INTRODUCTION Geographical Distribution, Host Range and Epidemics. Since the time when the first virus disease in papaya was described in 1929 from Jamaica (Smith, 1929), a number of diseases in papaya caused by different groups of plant viruses have been reported from papaya-producing areas in the world as a major limiting factor for papaya cultivation and papaya production (Adsuar, 1946; Capoor and Verma, 1948; Harkness, 1960; Lastra and Quintero, 1981; Wan and Conover, 1983; Chandra and Samuel, 1999). Among the different viruses infecting papaya, papaya ring spot diseases caused by Papaya Ring Spot Virus (PRSV) of family Potyviridae and papaya leaf curl disease caused by Papaya Leaf Curl Virus (PLCV) of family Geminiviridae cause major threat for papaya production in India and other papaya growing countries (Chitra et al., 2019; Reena et al., 2022). Out of PRSV and PLCV, latter is an important constraint to papaya production in particular, as it causes one of the most predominant and economically important diseases affecting several varieties of papaya in the Indian subcontinent. Papaya leaf curl disease was reported for the first time in India in 1939 by Thomas from Madras. The virus causes devastating disease of papaya characterized by upward or downward leaf curling, leaf crinkling and crumpling of leaves, vein clearing and thickening, yellow mottling, leaf puckering, blistering of leaves and general deformation of leaves. In the later stages of the infection, plants appear stunted with shortened internodes and show impaired fruit setting, leading to complete crop loss (Singh-Pant et al., 2012). In recent years, incidence and severity of leaf curl disease is increasing in papaya fields in India (Nariani, 1956; Saxena et al., 1998a-c) resulting in severe economic losses. In recent years, incidence of PLCV infection has been reported from different regions of the world in papaya as well as in a number of species of agricultural, horticultural importance and in a number of weed species too. Table 1 shows the countries from where the PLCV has been reported till date.Earlier epidemics of PLCV were reported from Asian and African Countries with majority reports coming from India, Pakistan, China, Bangladesh, Thailand, Sudan, Egypt, and some other countries. The papaya leaf curl virus (PLCV) is a monopartite begomovirus species belonging to Genus Begomovirus, Family Geminiviridae and is transmitted by white fly Bemisia tabaci (Gennadius) as the vector for virus transmission (Nariani, 1956). It has a circular, single stranded DNA (2.7Kbapprox.) called DNA-A (Saxena et al., 1998b). The PLCV genome is occasionally found to be associated with smaller DNA molecule called Alpha and Beta Satellites of 1.3Kb size approximately (Saxena et al., 1998a-c). PLCV has been recently reported to diversify its host range from Papaya to seasonal vegetable crops and other fibre crops (Varun and Saxena 2018). Generally, the Gemini viruses associate with a specific host for its multiplication and hence, infection. However, the host specificity is now being reported by many workers to be evolving into host non-specificity or a broader range of host specificity indicating expanding viral genome base over time. Such a host range expansion indicates evolution in the viral genome to diversify and thus establish infection into other crops which were earlier considered begomoviral non-host species. The situation has been rapidly changing as the virus is now fast spreading to larger stretch of these geographical regions and expanding its host range. The virus has an extensive host range now and is found in a number of host plants including many weeds. The virus infects Nicotiana tabacum (tobacco), Lycopersicon esculentum (tomato), Crotalaria juncea (sun hemp), Capsicum spp. (Chilli), Petunia, Zinnia, and Daturastramonium, Ageratum, Cucurbits, Soybean (Jaidi et al., 2015); Jatropa (More et al., 2019) and other vegetable crops. The host range extension also indicates virus selecting for alternate hosts as a survival mechanism. Further, switching of the PLCV between perennial host (such as papaya) to seasonal host (such as agricultural crops) has enabled the virus to be a bigger threat to the crop growers and to the crop yield over time. Also, viral infection and viral load in weeds or in agricultural or horticultural crops may play a possible role as a reservoir host in transmission of this virus. The expansion of the virus is thus becoming rapid and effective over time and space. The present paper discusses the expanding host range of PLCV over the last two decades into non-papaya host species and tries to discern underlying patterns of their rapid host expansion in the last two decades. MATERIALS AND METHODS Begomoviral PLCV isolates (full length and betasatellites) reported from non-papaya host were obtained from NCBI GenBank Database. NCBI website was accessed between 01.06.2021 to 25.10.2021. Full-length DNA-A genomic component sequences and Beta-satellite sequences are available for isolates of PLCV infection reported from different cultivated plant species as well as weeds. Table 2 summarizes the various full length begomoviral PLCV isolates reported from host species other than Papaya. Table 3 summarizes the PLCV Betasatellites isolates reported from non-papaya host species. Nucleotide sequence used in the study. A total of 109 full length begomoviral PLCV isolates were obtained from NCBI GenBank (www.ncbi.nlm.nih.gov/nucleotide). Out of these 109, only 09 sequences belonged to PLCV reported from Papaya from different regions. These09PLCV isolates from papaya were randomly selected as reference sequence for the purpose of standard referencing and comparison based on sequence similarity. Remaining 100 sequences are full length begomoviral PLCV isolates reported from non-papaya host species as mentioned in Table 2. The accession number and the species from where these are reported is mentioned in Table 2. These 109 sequences were subjected to same treatment and were used in all downstream analyses. Percent Identity Matrix(PIM) Analyses. The 109 full length begomoviral PLCV sequences were subjected to percent identity matrix analyses using Mega 11.0 version (available at www.megasoftware.net; Tamura et al., 2021). The percentage identity so obtained was manually color coded using the scheme: Green color for all the pairs with 97% and above identity, Yellow color for all the pairs with 96.99%-90% identity, Blue color for 89.99%-.80% and Red color for all the pairs for 79.99% and less. Multiple Sequence Alignment. The entire dataset of 109 full length begomoviral PLCV sequences as mentioned earlier were converted to FASTA format and subjected to multiple sequence alignment (MSA) using Clustal W package (Thompson et al., 1994) built in the MEGA 11.0 software (Tamura et al., 2021). The sequences were truncated at the start of the sequences for making an equal length alignment with a total of 2937 positions (including gaps). Phylogenetic Tree Construction. The multiple sequence alignment file was subjected to phylogenetic and molecular evolutionary analyses using MEGA version 11 (Tamura et al., 2021). The parameters deployed in phylogenetic analyses were: Statistical method: (ML) Maximum Likelihood; Test of Phylogeny: Bootstrap Method; No. of Bootstrap Replications: 1000; Substitutions Type: Nucleotides, Model/Method: Tamura-Nei Method; Rates among sites: Gamma Distributes with invariant sites (G+I); No. of discreet gamma categories: 5; Gap/missing data treatment: use all sites; Select codon positions: Tick all; ML Heuristic method: Nearest-Neighbour Exchange (NNI); Initial Tree for ML: Make initial tree automatically; Branch Swap Filter: None; Number of threads: 3. It took nearly 38 hours to complete the phylogenetic tree construction. The ML tree thus obtained was saved in PNG format for further analyses. RESULTS AND DISCUSSIONS Begomoviral PLCV reported from non-papaya host species. Table 2 depicts the begomoviral PLCV sequences reported in the NCBI GenBank database that have been found to cause leaf curl disease of plant species other than papaya. PLCV, like most monopartite begomoviruses is involved in the major disease complexes affecting various crops in the “Old World” causing diseases in a number of plants belonging to (but not limited to) families including Asteraceae, Amaranthaceae, Apocynaceae, Solanaceae, Euphorbiaceae, Fabaceae, Gentianaceae, Malvaceae, Passifloraceae, Saliacaceae, Acanthaceae, Brassicaceae. The effects are economically most devastating in Solanaceae. PLCV complex initially reported in Papaya crops is now affecting vegetable and other crops along with a number of weeds. It has been documented that PLCV infects 29 different plant species as reported in Table 2. These reports of PLCV infecting non-papaya species has been reported from Bangladesh, China, India, Indonesia, Iran, Italy, Malaysia, Pakistan, Sri Lanka, Spain, Taiwan, Thailand, Tunisia & Vietnam. Table 3 summarizes Betasatellites associated with PLCV reported from non-papaya host species. These species from where DNA Betasatellites have been reported belong to agriculturally important species such as grain legumes (Vigna radiata),Tomato, and some weed species such as Parthenium hysterophorus (Kumar et al., 2016) and Pseuderanthemum reticulatum. In 2003, the association of PLCV with leaf curl disease on non-papaya host was reported for the first timeby Mansoor et al. (2003a-b). Subsequently, over the period 2003-2020, PLCV was identified in 29 different species belonging to 12 angiosperm families. Out of these, most reports belong to Ageratum conyzoides (Asteraceae), Capsicum annuum (Bell pepper) (Kim et al., 2018), Lycopersicon esculentum (Solanaceae), Nicotiana sp., (Solanaceae). These reports are mainly from India, China and Pakistan. Meanwhile, this virus has spread to various parts of the Indian sub-continent, expanding its host range drastically. Between 2000 and 2020, besides tomato, PLCV has been reported to be associated to infections of various hosts such as Amaranthus cruentus, Calendula officinalis, Calotropis procera, Capsicum sp., Catharanthus roseus, Codiaeum variegatum, Corchoropsis timentosa, Crotalaria juncea, Croton bonplandianum, Cyamopsis tetragonoloba, Eclipta prostrata, Euphorbia pulcherrima, Eustoma grandiflorum, Capsicum annuum (Bell pepper), Glycine max, Gossypium sp., Vigna radiata, Kalimeris indica, Nicotiana glutinosa, Nicotiana tabacum, Parthenium hysterophorus, Passiflora sp., Physalis peruviana, Populus alba, Pseuderantheum reticulatum, Raphanus sativus and Rhynchosia capitata (Table 2). This expansion of the PLCV into a number of plant families affecting so many species of economic importance is alarming. Over two decades, this virus in particular has managed to infect such a range of species belonging to 12 angiosperm families suggest higher rates of recombinations and massive spread of the virulent strain by the vector Bemisia tabaci. The virus in itself can now be considered a broad-spectrum generalist as it is now found in large phytogeographical region infecting so many host species that it seems tolose its host specificity. Such a huge expansion can also indicate that the begomoviral complex may become more detrimental to the original host Papaya which is under threat due to a number of viral diseases apart from PLCV. Percent Identity Matrix Analyses of the Begomoviral PLCV reported from non-papaya host species along with selected begomoviral PLCV reported from papaya. S1 (supplementary table) shows the percent identity matrix of the 109 full length begomoviral PLCV sequences reported from non-papaya host species as well as from papaya. As mentioned earlier, the percentage identity so obtained was color coded as per the following scheme: green color for all the pairs with 97% and above identity, Yellow color for all the pairs with 96.99%-90% identity, Blue color for 89.99%-.80% and Red color for all the pairs for 79.99% and less. Accordingly, a general study of S1indicates that the entire dataset of the sequences can be divided into following 02 sub-groups: a.) China Group that contains sequences reported from China, Vietnam, and Taiwan (Sr. No. 1-54) of S1and b.) Indian Groupthat contains sequences reported from India and Pakistan (Sr. No. 55-109) of S1. Within the China group (Sr. 1-55), majority of the pair-wise percent sequence identity lies in the yellow coded regionindicating that most sequences within the China group have percentage identity of 90% to 96.99%. It is worth mentioning that within the China group, the sequences have been reported from a number of different non-papaya host species such as Tomato, Ageratum, Eclipta, Tobacco, Passiflora, Corchoropsis, Euphorbia which are having a sequence identity lying in yellow color-coded region i.e., 96.99%-90% identity. This is followed by Red color coded region i.e. an identity percent of 79.99% and less. These values clearly indicate sequence divergence to a large extent within the China group indicating multiple parental types of PLCV strains from where they have spread into multiple host species. Interestingly, within the Indian group, majority of the pair-wise percent sequence identity lies in the redcolor-coded region i.e. percentage identity of 79.99% or less. This is followed by second largest set of pair wise similarity lying in the blue color-coded region i.e. percentage identity in the range of 80-89.99% (S1). This result indicates very high divergence mainly due to higher frequency of recombination between very different parental types of PLCV strains that have resulted in a such a lower range of percentage identity within the Indian group (Sr. No. 55 onwards) of S1. It further indicates that not only the Indian group is slightly more divergent in its sequence composition within, but it is also more divergent than the China group, as the latter has most pairwise identity lying in yellow followed by red region. Also, within the Indian Group, the non-papaya host species reported are way more than the China Group with Tomato, Jatropha, Calendula, Croton, Radish, Cluster bean, Populus, Codiaeum, Crotalaria, Rhynchosia, Cotton, Physalis, Cestrum, Capsicum, Catharanthus, Soybean, Cape Gooseberry, Aster, Amaranthus as the major host species reported from India and Pakistan. This trend indicates geographical distribution as one of the factors behind such a stratification of the entire dataset into China and Indian group, with China, Vietnam and Taiwan grouping together and India and Pakistan grouping as one in the pairwise sequence identity matrix analysis. Further, the occurrence of lower percentage identity is suggestive of higher recombination rates and multiple parental types being responsible for the outbreak into non-papaya host species. Multiple Sequence Alignment of the Begomoviral PLCV reported from non-papaya host species and papaya. Multiple Sequence Alignment of approx. 2736 bp long nearly full length 109 begomoviral PLCV (data not shown) revealed multiple conserved sites ranging from 10-20 bases at different intervals flanked by hypervariable regions. These highly conserved regions belong to Coat Protein (CP) or the AV1 genes and other highly conserved Inter-genic Regions (IR)/ Common Regions (CR), containing repeat sequences or Iterons and conserved putative regulatory TATA boxes regions as has been reported by other workers (Singh-Pant et al., 2012; Stanley, 1995; Hanley-Bowdoin et al., 1999). Phylogenetic analyses of the full length Begomoviral PLCV sequences used in this study. Fig. 1 is the Maximum Likelihood (ML) based phylogenetic tree obtained from MEGA 11.0 (Tamura et al., 2021). In the phylogenetic tree so obtained, in a similar fashion as was indicated in the percent identity matrix (S1), here also the entire dataset could be sub-grouped into two major clusters i.e.the sequences reported from China, Vietnam and Taiwan hereinafter referred to as China Group and sequences reported from India and Pakistan hereinafter referred to as the Indian Group of begomoviral PLCV. The tree indicated a similar trend of sequences from different host species reported from China, Vietnam and Taiwan grouping together in multiple clusters with branches and sub-branches. Interestingly, in majority of the cases although with some exceptions, all begomoviral PLCV sequences reported from a single (non-papaya) host species were together suggesting common lineage or proximity with each other in space and time during vector mediated transmission occurring between different host species present proximally resulting in such trends. Similarly, all the sequences reported from different host species (excluding papaya ones) from India and Pakistan clustered together in a similar fashion, where in majority of cases, sequences from same species were clustered together but in some cases sequences from different species also shared the same node suggesting close relationship mainly due to direct vector transmission of the PLCV strain between the different host species. A novel strain PLCrV clustered together in the middle of the tree with no sub-branching with any other PLCV and shared the common node with the Indian group of sequences. This indicates that PLCrV is highly distinct from the rest of the begomoviral sequences. Within the Indian group, two distinct clusters can be observed, one containing majority of sequences reported from different host species in India and the other consisting of sequences reported from multiple different host species reported from Pakistan. Although in both these distinct group within the Indian group, cross-contamination of either of the two sub-groups is commonly seen. Yet, it is conclusive evidence of certain sequences being phylogenetically and molecularly more similar or parallelly evolved than the China group. Recombination in begomoviruses as a cause and effect of host expansion. In Geminiviruses, genomic diversification and evolution has been driven by recombination events. Such phenomenon results in emergence of new recombinant viral strains. With recombinant strains comes greater biological fitness in view of higher degree of virulence as compared to their parental types from where they have been derived. Such recombinant viral strains naturally outcompete the host plant resistance strategies (Monci et al., 2002). Natural genetic recombination events are common in geminiviruses, and these occur among strains, species and even within and across genera although rarely (Bisaro, 1996; Padidam et al., 1999). It has also been reported that in many cases, geminiviruses occur as mixed infections with two or more virusesleading to increased viral DNA accumulation and increased symptom severity (Harrison and Robinson, 1999). Vector mediated transmission mainly by Whitefly is another factor contributing to recombination and its increasing host range on which it can feed on increases virus spread into a large number of species (Padidam et al., 1999). Recombination events has negative effects on the quality of the reconstructed phylogenetic tree (Posada and Crandall, 2002; Ruths and Nakhleh, 2005). Hence recombination detection must be done to ensure accurate phylogenetic analyses and to understand parental type recombination and mechanisms of virus diversification.