Single-stranded DNA (ssDNA) viruses of the family Geminiviridae, encompassing either one or two viral components encapsulated within geminate icosahedra, serve as causal agents of plant diseases prevalent in tropical and subtropical regions (Zerbini et al., 2017). The Geminiviridae family, comprising 14 genera, holds the distinction of being the largest family of plant viruses. These viruses are capable of causing devastating yield losses in both monocotyledonous and dicotyledonous crops (Fiallo-Olivé et al., 2021). With over 500 species, the Geminiviridae family boasts an extensive repertoire of viruses. The Begomovirus genus, with its two lineages, Old World (OW) and New World (NW), stands out as the most species-rich group (Fiallo-Olivé and Navas-Castillo, 2020). Begomoviruses exhibit two genome configurations: bipartite, consisting of two components (DNA-A and DNA-B) each approximately 2.6 kb, and monopartite, featuring a single component (DNA-A) similar to that of the bipartite counterpart ranging from 2.7-2.8 kb. While the majority of begomoviruses conform to a specific genomic structure based on their geographic origin, there are intriguing exceptions. Notably, there are only a limited number of instances of bipartite begomoviruses indigenous to the OW, and conversely, monopartite begomoviruses are rarely found native to the NW (Romay et al., 2019).

In the OW, both monopartite begomoviruses and the DNA-A component of bipartite begomoviruses harbor six open reading frames (ORFs). Two genes encoded by the virion-sense DNA strand include the coat protein (CP) and the AV2/V2 protein, while four genes encoded by the complementary-sense DNA strand include the replication-associated protein (Rep), the transcriptional activator protein (TrAP), the replication enhancer protein (REn), and the C4 protein (Hanley-Bowdoin et al., 2013). Besides the six established ORFs, recent studies have identified a set of novel small ORFs encoded within the monopartite begomovirus genome, including V3, C5, C6, and C7 (Gong et al., 2021; Liu et al., 2023). The DNA-B component of bipartite begomoviruses also possesses two ORFs, the nuclear shuttle protein (NSP; BVI) encoded by the virion-sense DNA strand and the movement protein (MP; BC1) encoded by the complementary-sense DNA strand (Fondong, 2013). Bipartite begomoviruses possess a common region (CR) spanning around 200 nucleotides (nt) in both DNA-A and DNA-B. This CR, housing the origin of replication, is a shared non-coding segment with a nona-nucleotide sequence (TAATATTAC). This CR is indispensable for replication and bidirectional gene expression. In the OW, many of the economically important plant diseases are caused by monopartite begomoviruses in association with single-stranded DNA-satellite components (betasatellites, alphasatellites, and deltasatellites) (Fiallo-Olivé and Navas-Castillo, 2020). Betasatellites, in particular, influence disease development by encoding the symptom determinant protein βC1 (Yang et al., 2019). Recently, another ORF βV1 has also been shown to act as a protein elicitor and pathogenicity determinant (Gupta et al., 2022). Alphasatellites and deltasatellites play crucial roles in the context of monopartite begomovirus infection by providing additional functions that contribute to the complexity of the infection process (Briddon et al., 2018).

The widespread distribution of begomoviruses is attributed to the widespread prevalence of the cryptic species complex of whiteflies (Bemisia tabaci), non-cultivated host plants, human activity, and the ability of the virus to evolve through recombination and the acquisition of new components (Gilbertson et al., 2015; Rojas et al., 2018). While the indigenous begomovirus species within a region exhibit genetic divergence from introduced species, they can evolve in parallel or through localized adaptations (Zhou et al., 2008). Intercontinental viral migrations have become increasingly evident in recent years. Mabvakure et al. (2016) observed the westward spread of tomato yellow leaf curl virus (TYLCV) across Africa in the 1990s, while Sobh et al. (2012) documented the arrival of northwestern squash leaf curl virus in the Middle East. Notable, Sattar et al. (2013) tracked the eastward expansion of cotton leaf curl disease (CLCuD) from the Indo-Pakistani subcontinent to China. More recently, OW watermelon chlorotic stunt virus (WmCSV) has made a dramatic cross-continental leap, reaching Mexico and the USA (Dominguez-Duran et al., 2018; Fontenele et al., 2021). Another noteworthy case is the identification of tomato leaf curl Palampur virus (ToLCPalV) in cucurbits, tomatoes, and melons in Iran (Heydarnejad et al., 2009) and most recently in Saudi Arabia (AlHudaib et al., 2022), indicating long-distance spread of begomoviruses. The ToLCPMV epidemic has inflicted severe damage on cucurbit production in Iran, leading to substantial economic losses for protected farms cultivating cucumbers (Heydarnejad et al., 2013).

Various monopartite and bipartite begomoviruses have been documented across the Arabian Peninsula, causing infections in diverse crops. Several begomoviruses pose significant threats to various vegetable crops in Saudi Arabia, with prominent examples including TYLCV, tomato leaf curl Sudan virus (ToLCSDV), WmCSV (Sattar, 2024), and cotton leaf curl Gezira virus (CLCuGeV) (Idris et al., 2014; Rezk et al., 2019; Sohrab, 2020). Most recently, ToLCPalV was first reported during a field survey conducted in the Al Ahsa region, in a mixed infection involving tomatoes and cucurbits (AlHudaib et al., 2022).

Cucurbits, belonging to the family Cucurbitaceae, are highly esteemed as vegetable crops. Among them, cucumber (Cucumis sativus L.) emerges as a noteworthy vegetable cultivated worldwide, boasting an estimated annual global production of approximately 93 million tons. In Saudi Arabia, a diverse range of vegetable crops is cultivated, with cucumber being extensively grown (Elzaki et al., 2022). In 2021, it covered an area of 2,357 hectares and yielded 0.18 million tons (https://www.indexbox.io/search/production-cucumber-and-gherkin-saudi-arabia/). Cucumbers are among the top choices for greenhouse cultivation in Saudi Arabia, occupying the second-highest position among all greenhouse vegetables (Mousa et al., 2019). During a survey of cucumber plants in both fields and greenhouses across the Al Ahsa region of Saudi Arabia, symptomatic plants were identified. Preliminary testing confirmed that these plants were infected with begomovirus. Despite considerable research efforts in this domain, conventional contemporary methods for detecting and characterizing the plant virome offer limited insights into the prevalent viral genomes (Idris et al., 2014; Shahid et al., 2021). Conventional approaches face obstacles in accurately evaluating the genetic diversity of begomoviruses within infected hosts due to the inherent selectivity of primers and the prevalence of particular viral genomes. The widespread importance of cucumber cultivation in Saudi Arabia prompted a comprehensive investigation into the potential of begomovirus infection on a broader scale, utilizing a next-generation sequencing approach. The study identified bipartite begomovirus WmCSV isolates from the symptomatic samples in mixed infections with both monopartite begomovirus TYLCV and bipartite begomovirus ToLCPalV. These mixed infections could involve either a single begomovirus species or both together.

In Saudi Arabia, small farms utilize diverse cultivation methods, including traditional open-field cropping and controlled greenhouse environments, for cucumber production (Mousa et al., 2019). Research conducted in the Western region has identified the presence of both ToLCSDV and tomato yellow leaf curl betasatellite (TYLCB) within these agricultural ecosystems (Sohrab et al., 2017). A paucity of research on begomovirus molecular characterization in Saudi Arabia has left a knowledge gap regarding economically important geminiviruses (Rezk et al., 2019; Sohrab, 2020). In a prior investigation employing a high-throughput sequencing approach, we successfully identified mixed infections of CLCuGeV associated with DNA-satellites and/or TYLCV and ToLCPalV infecting tomato and muskmelon crop plants in Saudi Arabian fields (AlHudaib et al., 2022).

The current survey in the Eastern region of Saudi Arabia, identified cucumber plants with characteristic begomovirus symptoms, including leaf yellowing and yellow mosaics. Initial analyses confirmed the presence of begomovirus/es in nine symptomatic cucumber plants from the Al-Hofuf region using begomovirus core CP amplification. However, no amplification was obtained from samples in the Qateef region. The subsequent high-throughput sequencing of eight selected cucumber samples from Al-Hofuf revealed diverse begomovirus sequences, with full-length sequences identified in each sample. Comparative sequence analysis, phylogenetic dendrograms and recombination analysis unveiled that cucumber samples were infected with either a single begomovirus genome or a mixture of multiple begomoviruses involving TYLCV, ToLCPalV and WmCSV. The phylogenetic dendrogram, shows that TYLCV isolates formed a clade with Boushehr and Iran strains of other TYLCV isolates from Saudi Arabia, Kuwait, and Iran. Nevertheless, TYLCV isolate 7CYT1 constituted a distinct monophyletic clade, separate from other TYLCV isolates ( Figure 3 ). These results suggest the possibility that TYLCV isolates may have originated from two independent introductions into Saudi Arabia.

The WmCSV isolates showed high mutual nt sequence identities, aligning with the DNA-A component of WmCSV isolate reported from squash plants in Saudi Arabia (Rezk et al., 2019). In recent years, the emergence of two begomoviruses has posed a significant threat to cucurbit cultivation in the Middle East. Specifically in cucurbits Lapidot et al. (2014) documented the invasion of a NW begomovirus, squash leaf curl virus (SLCV), and an OW begomovirus, WmCSV. The phylogenetic dendrograms in the current study revealed distinct clades for DNA-A and DNA-B sequences of WmCSV isolates from Saudi Arabia separate from other WmCSV isolates, suggesting its successful establishment in the region, after its introduction. Previous reports of WmCSV from cucurbit crops support this speculation (Al-Saleh et al., 2014; Sattar, 2018; Rezk et al., 2019). It is also evident that WmCSV has now been spreading into the cucurbit fields in Saudi Arabia. This unique evolutionary trajectory indicates that WmCSV has been adapted and diversified within Saudi Arabia, highlighting its potential for long-term persistence in this region.

The DNA-A of ToLCPalV isolates exhibited high nt sequence identities with ToLCPalV isolates reported from Saudi Arabia and Iran. These isolates, along with other ToLCPalV isolates from Saudi Arabia, formed a distinct clade in the phylogenetic dendrogram. Conversely, the DNA-B isolates displayed varied nt sequence identity with the DNA-B of ToLCPalV isolates from Saudi Arabia and Iran. Furthermore, these isolates segregate into two independent clusters in the phylogenetic dendrogram. Isolates 4CSPb1 and 4CSPb2 clustered with ToLCPalV DNA-B from Saudi Arabia, Iran and Iraq, while isolates 1CKPb1 and 1CKPb2 formed a distinct clade ( Figure 3 ). ToLCPalV, a bipartite begomovirus, is extensively distributed in India and Pakistan, where it has been documented infecting various host plants such as tomato, cucurbits (Ali et al., 2010; Namrata et al., 2010; Shafiq et al., 2019; Hanamasagar et al., 2021), and more recently, a weed (Sattar et al., 2022). It was first reported in 2009 from Iran (Fazeli et al., 2009), where it has been identified infecting common bean, cucumber, melon, pumpkin, and watermelon crops (Heydarnejad et al., 2009, 2013; Esmaeili et al., 2015). The discovery of ToLCPalV in Saudi Arabia suggests a wider geographic distribution than previously understood. A detailed analysis revealed that the ToLCPalV isolates prevailing in the Arabian Peninsula differ significantly from those in Southeast Asia ( Figure 3 ). Both groups are separate in the phylogenetic dendrogram, suggesting their independent evolution from a common ancestor and parallel establishment in different niches. It is more probable that the introduction of these viruses into the Saudi Arabian agroecosystem originated from Iran.

In this current investigation, the complete genomes of TYLCV, ToLCPalV and WmCSV were assessed for genomic variations present in their respective genetic compositions. Surprisingly, our findings revealed that the analyzed begomoviruses primarily undergo evolution influenced by strong negative pressure. Notably, no instances of recombination were observed in the DNA-A components, except for TYLCV isolates and the DNA-B of WmCSV. The TYLCV, ToLCPalV, and WmCSV begomovirus populations exhibited a high level of genetic diversity indices, with ToLCPalV DNA-B displaying the highest genetic diversity among all the genomic components of the studied begomoviruses. In contrast, WmCSV DNA-A displayed the lowest genetic diversity indices, indicating that low level of genetic differentiation is operating in this population. Furthermore, within the examined datasets of begomovirus populations, the mean number of segregating sites (θw) and the negative TD values consistently suggest that these populations are undergoing diversification to different extents, primarily driven by purifying selection. Another possible notion is that these begomovirus populations may have recently expanded, as opposed to undergoing neutral selection (Srivastava et al., 2022). The presence of low-frequency alleles within these populations could potentially account for the notably negative TD and FLD values (Farooq et al., 2021). The genetic variations (π and θw) observed in the populations of TYLCV and ToLCPalV surpass those found in the WmCSV population. These results highlight significant sequence divergence and a greater prevalence of unique mutations within the TYLCV and ToLCPalV populations, underscoring their pivotal roles in their emergence. Despite the prevailing notion that DNA-A components predominantly contribute to begomovirus evolution, some recent studies suggest that DNA-B can also play a pivotal role in begomovirus evolution (Sattar et al., 2022). Our findings revealed remarkable genetic diversity indices in the DNA-B of ToLCPalV and WmCSV compared to their DNA-A counterparts, supporting the speculation that DNA-B may actively assist the virus in adapting swiftly, particularly in response to pronounced negative pressure.

Our findings regarding the ORFs encoded by begomoviruses revealed distinct genetic diversity indices for each ORF, highlighting the non-uniform distribution of nt diversity and genetic mutations across their sequences. Specifically, AC1 (C1 or Rep) demonstrated the highest values across various genetic diversity indices, whereas AV2 (V2) exhibited the lowest values in all datasets. Previous studies have consistently reported higher nt variability in the Rep genes of begomoviruses (Lima et al., 2017). Our findings further support the non-random distribution pattern of nt diversity and genetic mutations observed within the ORFs of various begomoviruses. Furthermore, when comparing populations ORFs using k values, it was observed that the Rep consistently exhibited the highest k values across all ORF datasets. This underscores a higher degree of genetic differentiation. In contrast, AC4 and V2 displayed a lower level of genetic differentiation. The consistent occurrence of negative values for both TD and FLD across all begomovirus populations and their corresponding ORFs highlights the conserved nature of their genes. These findings are likely in populations where predominant nt variations are transient and subsequently eradicated by purifying selection (Mishra et al., 2022). Furthermore, varied dN/dS ratios observed in distinct begomovirus-encoded ORFs suggest the influence of diversifying selection. Notably, the elevated dN/dS ratio observed in AC4 (C4) implies that this particular gene is undergoing evolution under rigorous purifying and negative selection. Similar observations have been previously identified in various viruses (Aguilar Rangel et al., 2023), as well as in different begomoviruses such as ChiLCV (Mishra et al., 2022) and ToLCPalV (Sattar et al., 2022). In our investigation, the majority of begomovirus components exhibited elevated NSSY rates. This aligns with previous research indicating that geminiviruses consistently demonstrate high NSSY rates, similar to many RNA viruses (Duffy and Holmes, 2009). While we employed both strict and relaxed molecular clocks to estimate the NSSY rate, the strict molecular clock produced more favorable results. This finding contrasts with a previous study that leaned towards the relaxed molecular clock (Srivastava et al., 2022).

The analysis of recombination revealed prevalent recombinants within the populations of TYLCV and WmCSV DNA-B. The presence of both intra- and interspecies recombination events was indicated by the identification of unknown recombinant parents, as well as parents from different species. The presence of well-supported recombination events in TYLCV isolates, involving multiple begomoviruses as major and minor parents, further strengthens the hypothesis that TYLCV has been introduced into Saudi Arabia on multiple occasions. High mutation rate and strong purifying selection likely drove the independent evolution of ToLCPalV in the absence of recombination, as evidenced by the nucleotide diversity indices and recombination analysis. This speculation is further supported by a study conducted by Sattar et al. (2022). Our study detected four instances of mixed infections in plants, where TYLCV and ToLCPalV co-occurred alongside WmCSV isolates. Dual and even triple begomovirus infections within single plants are well-documented (Leke et al., 2011; Charoenvilaisiri et al., 2020; Batista et al., 2022; Rahman et al., 2023). These co-infections potentially contribute to the emergence of novel recombinant or pseudo-recombinant begomoviruses. This, coupled with a large vector population, can significantly enhance intra-regional genetic exchange among begomoviruses (Ouattara et al., 2022). Geminiviruses replicate through a rolling-circle mechanism, where the Rep protein binds to specific iteron sequences. In bipartite begomoviruses, similar iteron sequences in DNA-A and DNA-B facilitate successful replication (Hanley-Bowdoin et al., 2013). This explains why, during mixed infections involving monopartite and bipartite begomoviruses, the DNA-B component might not be efficiently trans-replicated by the monopartite begomovirus genome (Ouattara et al., 2022).

Overall, the comprehensive analysis of high-throughput sequencing data and comparative sequence analysis sheds light on the diversity and coexistence of begomovirus genomes in cucumber crops in Al-Ahsa, Saudi Arabia. The identified strains and isolates contribute valuable information to the understanding of begomovirus dynamics in the region.

Our study provides insights into the begomovirus dynamics in cucumber crops in Al-Ahsa, Saudi Arabia. The introduction of diverse begomoviruses into the Saudi Arabian agroecosystem raises concerns for agriculture, with potential factors including extensive trade, cross-border travel, and various transportation means contributing to the overland introduction of these viruses. The study revealed that TYLCV and ToLCPalV isolates originated from multiple introductions into Saudi Arabia. The begomovirus populations in our study underwent evolution under strong purifying selection and low recombination rate. Additionally, mixed infections involving TYLCV, ToLCPalV, and WmCSV in the same plants may contribute to the emergence of novel recombinants. This study contribute valuable information for future research, disease management, and the development of strategies to mitigate the impact of begomoviruses on agriculture in the Middle East.