirus by PCR were subjected to a second analysis with specific primers for WfaBV3 and OYMMV by using the sequence of the CP of each virus (KT099127.1 and HM035059.1). Specific primers were designed with the NCBI primer-blast tool: VEM-3-F 5’-AGTCCTACGAGCAACGTCAC-3’/VEM-3-R 5’-TCTCGTACTTCGCAGCTTCC-3’ and OYMMV-F 5’-AAAGGTGAGCCGCAA TGCTA-3’/ OYMMV-R 5’- GTCGCGTAGG TCGTTCTTCA-3’, which amplified a product size of 438 and 463 bp, respectively. The reaction mixture consisted of 2 μL reaction buffer (5X Green GoTaq^®), 0.6 μL of MgCl₂ (25 mM), 0.2 μL of dNTPs (10 mM), 0.6 μL (10 μM) of each primer, 4.9 μL of ultra-pure water, 0.1 μL of GoTaq^® DNA Polymerase (5U/μL PROMEGA) and 1 μL of DNA (20 ng/μL) in a final volumen of 10 μL. PCR was performed as follows: initial denaturation at 95 °C/5 min, 40 cycles at 95 °C/30 s, 57 °C/30 s, 72 °C /30 s and a final extension cycle of 72 °C/5 min for both viruses. Three samples of roselle positive to OYMMV and three positive to WfaBV3, as well as five weeds positive to OYMMV were sequenced (Macrogen Inc., Korea). To corroborate the specificity of the viruses, tomato plants infected with Tomato severe leaf curl virus and pepper plants infected with either Pepper golden mosaic virus or Pepper huasteco yellow vein virus, respectively, were analyzed by PCR with both VEM-3-F/VEM-3-R and OYMMV-F/OYMMV-R. Seeds from each of three roselle cultivars and from five weed species tested positive to begomoviruses were collected and analyzed as follows: a) Total nucleic acids were extracted from 10 seed samples of each roselle cultivar (10 seeds per sample) and four seed samples of each weed (10 seeds per sample too) and analyzed by PCR with universal primers for begomoviruses as previously described. In addition, the weed seed was analyzed with specific primers for OYMMV. b) One hundred seeds from each roselle cultivar and 40 seeds from each of the five weeds were planted in trays containing sterile soil. To avoid contamination by a virus present on the seed coat, seeds were surfaced sterilized with 10% sodium hypochlorite and rinsed with distilled water (Kim et al., 2015). The trays were kept at 27º C and a 16 h photoperiod (^{Albrechtsen 2006}). As controls, 200 seeds of each roselle cultivar were sown from roselle plants that were negative to begomoviruses. Three months after seeding, young leaves were harvested and total nucleic acids were extracted from ten of seedlings of each roselle cultivar. In the case of the weeds, young leaves of ten seedlings of each of the five species were randomly selected at 40 days after seeding. Roselle and weed seedlings were analyzed by PCR with universal primers for begomoviruses as described previously. Additionally, young leaves of weeds seedlings were analyzed with specific primers to OYMMV. Extraction of total nucleic acids from seeds and seedlings was performed according to ^{Dellaporta et al. (1983}), with some modifications. The products were sequenced, edited and compared with those at the GenBank^® database as previously described. Total nucleic acids from young leaves of roselle seedlings were subjected to RCA with the TempliPhi 100 Amplification^® kit (Amersham Biosciences) following the manufacturer’s instructions with the purpose of enriching the circular DNA that could be present and verified by 0.8% agarose gel electrophoresis.

A total of 34 roselle plants with yellowing or mosaic and 10 with no symptoms were collected. Additionally, six weeds with yellowing and foliar distortion (Figure 1) and six with no symptoms were collected. All symptomatic roselle plants amplified the expected fragment (550 bp) for begomovirus and 26 of them had a similarity of 97-98% with Whitefly-associated begomovirus 3 (WfaBV3) (Accession number MF632074, MF632075, MF632076 and MF632077). These 26 symptomatic roselle plants were analyzed by PCR with VEM-3-F/VEM-3-R primers and all amplified the expected product of 438 bp. Sequence of the latter (Accession number MH090700) had a similarity of 98% with Whitefly-associated begomovirus 3. ^{Rosario et al. (2015}) reported this begomovirus in Bemisia tabaci and it was named VEM-begomovirus 3 because it had not previously been detected in plant species, however, the name of this species at the present time accepted by the ICTV is Whitefly-associated begomovirus 3. The remaining eight plants showed a 93-94% similarity with OYMMV (Accession number MF315084, MF315085, MF315086 and MF315087). These eight symptomatic roselle plants were analyzed by PCR with OYMMV-F/OYMMV-R primers and all amplified the expected product of 463 bp. Sequence of the latter (Accession number MH090684) had a similarity of 92% with OYMMV, a virus previously reported by ^{Velázquez et al. (2016)} in roselle cultivars in the same area. In the case of begomoviruses it has been established that a sequence with a similarity ≥ 91% with the whole genome or part of the DNA-A component, it means that it is the same species, whereas if it has a similarity <91% then it should be considered a new species (^{Brown et al., 2015}). The CP gene is the most highly conserved gene in the family Geminiviridae (^{Wyatt and Brown 1996}). This gene sequence, which effectively predicts discrete strains, species, and taxonomic lineages of begomoviruses, has been accepted by the ICTV as a desirable marker for virus identity when a full-length genomic sequence is not available (^{Brown et al., 2001}). There was no amplification with primers VEM-3-F/VEM-3R and OYMMV-F/OYMMV-R in the tomato sample infected with Tomato severe leaf curl virus nor in the pepper plants infected with Pepper golden mosaic virus and Pepper huasteco yellow vein virus, respectively. Roselle plants positive to OYMMV initially showed venial chlorosis and then a yellow mosaic that covered the entire leaf surface. In the case of the roselle plants positive for WfaBV3, a mosaic was always observed and it became more evident over time. Six weed species belonging to two families associated with roselle were identified, with the Malvaceae family having the highest number of species: Sida aggregata K., S. collina S., S. haenkeana (C.) Presl., S. acuta Burm. f., Malachra fasciata Jacq. and Euphorbia heterophylla L. These results are similar to those reported in other studies in which different begomoviruses associated with weeds belonging to the Malvaceae family have been found (^{Hernandez-Zepeda et al., 2007}; ^{Nascimento et al., 2016}). The expected fragment for begomoviruses (550 bp) was amplified from all symptomatic weed samples, as well as that of the 18s rRNA gene (844 bp) used as the internal control; the latter is essential for the detection of false negatives, DNA degradation or presence of inhibitors in the PCR (^{Gambino and Gribaudo 2006}). In Sida aggregata, S. collina, S. haenkeana, S. acuta and Malachra fasciata expected PCR product of 463 bp obtained with primers OYMMV-3-F/OYMMV-3-R was observed (Data not shown) and their sequences were 93 to 95 % similar for OYMMV. In Euphorbia heterophylla the begomovirus Euphorbia mosaic virus was detected. From the positive weeds to OYMMV, only Sida acuta had previously been reported as a host of this virus (^{Hernandez-Zepeda et al., 2007}), while, S. aggregata, S. collina, S. hankeana and Malacra fasciata are reported for the first time as hosts. Most of the weeds have a high adaptability to the environment and several of them, have been reported as hosts of a large number of begomoviruses and they are considered an important factor in epidemiological studies (^{Prajapat et al., 2014}). In addition, to know the range of alternative virus hosts is essential for designing effective virus management strategies (^{Kai-Shu et al., 2011}). Transmission of OYMMV by seed of weeds favoring its continous permanence in the field and seedlings originated from them are a potential reservoir for viruliferous whitefly colonies in the early stages of the crop. None of the weed species analyzed in the present study was positive to WfaBV3 though this virus was detected in roselle plants with mosaic. It is possible that WfaBV3 might be limited to the Hibiscus genus, like in the case of SPLCV, which was found only in plants of the Ipomoea genus from a total of 111 species belonging to 30 families (^{Kai-Shu et al., 2011}). WfaBV3 and OYMMV were not detected by PCR in the different roselle cultivars in both seeds and seedlings and only amplification of the 18s rRNA gene was obtained, so false negatives or the presence of PCR inhibitors were excluded. No amplification was obtained by RCA, which supports the previous results of non-transmission of these begomoviruses by roselle seed. These results differ from those obtained for the begomoviruses Sweet potato leaf curl virus and Mung bean yellow mosaic virus that were detected in asymptomatic seedlings by PCR; however, in the field such seedlings may be permissive and act as a source of inoculum for the infection of healthy seedlings (^{Kim et al., 2015}; ^{Kothandaraman et al., 2016}). OYMMV was detected in seed of Sida aggregata (Accession MF632078, MH090690), S. collina (Accession MF632079, MH090691), S. haenkeana (Accession MF632080, MH090692), Sida acuta (Accession MF632081, MH090693) and Malachra fasciata (Accession MF632082, MH090694) with both universal primers for begomoviruses and specific primers for OYMMV. OYMMV was detected by PCR with OYMMV-3-F/OYMMV-3-R primers in seedlings of the five weed species in different relative rate (positive seedlings/analyzed seedlings; Accession number): Sida aggregata (3/10; MH090695), S. collina (2/10; MH090696), S. haenkeana (1/10; MH090697), Sida acuta (5/10; MH090698) and Malachra fasciata (1/10; MH090699) and no symptoms were observed in them. Similarly, asymptomatic seedlings of Ipomoea batatas and Vigna mungo were positive for Sweet potato leaf curl virus and Mung bean yellow mosaic virus, respectively (Kim et al., 2015; Kothandaraman et al., 2016). Detection of OYMMV in seed and seedlings of five weed species suggests that this virus is inside the seed and it can be transmitted from one generation to another through the embryo (Kim et al., 2015). It is known that many viruses that are transmitted by seed do so only in certain plant species and not others, such as the TYLCV that is transmitted by seed of Solanum lycopersicum (^{Kil et al., 2016}) but not of Nicotiana benthamiana (^{Rosas-Diaz et al., 2017}). The latter could explain the detection of OYMMV in weed seeds but not in roselle WfaBV3 was detected in roselle plants exhibiting mosaics, whereas OYMMV was associated to plants showing yellow mosaics. Five species of Malvaceae considered as weeds were identified to harbor OYMMV and it was transmitted by seed in all them. WfaBV3 was not detected in weeds and there was no transmission of this virus or OYMMV to roselle seeds nor seedlings. To our knowledge, this is the first report of four new weed hosts of OYMMV and its transmission by seed.

Figure 1 Symptoms in weeds associated to a roselle crop. A) Sida acuta, B) S. haenkeana, C) S. collina, D) S. aggregata, E, F) Malachra fasciata.