The bipartite begomovirus PepYLCIV was first identified in Indonesia; its distribution has been confirmed in various parts of the country, including Sumatra, Java, Bali, and Sulawesi (Koeda et al. 2016; Selangga et al. 2021; Taufik et al. 2023; Wahyono et al. 2023). The disease caused by PepYLCIV is a serious threat to Solanaceae crops, with production losses of up to 100% (De Barro et al. 2008). In 2017, a recombinant begomovirus species, PepYLCAV, was isolated from pepper, tomato, and tobacco (N. tabacum) plants in multiple fields in Aceh Province, Northern Sumatra, Indonesia (Kesumawati et al. 2019). Recombinants more virulent than their parental viruses have been reported in previous studies on cotton leaf curl disease in India–Pakistan (Farooq et al. 2021; Qadir et al., 2019) and tomato yellow leaf spot virus infections in Brazil (Calegario et al. 2007). In this study, PepYLCAV was more virulent than the putative parental virus PepYLCIV during infections of multiple plant species.
Frequent sampling and diagnosis are required to confirm the replacement of a parental virus by a recombinant virus in nature (Bellabess et al. 2015; Monci et al. 2002; Tsai et al. 2011). Because PepYLCIV infections have been detected in almost all pepper-producing regions in Indonesia (Wahyono et al. 2023), our analysis of a recombinant PepYLCAV may be useful for clarifying the possible replacement of phytopathogenic viruses by a novel begomovirus. All of the samples collected from 2012 to 2016 were infected with PepYLCIV alone, but PepYLCAV infections were detected in 2017, 2018, and 2019. Intriguingly, the mixed infection rate (i.e., both PepYLCIV and PepYLCAV) ranged from 37–46% of the plants collected in this period, suggesting PepYLCIV co-existed with PepYLCAV. A similar trend was reported for the shift in the predominant begomovirus species from tomato leaf curl Taiwan virus to tomato yellow leaf curl Thailand virus, with these two begomoviruses subsequently co-existing in Taiwan (Tsai et al. 2011). Currently, we rely on PCR primer-based begomovirus detection to determine the infection rate of both viruses, but this approach cannot fully reveal the mutation and recombination of the other parts of the viral genome. Therefore, a reliable method for analysing the full-length genome sequences of begomoviruses in many samples collected from different geographical locations must be developed to more precisely characterise the shift in viral populations.
Recombination reportedly enhances the ability of begomoviruses to adapt to diverse plant hosts and environmental conditions, which is favourable for their emergence and enhancement of pathological features, potentially leading to an increased host range (Fiallo-Olivé and Navas-Castillo 2023). According to an earlier recombination analysis, PepYLCAV was predicted to have PepYLCIV as a major parental donor and pumpkin yellow mosaic virus (PuYMV) and tomato leaf curl New Delhi virus (ToLCNDV) as minor parental donors of DNA-A and DNA-B, respectively (Kesumawati et al. 2019). Notably, in a recent study, PepYLCAV asymptomatically infected eggplants that were not infected with PepYLCIV, suggesting PepYLCAV has a wider host range than PepYLCIV, possibly because of recombination (Kikkawa et al. 2023). Because the putative minor parents (PuYMV and ToLCNDV) were isolated from cucurbits (Kesumawati et al. 2019; Yamamoto et al. 2021), PepYLCIV adapted to Solanaceae crops and a begomovirus adapted to cucurbit crops may have co-infected a certain host, which resulted in the emergence of PepYLCAV. The expansion of the host range of PepYLCAV to include solanaceous and cucurbitaceous plants is currently being investigated.
Among the Ty genes identified in tomato, Ty-1 is the most widely used for controlling TYLCV in commercial F1 hybrid tomato cultivars in Europe, the US, and Japan (Koeda et al. 2020; Lapidot et al. 2014). On the basis of genotyping analyses conducted using RDR-linked markers, almost all TYLCV-resistant tomato cultivars in Japan possess Ty-3a, which is a Ty-1 allele (Koeda et al. 2020; Saito et al. 2008; Suzuki et al. 2019). However, we recently used newly developed RDR in-gene markers to reveal the presence of Ty-1 in commercially available TYLCV-resistant tomato cultivars in Japan (Koeda and Kitawaki 2024). The genotyping of the RDR allele in the current study showed that MP also has Ty-1 instead of Ty-3a. We previously detected moderate symptoms in MP plants infected with the bipartite tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) and the monopartite lisianthus enation leaf curl virus (LELCV) (Koeda et al. 2020; Taniguchi et al. 2023). Thus, Ty-1-based resistance is only partially effective against highly virulent begomoviruses, including PepYLCAV, TYLCKaV, and LELCV from Southeast and East Asia. Interestingly, Ty-1-based resistance is fully effective against the putative parent PepYLCIV, but it was overcome by the recombinant PepYLCAV. Genotyping using the in-gene marker for the begomovirus resistance gene pepy-1 (Koeda et al. 2021) indicated that more than 80% of pepper plants cultivated in Indonesia in 2023 carried the begomovirus resistance allele (pepy-1), reflecting the extensive use of this resistance gene in Indonesia (data not shown). In addition, begomovirus-resistant cultivars bred via conventional phenotype-based selection are widely grown in Indonesia. Researchers have also attempted to apply marker-assisted breeding technology to develop Indonesian tomato cultivars carrying Ty genes (Ty-1, ty-5, and Ty-6) (Saputra et al. 2020). The extensive use of begomovirus resistance genes in pepper and tomato plants may have triggered the emergence of resistance-breaking PepYLCAV through positive selection. Although there are no reports of TYLCV isolates that can overcome Ty-1-based resistance, several TYLCVs that have adapted to Ty-1-based resistance have been identified, including IS76-like TYLCV-ILs, which were initially isolated in Morocco and have recently been detected in southern Spain (Belabess et al. 2015, 2016; Fortes et al. 2023). Recombination between TYLCV and other begomovirus species may lead to the emergence of resistance-breaking recombinants like PepYLCAV.
Our previous recombination analysis of the PepYLCAV genome sequence indicated that the recombination breakpoints in the longest fragment were located in the ORFs encoding AC1 and AC4 as well as in the intergenic region (Kesumawati et al. 2019). There is currently interest in the non-synonymous mutations in these PepYLCAV factors that are related to enhanced virulence. Several studies showed that AC1/C1 (Rep) is associated with virulence and increased symptom severity (Rodríguez-Negrete et al. 2013; Wang et al. 2020). The carboxyl terminus of AC1 (Rep) reportedly explains why a Col strain of the Sri Lankan cassava mosaic virus (SLCMV) is more virulent than its putative parent SLCMV-HN7 (Wang et al. 2020). The geminivirus C1 (Rep) protein decreases the expression of the genes encoding plant maintenance DNA methyltransferase 1 and chromomethylase 3, thereby functioning as a transcriptional gene silencing (TGS) suppressor (Rodríguez-Negrete et al. 2013). In addition to AC1/C1, AC4/C4 should also be analysed as a virulence factor because earlier research revealed a putative mutation to cotton leaf curl Multan virus C4 suppresses both post-transcriptional gene silencing and TGS via the inhibition of S-adenosyl methionine synthetase (Ismayil et al. 2018). Moreover, the second glycine in ageratum leaf curl Sichuan virus C4 is important for pathogenicity (Li et al. 2020). In the present study, we observed PepYLCAV is more virulent than its putative major parent PepYLCIV. We are currently investigating whether a specific non-synonymous amino acid mutation encoded by a particular ORF is associated with enhanced fitness and increased virulence.
Considered together, our study results indicate that PepYLCAV is a novel bipartite begomovirus species that emerged in 2017 in pepper plants cultivated in Indonesia. Its virulence exceeds that of its putative parent (PepYLCIV), resulting in increased infection rates, viral DNA accumulation, disease symptom severity, and growth inhibition in multiple Solanaceae plants. The replacement of PepYLCIV by PepYLCAV after 2017 highlights the importance of routine field inspections and studies conducted to clarify the adverse effects of recombinant begomoviruses on agricultural production. Finally, we revealed that Ty-1-mediated resistance is effective against an infection by PepYLCIV alone, but is less effective against an infection by PepYLCAV alone. To the best of our knowledge, this is the first study to identify a novel recombinant begomovirus species in Indonesia that is more virulent than the other predominant species (PepYLCIV).