Potyviridae is the largest family of RNA plant viruses, characterized by single-stranded, positive-sense RNA, monopartite or bipartite genomes, and flexuous, filamentous particles (Inoue-Nagata et al. 2022). Approximately 30 plant families are known to be infected by potyviruses, and 19 potyvirus species specifically infect legume crops (Chatzivassiliou 2021; Inoue-Nagata et al. 2022). Common bean (Phaseolus vulgaris), cowpea (Vigna unguiculata), and soybean (Glycine max) are among the important legume crops primarily infected by potyviruses, including bean common mosaic virus (BCMV; Potyvirus phaseovulgaris), bean yellow mosaic virus (BYMV; Potyvirus phaseoluteum), clover yellow vein virus (ClYVV; Potyvirus trifolii), and soybean mosaic virus (SMV; Potyvirus glycitessellati) (Jha et al. 2023).
Yambean mosaic virus (YBMV; Potyvirus pachyrhizus) was first reported in Peru, isolated from yambean (Pachyrhizus spp.) field trials exhibiting mosaic symptoms, with or without leaf deformation (Fuentes et al. 2012). Additionally, calopo (Calopogonium mucunoides) and jack bean (Canavalia ensiformis) from Australia (de Sá Andrade Medeiros et al. 2019, Filardo et al. 2023), and senna (Senna hirsuta) from Nigeria (Ekpiken et al. 2023) have been reported as new hosts for YBMV. However, there is no evidence that YBMV infects butterfly peas (Clitoria ternatea).
Butterfly peapossesses various advantages in traditional medicine, agriculture, and the food industry (Oguis et al. 2019). In addition to other plant pathogens such as fungi, bacteria, and nematodes, butterfly pea is a host for several significant plant viruses, such as BCMV (Lima et al. 1993), clitoria yellow mottle virus (CliYMV; Tobamovirus clitoriae) (Wei et al. 2012), and papaya ringspot virus (PRSV; Potyvirus papayanuli) (Sultana et al. 2019). In Thailand, butterfly pea is an important herb, particularly valued for its flowers, which are widely used as a natural food coloring (Hasanah et al. 2023). Despite its importance, there have been few studies on the plant pathogens affecting butterfly pea and no reports of plant viruses infecting butterfly pea in Thailand. This study presents the first report of YBMV infecting butterfly peas, along with its molecular characterization.
Butterfly pea plants showing severe mottling, vein banding, blistering, and wrinkling similar to potyvirus infection were observed in San Sai district (18.89403° N, 99.01058° E), Chaing Mai province, in January 2024 (Fig 1). A total of five samples, three symptomatic leaf samples and immature and mature seed samples from suspected plants were subjected to molecular detection. One positive sample was further used for mechanical inoculation.
Total RNA was extracted from leaf and seed samples using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. Group-specific degenerate primers for potyvirus, CPUP (5’-TGAGGATCCTGGTGYATHGARAAYGG-3’) and P9502 (5’-GCGGATCCTTTTTTTTTTTTTTTTT-3’), were used to amplify the coat protein (CP) gene (the 3’-terminal region) (~750 bp) (van der Vlugt et al. 1999). RT-PCR reactions were performed to amplify the viral genome using the RevertAid First Strand cDNA Synthesis Kit (Thermo ScientificTM, USA). cDNA synthesis was performed using 1 µg of RNA as a template in a final reaction volume of 20 µl, following the manufacturer's instructions. The initial steps included incubation at 65°C for 5 minutes, 42°C for 60 minutes, and 70°C for 5 minutes. PCR amplification was optimized as follows: denaturation at 95°C for 2 minutes, followed by 35 cycles of 95°C for 30 seconds, 57°C for 1 minute, and 72°C for 1 minute and 45 seconds, with a final extension at 72°C for 7 minutes (van der Vlugt et al. 1999). The sizes of the PCR amplicons were estimated using 1.5% agarose gel electrophoresis in TBE buffer. Selected amplified products were sent for Sanger sequencing at Macrogen in Seoul, Korea. The BLAST search was performed on the NCBI BLAST platform (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Manual improvements were made using BioEdit v. 7.2.3 (Hall et al. 2011) where necessary. Maximum likelihood (ML) analysis was conducted using IQ-TREE (Trifinopoulos et al. 2016) with the ML+rapid bootstrap setting and 1,000 replicates. The generated tree was visualized using FigTree v.1.4.0 (Rambaut 2012), and the final layout was completed with Adobe Illustrator® CS5 (Version 15.0.0, Adobe®, San Jose, CA).
Mechanical inoculation was performed using five 2-week-old seedlings of each butterfly pea, senna (Senna occidentalis), and yardlong bean (Vigna unguiculata subsp. sesquipedalis). Symptomatic leaves (MJU-L1) were ground in 0.05 M potassium phosphate buffer (pH 7.2) and used to inoculate selected leaves of each test plant. The test plants were placed in the dark for 24 hours and then transferred to a growth chamber in the greenhouse, maintained at 25-30°C under ambient light conditions, for continuous observation. Control plants were mock inoculated using the same procedures. Test plants showing symptoms were subsequently subjected to molecular detection as described.
Except for the mature seed sample, three leaf samples and one immature seed sample were positive for potyvirus-specific primers (Table 1, Fig. 2). The two obtained sequences (PQ238432; MJU-L1: Source, PQ238433; HGR2: Senna test plant) were 99.4% identical (with a 4-bp difference) and had 100% similarity in their amino acid sequences. Both sequences were 98.96% (PQ238432) and 99.25% (PQ238433), similar to the YBMV isolate BR-6 (MZ018226). The ML phylogenetic analysis based on the CP gene showed two main clades: BCMV and YBMV. Within the BCMV clade, the subclades BCMV, BCMV-BIC, and BCMV-PSt were identified. Our two isolates clustered with the YBMV isolate BR-6, showing strong statistical support (94% ML) within the YBMV clade (Fig. 3). The isolates YBMV-VN/BB2-5 (DQ925422), YBMV-VN/YB2 (DQ925425), and YBMV-Bogor (AB289438), originally identified as BCMV from black bean, yardlong bean, and yambean, respectively, clustered within the YBMV clade, aligning with the phylogenetic results reported by Damayanti et al. (2008), Ha et al. (2008), and Fuentes et al. (2012).
Since BCMV, blackeye cowpea mosaic virus (BlCMV), and peanut strip virus (PStV) have already been revisited, the results indicate that this separation reflects evolutionary divergence and the emergence of a unique viral entity within YBMV (Tang and Feng 2022). The observed genetic diversity among YBMV isolates from various localities suggests that the virus may have undergone multiple introductions or evolved locally (Zou et al. 2020). However, we need additional isolates from different hosts and locations to draw definitive conclusions about the nucleotide differences in our two isolates.
Mechanical inoculation resulted in a distinct mosaic pattern in butterfly pea (5 symptomatic plants out of 5 inoculated) and senna (5 out of 5) 30 days post-inoculation (dpi). Yardlong beans (3 out of 5) showed thickening and wrinkling of the leaves at 7 dpi, which became more pronounced over time (Fig. 1). All symptomatic plants tested positive with potyvirus-specific primers. The results indicate that YBMV exhibits distinct symptomatic expressions, including mosaic patterns, leaf wrinkling, and chlorosis, which are consistent with typical potyvirus symptoms across a wide host range. The variation in symptom severity across different host plants highlights the complexity of YBMV-host interactions and the potential impact on leguminous crops (Ekpiken et al. 2023). This emphasizes the need for vigilant monitoring of the appearance of virus symptoms and possible management practices in legume cultivation (Filardo et al. 2023).
To confirm the presence of potyviruses in immature and mature seeds from visibly infected plants, we obtained a positive result only in the immature seed samples, while the mature seeds, which are essential as planting material, tested negative. The negative results for the mature seeds require re-evaluation to determine whether they are due to insufficient RNA extraction, the impact of seed soaking, or differences related to the growth stage. One of the primary issues with plant virus management is the risk of overlooking infected or contaminated seeds, which could lead to viral disease epidemics and threaten food security. Bowers and Goodman (1979) reported that although immature seeds can harbor the virus, mature seeds may contain undetectable amounts of the virus in soybeans. Wang and Maule (1994) found pea seed-borne mosaic virus (PSbMV) in the testa tissues of immature pea seeds, noting that the amount of the virus decreases as seeds mature, leading to a distribution limited to patches in older seeds. The distribution pattern may be specific to the type of virus due to different viral replication strategies, or it might also depend on the host species. In-host virus movement modeling studies suggest that less virulent viruses might have a higher chance of being vertically transmitted (Cobos et al., 2019). Seed transmission of potyviruses is influenced by a combination of viral genetic factors, host plant genotype, environmental conditions, timing of infection, and interactions with vectors (Simmons and Munkvold 2014). Interestingly, Dall et al. (2019) noted that many records of potyviruses can only logically be explained by seed transmission. They suggest that potyviruses may be more frequently seed-borne than published data indicate, likely due to limited seed testing. However, there are no previous reports on the seed-borne nature or seed transmission potential of YBMV. This study, however, provides initial evidence suggesting that YBMV may be seed-borne. Further experiments are needed to investigate insect transmission, potential seed transmission, and the alternative host range of YBMV in Thailand to support sustainable virus management strategies for legumes.