Introduction

The okra, bhendi, angú or gombo (Abelmoschus esculentus L. Moench, Malvaceae family), an African originated crop, is a valuable vegetable in tropical and subtropical regions. This vegetable is most popular in India, Nigeria, Pakistan, Cameroon, Iraq and Ghana, and other countries. India is the largest producer of okra where it is widely grown throughout the year and occupies the first place with 73.25% of total world production (Biswas et al. 2018). Okra is considered an important component of balanced foods due to its dietary fibers and amino acid composition that is rich in lysine and tryptophan (Hughes 2009). Its fruits are harvested immature and are commonly consumed in salads, soups and stews. The roots and stems are used to clean sugarcane juice during the preparation of brown sugar, and the seeds have also gained a lot of interest for their oil (30–40%) and protein (15–20%) content (Gemede et al. 2015; Shetty et al. 2013). Okra also contains considerable amounts of iron, calcium, manganese, magnesium and vitamins A, B, C and K. It has been found to have several ethno-pharmacological and medicinal properties against cancer, high cholesterol and diabetes mellitus (Mishra et al. 2017; Sabitha et al. 2011).

In Mexico, the interest for this vegetable is commercial and the production is mainly exported to the USA, Canada and Japan. The species is cultivated in several tropical and subtropical states such as Guerrero, Michoacán, Morelos, Tamaulipas and Yucatán. The annual production was estimated at 33,129 tons harvested from a total area of 3518 hectares (SIAP 2019). The production and cultivated area of okra has been fluctuating particularly in the state of Guerrero, due mostly to viral diseases, thus reducing the economic value of the crop. Virosis has been reported to be caused by Okra yellow mosaic Mexico virus (OYMMV), initially named Okra yellow mottle virus (De La Torre-Almaráz et al. 2004). In addition to the detrimental effect on yield, viral diseases reduce the quality of the fruits, which compromises their exportation. OYMMV has been one of the main causes of reducing the crop in the states of Guerrero and Morelos (Díaz-Franco et al. 2007).

The Begomovirus genus includes the largest number of species in the Geminiviridae family, currently established with 409 species recognized by the International Committee on Taxonomy of Viruses (ICTV) (https://talk.ictvonline.org/taxonomy/). Begomoviruses infect only dicotyledonous hosts. In the tropical and subtropical region, begomoviruses have emerged as major threats to the cultivation of many economically important crop species, such as radishes, tomatoes, cotton, among others (Fauquet et al. 2008). The cultivated okra is susceptible to at least 27 begomoviruses, of which Yellow Vein Mosaic Disease (YVMD) and Okra Enation Leaf Curl Disease (OELCD) affect its production, yield and quality most severely (Mishra et al. 2017; Sanwal et al. 2016; Venkataravanappa et al. 2013). Begomoviruses are transmitted by the whitefly Bemisia tabaci, a complex of cryptic species (Brown 2007; Venkataravanappa et al. 2015). Some begomoviruses are transmitted by mechanical inoculation, although most require an Agrobacterium-mediated transfer of cloned genomic DNA or biolistic supply of cloned genomic DNA for experimental transmission (Rojas et al. 2005).

Begomoviruses have monopartite or bipartite genomes and are found in the Old World (both genome types) and in the New World (mostly bipartite genomes, including a recently described monopartite genome virus) (Brown et al. 2000, 2015). The bipartite begomovirus genomes consist of two components, called DNA-A and DNA-B, each one of 2.5–3 kb (Brown et al. 2015). The DNA-A component encodes the factors necessary for viral encapsidation, replication and suppression of host defense. Meanwhile, DNA-B encodes essential factors for viral movement, host range determination and symptom expression in host plants (Rojas et al. 2005). The DNA-A and DNA-B components share approximately 200 nucleotides within the intergenic region called common region (CR) (Hanley-Bowdoin et al. 1999).

Over the past decades, virus detection and identification has been attempted with microscopy techniques, serological methods, cross-reaction of antibodies, host plants and type of vectors, but these methodologies have various disadvantages such as poor specificity, difficulty in separating closely related viruses, excessive costs and long experimentation periods (Rojas et al. 2005). However, the development of technologies such as PCR sequencing, rolling circle replication (RCA) (Haible et al. 2006; Inoue-Nagata et al. 2004) and next generation sequencing has allowed a remarkable advance in the demarcation and classification of virus species. Thus, the present study aimed to identify viruses as potential causal agents involved in the yellow mosaic in okra crop by means of transmission tests and DNA-A component sequencing.

Materials and methods

Plant sampling

Plant materials with yellowing and/or mottled symptoms were collected from okra crops in Iguala, Guerrero, Mexico, in two different years (1999 and 2007). The incidence of the disease ranged from 6 to 46%, with a tendency to increase towards the stages of formation and ripening of the fruits. The collected plants were kept in greenhouse conditions to carry out the different tests of transmission and identification of vectors.

Mechanical transmission

Symptomatic okra leaves were macerated in 1 M potassium phosphate buffer, pH 7. Next, the macerate was scrubbed on healthy leaves of okra plants 30 days after emergence, previously sprinkled with 600 mesh carborundum. Similarly, the mechanical transmission test was performed on leaves of the following indicator plants: Chenopodium album, Ch. amaranticolor, Ch. quinoa, Cucurbita pepo, Nicotiana benthamiana, N. clevelandii, N. glutinosa, N. occidentalis, N. rustica, Capsicum annuum and Datura stramonium, and in leaves of plants sensitive to geminivirus: Lycopersicon esculentum, Phaseolus vulgaris, Vicia faba, Abelmoschus esculentus, Gossypium hirsutum and Hibiscus sabdarifa.

Seed transmission

Okra seeds collected from healthy and symptomatic plants (150 and 50 seeds, respectively), obtained from the same area of the State of Guerrero, were sown under greenhouse conditions. The seedlings were maintained and observed for 30 days.

Graft transmission

Buds from symptomatic okra plants were grafted into 8 healthy three-month-old okra plants and into 5 Nicotiana occidentalis plants. The grafted plants were covered with a plastic bag to maintain relative humidity and kept for 30 days in the greenhouse at a temperature of 25–30 °C and free of vector insects.

Vector transmission

A wooden cage containing 10 okra seedlings of two true leaves was used to introduce whiteflies, captured from the same infected field of okra. Symptomatic okra plants were also included to allow whiteflies feed and acquire the virus. The greenhouse temperature was maintained between 35 and 40 °C during the day and between 25 and 30 °C during the night.

Vector identification

The species involved in the transmission of the virus causing the symptoms described in okra was identified in pupae of the fourth instar of whiteflies collected from the infected crops. For this purpose, a rinse with lactophenol was performed, heating for 5 min; and the pupae were then stained with acid fuchsine (0.5 g of acid fuchsine in 100 mL of 70% alcohol) and covered with coverslips. Subsequently, the assemblies were heated for 5 min. Fifty assemblies were prepared, and the preparations were observed under a light microscope La A1 (Zeiss, Germany), using the keys of Martin (1987).

DNA isolation and geminivirus detection

Total DNA extraction was performed from leaves of healthy and symptomatic okra plants retrieved from the field using the method of Dellaporta et al. (1983). DNA quality was determined by 1% agarose gel electrophoresis and quantified by spectrophotometry (ND-1000 Thermo scientific, USA). For plants analyzed in the first sampling, several genomic regions of viruses were amplified using specific primers for geminivirus. The primer pairs PAL1v1978/PAR1c496 and PBL1v2040/PCRc (Rojas et al. 1993), 260/261, 240/241, 423/424 and 425/426 (Torres-Pacheco et al. 1996) were evaluated in order to detect any virus already reported in Mexico. For the plants in the second sampling, the primer pairs used were PAL1v1978/PAR1c496 and OYMMSA-R1/OYMMSA-FI (Table 1); this last pair was designed for this study.

Table 1 Primers and PCR products used to detect geminivirus in okra and chili
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The PCR reaction mixture contained 100 ng of DNA, 1.5 U of DNA Taq polymerase (Gibco, USA), dNTPs (200 µM each), MgCl2 (2.5 mM), reaction buffer 1x, primers (10 pM each) and nuclease-free water, adjusting to a final volume of 25 µL. A DNA Thermal Cycler 480 (PerkinElmer, USA) was used, and the amplifying conditions were as follows: for the PAL1v1978/PAR1c496, 423/424, 425/426 and 260/261 pairs, one cycle of 94 °C for 5 min; 30 cycles (94 °C for 1 min; 50 °C for 1 min; 72 °C for 2 min) and one final extension cycle at 72 °C for 5 min, was applied. For the primer pair 240/241, the same program was used, except the alignment temperature (45 °C). For the OYMMSA-R1/OYMMSA-FI primer pair, the alignment conditions were 52 °C for 2 min, and for PBL1v2040/PCRc1, the primer alignment was 55 °C for 2 min. The PCR products were electrophoresed on 1.2% agarose gels, stained with ethidium bromide and documented with a Kodak ID system. DNA from chili plants (Capsicum annuum L.) affected by geminivirus, provided by the Diagnostic Laboratory of the General Directorate of Plant Health, was included as a reference in the PCR assays. The negative control consisted of molecular grade water. 1 Kb (Gibco, USA) molecular weight marker was used as a reference. Primer pairs PAL1v1978/PAR1c496 (Rojas et al. 1993) and OYMMSA-R1/OYMMSA-FI were used to amplify and sequence the DNA-A component.

DNA-A component sequencing and assembly

DNA-A component sequencing was carried out by Macrogen (Seoul, Korea) using the 1100 bp fragment obtained by the pair primer PAL1v1978/PAR1c496, and approximately 1550 bp fragment obtained by the pair primer OYMMSA-R1/OYMMSA-FI, which were designed from the ends of the 1100 bp fragment referred above, using the Primer3 program (Koressaar and Remm 2007). For the editing and assembly of the sequences, BioEdit software (Hall 1999) and MEGA3 (Kumar et al. 2004) software were used. The sequences obtained were deposited in the Genbank-NCBI database (Table 2).

Table 2 Complete DNA-A genome sequences used for demarcation and phylogenetic analyses
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Pairwise alignment and demarcation analyses

The DNA-A sequences were compared against public databases using the BLASTn/ “non-redundant nucleotide” tool to identify species whose members have more similar sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The demarcation of the new species and the new strain was evaluated based on the sequence identities of the complete DNA-A genomes, applying the demarcation threshold criteria of 91% and 94% for begomovirus species and strains, respectively (Brown et al. 2015). For this purpose, Muscle, ClustalW and Mafft algorithms implemented in the SDTv1.2 software (Muhire et al. 2014) were used. The three complete DNA-A sequences obtained in this study, together with all the sequences belonging to the species Okra yellow mosaic Mexico virus and Chino del tomate virus (to which the new sequences belong) reported for Mexico (October 2019), were analyzed and compared. Also, two sequences of Sida mosaic Sinaloa virus were used because of their phylogenetic proximity as identified by BLASTn search.

Phylogenetic analysis

The 15 DNA sequences indicated in Table 2 were aligned using the ClustalW tool implemented in MEGA X (Kumar et al. 2018). The most appropriate nucleotide substitution models were chosen using the tool implemented in MEGA X. The maximum likelihood and neighbor-joining phylogenetic trees were generated using MEGA X, and the internal branches were evaluated by 1000 bootstrap replications.

Recombination analysis

The recombination analysis was carried out to detect putative recombination events and possible parents in the sequences obtained in the present study. The multiple sequence alignment file of all begomoviruses reported for Mexico (https://talk.ictvonline.org/ictv_wikis/geminiviridae/m/files_gemini/7195) was exported to the RDP4 version 4.97 software, which includes seven automated algorithms (RDP, GENECONV, Bootscan, Maxchi, Chimaera, SiSscan and 3Seq). The most acceptable cut-off value corrected by Bonferroni (P = 0.05) was applied to estimate recombination events (Martin et al. 2015). Each event was verified from the distribution plots of the breaking point and comparing the UPGMA phylogenetic trees generated with genetic regions of major and minor parents. To exclude the possibility of false positives in the detection of recombination, only recombination events detected by five or more methods were considered as significant.

Results

Disease description

Symptoms of a disease in mature leaves and young shoots were recorded in okra cultures in Iguala, state of Guerrero, Mexico; these symptoms consisted of yellow mosaics with shortening of the leaf lamina and white mosaics, while the fruits presented chlorotic mottling (Fig. 1).

Fig. 1
figure 1

Symptoms of mosaic and chlorotic mottling on leaves and fruits of okra

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Transmission tests

Seed and mechanical transmission tests of the disease to indicator and sensitive plants were negative, since the symptoms observed in okra plants in the field were not reproduced. However, virus transmission was possible by grafting and by vectors, since the typical symptoms of the disease were observed after 45 days under greenhouse conditions.

Vector identification

Of the 50 assemblies, Trialeurodes vaporariorum West. (greenhouse whitefly) was identified. The observed larvae were characterized by presenting typical submarginal row of papillae, caudal mushroom and basiform orifice.

PCR amplification and sequence comparison

Initially, geminivirus detection tests were performed by the primers that were available for Mexican geminivirus for some crops (Rojas et al. 1993; Torres-Pacheco et al. 1996). The results obtained indicated that the viruses isolated were geminiviruses although different from those previously reported (before 2010, date of the last determinations). Using primers designed in the present study, OYMMSA-R1 and OYMMSA-FI, a 1550-bp genomic fragment, corresponding to the DNA-A component, was amplified. Meanwhile, the primer pair PBL1v2040/PCRc1 amplified a partial region of ≈0.6 kb of the DNA-B component, corresponding to a section of the BL1 reading frame (involved in infectivity) and the intergenic region (Hanley-Bowdoin et al. 1999).

The complete DNA-A component sequences of the three isolates were made up of 2609 nucleotides. The sequences were deposited in Genbank database under accession numbers “AY751753,” “HQ020409” and “HQ116414.” The DNA-A genome of the three isolates consists of five open reading frames (ORF), of which one is in the sense orientation (+) and four are in antisense orientation (−); the ORFs had similar sizes between species (Fig. 2; Supplementary Data S1). The DNA-A component of the first isolate (AY751753) was compared against the available nucleotide sequences database using the BLASTn tool, and the search results showed a similarity of 89.76% to Chino del tomate virus (accession No. DQ347945.1), its closest relative. The second begomovirus (HQ020409) was found to be 93.76% similar to the DNA-A component of Okra yellow mosaic Mexico virus (HM035059.1). The third isolate (HQ116414) was found to be 94.6% similar to the DNA-A component of the Okra yellow mosaic Mexico virus (HM035059.1). Isolates 2 (isolate 13–14) and 3 (isolate 15–16) share a level of similarity of 99.04%.

Fig. 2
figure 2

ORFs composition of complete DNA-A component of three okra begomovirus isolates. a Okra mottle Mexico virus (accession No. AY751753), b Okra yellow mosaic Mexico virus (accession No. HQ020409), c Okra yellow mosaic Mexico virus (accession No. HQ116414). AV1: Coat protein. AC1: Replication initiator protein. AC2: Transcription activator protein. AC3: Replication enhancer protein. AC4: Symptom expression protein. The arrows indicate the orientation of ORFs

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The identity percentages between all the sequences obtained in this study and those reported in Genbank for Chino del tomate virus and Okra yellow mosaic Mexico virus were pairwise compared following the guidance proposed by Brown et al. (2015) and applying Muscle, ClustalW and Mafft algorithms implemented in the SDTv1.2 software (Muhire et al. 2014). The results indicated that the three virus isolates studied here are considered as follows: (1) isolate 15–16 belongs to an already reported strain of the OYMMV species; (2) a new separate viral strain (isolate 13–14) of the OYMMV species whose accepted name is Okra yellow mosaic Mexico virus—[Mexico:Isolate 13–14:2010]; and (3) a new viral species separated and tentatively named Okra mottle Mexico virus, OMoMV—[Mexico:2005]. The phylogenetic analysis revealed that the DNA-A component of the new species, reported here, was located in a separate and distinct clade from other known Mexican begomoviruses, and that the relative close species is Chino del tomate virus (Fig. 3).

Fig. 3
figure 3

Phylogenetic tree based on complete sequences of the DNA-A component of the related begomoviruses Chino del tomate virus, Okra yellow mosaic Mexico virus and Sida mosaic Sinaloa virus reported for Mexico. The maximum likelihood method and the TN93 + G substitution model were applied. The numbers in the nodes represent the bootstrap values estimated from 1000 repetitions. The key in parentheses indicates the sequence accession number in Genbank. The sequences generated in the present study are preceded with a full black rectangle

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By individually comparing the predicted amino acid sequences for the different inferred proteins from the new OMoMV against their relatives begomoviruses (Supplementary Data S2), ORF AV1 was found to be more similar to that of Chino del tomate virus (accession No. AF101476, DQ885456, AF226664 and AF226665), while ORFs AC1 and AC4 were more similar to those of Chino del tomate virus (accession No. AF101476). ORF AC2 was more similar to that of Okra yellow mosaic Mexico virus (accession No. HQ116414, HQ020409 and HM035059), and ORF AC3 was more similar to that of OYMMV (accession No. HQ020409). On the other hand, the common region (CR) was more similar to that of Chino del tomate virus (accession No. AF101476, DQ885456, AF226664 and AF226665).

Recombination analysis

Recombination analysis was carried out to identify putative recombination events and breakpoints between DNA-A component sequences of 66 species of begomoviruses reported for Mexico. Out of the 47 total recombination events detected, 29 events were significant. For the three new sequences reported in this study, the Okra mottle Mexico virus sequence (accession No. AY751753.2) presented one putative recombination event of 93 bases in the ORF AV1 + CR region. The viruses Chino del tomate virus (accession No. AF226665, AF101476, AF226664, DQ347945 and DQ885456), Okra yellow mosaic Mexico virus (accessions No. GU990614, GU990613 and GU990612) and Corchorus yellow spot virus (accession No. DQ875868) were identified as possible major parents (Supplementary Data S3). On the other hand, the two sequences of Okra yellow mosaic Mexico virus reported here (accession No. HQ020409 and HQ116414) presented the same recombination event of 306 bases in the CR + ORF AV1 region, but it was not possible to identify any major parent.

Discussion

At the beginning of this century, De la Torre-Almaráz et al. (2004) reported the presence of mottled and yellow leaf mosaic, deformation and reduction of the leaf lamina, and fruits with elongated stripes, as well as small bumps in the epidermis of okra plants; these symptoms were similar to those observed in the present study (Fig. 1). This disease decreases yield, productivity and, also it reduces fruit quality since fruits with visible symptoms are discarded (De la Torre-Almaráz et al. 2004). Similar disease was reported by Hernandez-Zepeda et al. (2010) in okra crops in Texas, USA, where they observed irregular yellow patches on leaves, distinctive yellow borders on leaf edges and chlorosis of subsequently developing leaves. These typical symptoms are indicators of presence of Begomovirus species (De la Torre-Almaráz et al. 2004; Hernandez-Zepeda et al. 2010).

The disease was not transmitted neither by seed nor by mechanical means. However, it was transmitted by grafting and insect vectors. The foregoing is consistent with the assertion that begomoviruses can be transmitted by grafting, but they are not conceived from transmission by seed or by mechanical inoculation (Mishra et al. 2017). The geminiviruses, to which begomoviruses belong, are closely related to the phloem, so they are not transmitted by sap (mechanical transmission) or by seeds (Gilbertson et al. 2015). It is likely that the limitation to the phloem reflects the co-evolution with the genetic elements of the phloem in angiosperm plants, in the sense that the necessary functions (replication, movement and transmission of insects) have been optimized for these cell types (Rojas et al. 2005; Wege et al. 2001). Although T. vaporariorum West. was identified from the 50 preparations carried out in the present research, it was impossible to conclude on the role of this insect in the transmission of these viruses in the okra. This is due to the lack of total control of the insects used for transmission tests, since they were sampled directly from the field with the possibility of co-habitation with other insects, especially B. tabaci. Hidayat and Rahmayani (2007) indicated that both species of whiteflies, B. tabaci and T. vaporariorum, are capable of transmitting Tomato leaf curl virus (ToLCV) and showed that B. tabaci is more effective as ToLCV vector in chili and tomato, qualifying it as a supervector (Gilbertson et al. 2015). Moreover, Czosnek et al. (2017) reported that begomoviruses can be ingested but not transmitted by T. vaporariorum. Likewise, Uchibori et al. (2013) indicated that begomoviruses are transmitted only by the B. tabaci whitefly, showing that Tomato yellow leaf curl virus (TYLCV) can enter the epithelial cells of the middle intestine of the B. tabaci vector but not those of T. vaporariorum. Exhaustive and detailed studies on efficiency of the transmission of begomovirus by T. vaporariorum are required.

By sequencing the entire component A and amplifying a region of component B, the genomic organization was deciphered as typical New World bipartite begomovirus (Brown et al. 2015; Hernandez-Zepeda et al. 2010). Moreover, the availability of a large number of DNA-A complete genome sequences of begomovirus isolates allows a useful and conveniently defined subdivision between species and strains (Fauquet et al. 2008). Therefore, the recent taxonomy of begomovirus has been based mainly on the comparison of available sequences through a solid statistical analysis without much discrepancy with the viruses’ biology; it was even shown that the taxonomy based on the complete sequences of the DNA-A component of related begomovirus isolates can accurately reflect the differences in their biology (Brown et al. 2015). ICTV taxonomic criterion, as reported by Brown et al. (2015), establishes that if the sequence of the complete DNA-A component of a new begomovirus shows a percentage of identity ≤ 91% with the closest begomovirus reported in the database, the new isolate could be considered a new species; and if it shows a percentage of identity ≤ 94%, the new isolate could be considered a new strain. This criterion allowed (1) to locate the isolate 15–16 in an already reported strain of OYMMV species; (2) to identify the isolate 13–14 as a new viral strain of the OYMMV species; and (3) to report a new separate viral species tentatively named Okra mottle Mexico virus, OMoMV—[Mexico:2005]. This species is related to the Chino del tomate virus within the begomovirus species reported for Mexico.

Genetic variation is common in begomovirus species, and recombination is considered one of the driving forces for their divergence and evolution (Biswas et al. 2018; Kumar et al. 2017; Lefeuvre and Moriones 2015; Venkataravanappa et al. 2013). The possible existence of recombination events in the reported species that infect okra in Mexico would be a factor of divergence and appearance of new strains and/or species of begomoviruses. On the other hand, the coexistence of several begomoviruses in the same crop may be due to the movement of viruses to new ecosystems by human activities, culture systems and to the role of whiteflies, which colonize many other plant species (Brown 2007). In addition, there is the possibility that different viruses produce a synergism that enhances the effects on crops. All this establishes the importance of further analyzing the possible phytosanitary implications of the presence of Chino del tomate virus, Okra yellow mosaic Mexico virus and Okra mottle Mexico virus in okra crops, as well as the risks of their propagation in new hosts. Recently, the OYMMV virus was reported in Roselle crops (Hibiscus sabdariffa L.) and in several weed species such as Sida collina, S. aggregata, S. acuta, S. haenkeana and Malacra fasciata (Ortega-Acosta et al. 2019; Velázquez-Fernández et al. 2016). Further exploration and cross-infectivity of these viruses in okra, and subsequent analysis of the recombinants, would provide a greater understanding of the diversity and evolution of begomoviruses in Mexico.

In the present study, the coexistence of at least two species of bipartite begomoviruses in okra was evident. Through PCR assays and complete sequences of the DNA-A component, it was possible to isolate and identify a new species of begomovirus tentatively named Okra mottle Mexico virus, OMoMV – [México:2005], and a new strain of the virus OYMMV – [Mexico:Isolate 13–14:2010]. Moreover, it was determined that the isolates obtained cannot be transmitted by seed or mechanically, but they can be transmitted by grafting and by whitefly. In addition, the results suggest that part of the genome of the new reported virus is a result of recombination with other viruses reported in Mexico.