Identification of Novel Begomoviruses Associated with Leaf Curl Disease of Papaya (Carica papaya L.) in India

Abstract

Papaya (Carica papaya L.) is one of the most important fruit crops grown in tropical and subtropical regions of the world. Papaya leaf curl disease is one of the greatest concerns next to Papaya ring spot disease for India and the world. A survey was conducted during the year 2019 to 2021 for assessing the leaf curl disease incidence in five major papaya-growing districts of Karnataka State, India. The incidence ranged from 10 to 21 percent, with plants expressing typical begomovirus symptoms. Thirty-two virus-infected papaya samples (PLC-1 to PLC-32), collected from different farmer’s fields, gave positive amplification for begomovirus detection. Based on the partial genome analysis, 13 representative papaya leaf curl isolates were selected for complete genome amplification by rolling circle DNA amplification (RCA). The RCA products were cloned, sequenced and analyzed. Based on the analysis and strain classification criteria for begomoviruses, five isolates (PLC-2, 3, 9, 11 and 18) were considered variants of Chilli leaf curl virus (ChiLCV). Isolate PLC-22 is considered a strain of ChiLCV, with 93.5% nt identity sharing. Similarly, isolate PLC-28 is considered a strain of Croton yellow vine mosaic virus (CYVMV), and isolates PLC-25 and PLC-31 were considered as strains of Papaya leaf curl virus (PaLCuV). Among the remaining four isolates, three (PLC-1, PLC-4 and PLC-7) share more than 91% nt identity among them and less than 91% nt identity with all other reported begomovirus isolates. Hence, they are considered to be isolates of the novel begomovirus, and the name Papaya leaf curl Bagalkote virus [India:Karnataka:Bagalkote:Papaya:2021] is proposed. One isolate (PLC-32) is also found to be distinct from all other begomovirus isolates, including the isolates in the current study also considered to be novel begomovirus, for which we propose the name Papaya leaf curl Haveri virus [India:Karnataka:Haveri:Papaya:2021]. The putative recombination analysis of all 13 papaya isolates showed that a major part of the viral genome was likely descended from the begomoviruses reported previously. This is the first report on the diversity and a distribution of the begomoviruses infecting papaya in Karnataka, India. The current investigation results revealed five major papaya-infecting begomoviruses (PaLCuBKV, ChiLCV, PaLCuV, CYVMV and PaLCuHV) in the sampled regions.

1. Introduction

Carica papaya, commonly known as “papaya”, L. belongs to the family, Caricaceae, and is a commercially cultivated species in tropical and subtropical regions of the world. Papaya is known to originate in southern Mexico and Costa Rica [1] and was introduced into India by Portuguese travelers [2]. It is cultivated in Brazil, China, India, Indonesia, Jamaica, Mexico, Nigeria, Peru, the Philippines, Taiwan, Thailand and the USA [3]. Owing to the realization of its importance in various industries, the cultivation of papaya has significantly increased across the globe. The current global production of papaya is about 13.89 Mt and is grown over an area of 4.68 lakh ha, with a productivity of 29.69 t/ha [3]. India is the largest producer of papaya, accounting for 44.04 percent of global production [4], which made papaya emerge as an alternative cash crop to a banana in India.

Many biotic and abiotic factors are threatening papaya production. Of these, viruses are a major bottleneck for the production of papaya, causing serious damage to the fruits and devastating the entire crop [5]. More than 29 viruses were reported to infect papaya throughout the world [6]. Among these viruses, Papaya ring spot virus (PRSV), Papaya leaf curl virus (PaLCuV) and Papaya mosaic virus (PMV) have gained global significance in all the papaya-growing countries. In India, PRSV is one of the greatest concerns, with the potential to cause 100 percent yield loss [7], followed by PaLCuV. A survey of the literature showed that the incidence of papaya leaf curl disease is greater in Asian countries (Bangladesh, China, India, Iran, Korea, Nepal, Oman, Pakistan, and Taiwan) [8,9,10,11,12,13,14,15,16]. Recently, the virus is also reported in the USA and Mexico, with disease incidence ranging from 40 to 100 percent [6,17]. Of the different species of Begomovirus, PaLCuV is more predominant on papaya. However, it also known to infect various crops like Rhynchosia capitata [18], Soyabean [18], Gossypium sp. [19], aster [20], grain amaranth [21], ornamental shrub Pedilanthus tithymaloides [22] and Aster alpines [23] and cause severe crop losses worldwide. The leaf curl disease of papaya was first recorded in India [24]. Later, the causal agent was identified as Begomovirus and shown to be transmitted by whitefly (Bemisia tabaci) as a vector [8,25,26].

The family, Geminiviridae, contains 14 genera, classified based on the host range, vector transmission and genome organization. The genus, Begomovirus, is the largest group of plant-infecting viruses. They have covalently closed circular DNA molecules of approximately 2.5 to 3.0 kb as their genome, which are encapsidated in geminate isometric icosahedral capsid (18 × 20 nm) particles [27]. The begomoviruses are divided into monopartite (single component homologous to DNA-A component of bipartite Begomovirus) and bipartite (contain two components, DNA-A and DNA-B) begomoviruses based on the number of genomic components the virus species has. The DNA-A component contains six to seven open reading frames (ORFs) in sense and antisense strands, which encode proteins responsible for viral encapsidation, replication, movement and insect transmission. DNA-B encodes two ORFs (BV1 and BC1), one each in sense and antisense strands, which are known to be involved in intracellular virus movement (Nuclear Shuttle Protein-NSP) and intercellular virus movement (Movement Protein-MP) within the plant [28,29].

Most of the Old World (OW) and several New World (NW) begomoviruses are commonly associated with three categories of novel satellite-like ssDNA molecules, known as alpha, beta and delta satellites, which depend upon their helper viruses for encapsidation, movement and insect transmission [30]. Symptoms of leaf curl disease, typical of begomovirus infection, were observed on papaya during a survey undertaken at different locations in Karnataka state, India. Currently, there is scant information available on the begomoviruses infecting papaya. Considering this backdrop, the present study was carried out to determine the prevalence of disease, characterization, diversity, recombination and evolutionary relationship of begomoviruses infecting papaya in Karnataka State, India. The aim of the current work was to determine the sequence diversity of begomoviruses associated with papaya and their recombination.

2. Materials and Methods

2.1. Survey and Collection of Virus Isolates

A roving survey was carried out at different locations and covered five districts (Bagalkote, Belagavi, Haveri, Kalaburagi and Vijayapura) of Karnataka, India, during the year 2019 to 2021. Its aim was to determine the prevalence and distribution of leaf curl disease on papaya. Disease incidence was estimated by counting the number of infected plants over the total number of plants in 25 × 25 mt micro plots within each field by visual examination. From the whole of these five districts, 32 papaya leaf samples showing leaf curl symptoms were collected from 15 locations in separate polythene bags, labeled, and brought to the Plant Pathology Laboratory, College of Horticulture, University of Horticultural Sciences, Bagalkote, India for further processing. Each sample was considered a separate isolate and was designated from PLC-1 to PLC-32 (Table S1).

2.2. DNA Isolation, Detection and Characterization of Begomovirus

Total genomic DNA from 32 symptomatic and one asymptomatic papaya samples was extracted using the CTAB method [31]. It was then quantified using a Thermo Scientific™ Nanodrop™ 2000 spectrophotometer (Waltham, MA, USA) and underwent electrophoresing on a 0.8 percent agarose gel. The extracted DNA was subjected to PCR for the detection of begomovirus using degenerative DNA-A and DNA-B specific primers, as described by Rojas et al. [32] (Table S2). Further, 13 representative samples were selected for whole viral genome amplification by the rolling circle amplification (RCA) method. Amplified RCA products (2 μL) were digested with BamH1 and Kpn1 restriction enzymes to obtain monomeric DNA-A/DNA-B components (~2.7 kb). Digested products were cloned into a pBluescript KS (+) cloning vector and transformed into the DH5α strain of Escherichia coli [33]. Plasmid DNA was isolated from the three clones obtained from each isolate by alkaline lysis method, and the presence of insert was confirmed by restriction digestion with a BamH1 restriction enzyme. Three clones from each sample were sequenced using an automated DNA sequencer from the ABI (Applied Biosystems) DNA sequencing facility at Medauxin Pvt. Ltd., Bengaluru, Karnataka, India.

2.3. Viral Genome Comparison, Phylogenetic and Recombination Analysis

Viral genome sequences, obtained after sequencing, were subjected to VecScreen software to remove vector sequences. The sequences obtained were assembled using BioEdit (version 7.2) (http://bioedit.software.informer.com) (accessed on 2 July 2022) [34]. Sequence similarities were checked at the NCBI database using BLASTn (http://www.ncbi.nlm.nih) (accessed on 2 July 2022), and sequences showing the highest blast score with present isolates were retrieved from the NCBI database for analysis. Open reading frames (ORFs) in the sequences were identified by the ORF finder available at NCBI (http://www.Ncbi.nlm.nih.Gov/gorf/gorf.html) (accessed on 2 July 2022). Pairwise nucleotide identity between sequences, obtained in the current study and reference sequences from GenBank (Table S3), were calculated by using Sequence Demarcation Tool (SDT version 1.2) (http://web.cbio.uct.ac.za/~brejn ev/) (accessed on 2 July 2022) [35]. Phylogenetic trees were generated using neighbor-joining method by MEGA11 tool with a 60 percent cut-off value and 1000 bootstrap replicates [36]. Recombination breakpoint analysis was performed between papaya isolates, along with selected begomoviruses, using different recombination detection methods (RDP, GENECOV, Bootscan, Max Chi, Chimera, Si Scan, and 3Seq) and integrated with RDP 5 by employing Bonferroni correction with a confidence value greater than 95 percent (p value 0.05) [37]. In order for begomoviruses to be considered recombinants, the recombination signal should be supported by at least three methods in order to be counted as such. In RDP analysis, the length of the window was set to 10 variable sites and the step size was set to one nucleotide. p Values were estimated by randomizing the alignments 1000 times.

3. Results

3.1. Survey for Incidence and Symptomatology of Papaya Leaf Curl Disease

Leaf curl symptomatic papaya plants were observed at all 15 different locations surveyed, distributed in five districts (Bagalkote, Belagavi, Haveri, Kalaburagi and Vijayapura) of Karnataka, India. Survey data indicated that the Red Lady variety of papaya was commonly grown in surveyed areas and was found to be susceptible to leaf curl disease caused by begomoviruses. Incidence of papaya leaf curl ranged from 10 to 21 percent. During the survey, infected papaya plants were observed from 35 days after transplanting to the harvesting stage of crop growth in different farmers’ fields. Infected plants exhibited different symptoms, including vein clearing, vein thickening, zigzag vein, vein enation, leaf rolling, downward leaf curling, severe leaf curl, yellowing, leathery leaf, distorted petioles and deformation, vein twisting and petiole bending (Figure 1). In addition, diseased fruits were undersized and distorted. Fruits from infected plants were reduced in number and never developed to full maturity, often falling from trees prematurely. During the survey, 32 infected papaya leaf samples presented with different kinds of symptoms were collected from 15 locations suspected to be infected by begomovirus (designated as isolate PLC-1 to PLC-32) and used for further characterization.

3.2. Detection of Begomovirus

Total DNA from 32 symptomatic and one asymptomatic papaya plant was extracted and subjected to PCR amplification using primers specific to the begomovirus. All the symptomatic samples resulted in an expected amplicon of 1.2 kb, specific to begomovirus, indicating the begomovirus association with all infected samples. Further, all samples showed negative amplification for the DNA-B specific primers, indicating that the begomoviruses associated with samples were monopartite. Amplified PCR products (1.2 kb) for begomovirus-specific primers were cloned and sequenced. Analysis partial viral genome of the 32 papaya leaf curl isolates (PLC-1 to PLC-32) had an identity percentage within the isolates that ranged from 80.7 to 99.3%. Out of 32 papaya leaf curl isolates, 25 isolates (PLC-2, 3, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 29 and 30) shared the maximum nt identity of more than 95% among them and were closely related with Chilli leaf curl virus. Two isolates, PLC-25 and PLC-31, shared nt identity of more than 95% identity between them and were closely related to Papaya leaf curl virus (PaLCuV). One isolate, PLC-28 was closely related to Croton yellow vine mosaic virus. Further, four isolates (PLC-1, PLC-4, PLC-7 and PLC-32) shared very low nt identity (less than 89% nt identity) with other isolates and were related to the Tomato leaf curl virus. Based on nt identity analysis of the partial genome of begomovirus isolates, 13 representative isolates (PLC-1, PLC-2, PLC-3, PLC-4, PLC-7, PLC-9, PLC-11, PLC-18, PLC-22, PLC-25, PLC-28, PLC-31 and PLC-32) were selected for complete viral genome amplification via the RCA method.

3.3. Characterization of DNA-A like Sequences

A complete genome of 13 begomovirus isolates (PLC-1, 2, 3, 4, 7, 9, 11, 18, 22, 25, 28, 31 and 32) from papaya was amplified, cloned and sequenced. It was determined to be from 2737 to 2773 nucleotides (nt) in length (Accession no OL630090 to OL677453). The genome structure of begomovirus isolates is typical to the Old World monopartite begomoviruses (Figure 2) encoding six ORFs, two in plus-strand and five in minus-strand of DNA-A component. Details of the coding capacity and length of ORFs were given in

Table 1. Genome organization of DNA-A component of begomoviruses isolated from papaya.

Begomovirus		PLC-1	PLC-2	PLC-3	PLC-4	PLC-7	PLC-9	PLC-11	PLC-18	PLC-22	PLC-25	PLC-28	PLC-31	PLC-32
		OL630090	OL630091	OL677445	OL677443	OL677444	OL677446	OL677447	OL677448	OL677449	OL677450	OL677451	OL677452	OL677453
Nucleotide length (nt)		2763	2760	2763	2757	2773	2762	2765	2763	2763	2773	2750	2772	2737
IR a	SSC	2612–2763 and 1–144	2609–2760 and 1–146	2612–2763 and 1–146	2616–2757 and 1–144	2617–2773 and 1–145	2611–2762 and 1–145	2613–2765 and 1–145	2612–2763 and 1–146	2612–2763 and 1–146	2617–2773 and 1–145	2615–2750 and 1–145	2614–2772 and 1–146	2585–2732 and 1–119
	PO	296	298	293	286	302	297	297	298	298	303	280	305	290
AV2 a	SSC *	144–491	147–503	147–503	145–501	146–502	146–502	146–502	147–503	147–503	146–502	146–502	146–502	120–458
	PO *	348	357	357	357	357	357	357	357	357	357	357	357	339
	PP *	115	118	118	118	118	118	118	118	118	118	118	118	112
	PWP *	13.1	13.6	13.5	13.5	13.6	13.6	13.6	13.7	13.7	13.7	13.6	13.2	12.7
AV1(CP) a	SSC	304–1074	307–1077	307–1077	305–1075	306–1076	306–1076	306–1076	307–1077	307–1077	306–1076	306–1076	306–1076	280–1050
	PO	771	771	771	771	771	771	771	771	771	771	771	771	771
	PP	256	256	256	256	256	256	256	256	256	256	256	256	256
	PWP	29.5	29.3	29.5	29.4	29.5	29.7	29.6	29.6	29.4	29.6	29.5	29.5	29.4
AC1(Rep) a	SSC	2611–1526	2608–1526	2611–1526	2615–1530	2616–1525	2610–1525	2613–1525	2611–1526	2611–1526	2616–1531	2616–1531	2613–1525	2584–1499
	PO	1086	1083	1086	1086	1092	1086	1089	1086	1086	1086	1086	1089	1086
	PP	361	360	361	361	363	361	362	361	361	361	361	362	361
	PWP	40.5	40.1	40.4	40.7	40.9	40.4	40.5	40.4	40.5	40.4	40.6	40.5	40.4
AC2 (TrAP) a	SSC	1623–1222	1623–1219	1623–1219	1627–1223	1628–1224	1622–1218	1622–1218	1623–1219	1623–1219	1628–1224	1628–1224	1628–1224	1596–1192
	PO	402	405	405	405	405	405	405	405	405	405	405	405	405
	PP	133	134	134	134	134	134	134	134	134	134	134	134	134
	PWP	14.9	15.2	15.3	15.2	15.2	15.3	15.3	15.2	15.2	15.2	15.3	15.3	15.2
AC3 (REn) a	SSC	1484–1077	1478–1074	1478–1074	1482–1078	1483–1079	1477–1073	1477–1073	1478–1056	1478–1074	1483–1079	1483–1079	1483–1079	1457–1047
	PO	408	405	405	405	405	405	405	423	405	405	405	405	411
	PP	135	134	134	134	134	134	134	140	134	134	134	134	136
	PWP	16.1	15.2	15.7	15.9	15.9	15.3	15.8	16.5	15.7	15.8	15.7	15.8	15.9
AC4 a	SSC	2454–2161	2457–2158	2454–2161	2458–2120	2459–2202	2459–2160	2462–2163	2460–2161	2460–2161	2459–2202	2459–2202	2459–2202	2432–2253
	PO	294	300	294	339	258	300	300	300	300	258	258	258	180
	PP	97	99	97	112	85	99	99	99	99	85	85	85	60
	PWP	10.8	11.3	11.1	12.6	9.7	11.3	11.3	11.3	11.3	9.4	9.2	9.5	6.9
AC5a a	SSC	612–295			1144–740							1145–741		791–531
	PO	318			405							405		261
	PP	105			134							134		86
	PWP	11.8			15.9							15.4		9.8
AC5b a	SSC				613–296							614–291
	PO				318							324
	PP				105							107
	PWP				11.8							11.9

* NT: Nucleotide (nt); SSC: Start–Stop codon (nt); PO: Predicted size of ORFs (nt); PP: Predicted size of protein (no. of amino acids); PWP: Predicted weight of protein (kDa). ^a Genes are indicated as IR: Intergenic region; AV1: Coat protein (CP); AV2: Pre-coat protein; AC1: Replication-associated protein (Rep); AC2: Transcriptional activator protein (TrAP); AC3: Replication enhancer (REn). The products encoded by ORFs AC4 and AC5 have yet to be named.

.

3.4. Relationship between Papaya-Infecting Begomoviruses with Known Begomoviruses

The complete genome sequences of 13 begomovirus isolates were compared with other begomoviruses sequences. Five isolates (PLC-2, PLC-3, PLC-9, PLC-11 PLC-18 and PLC-22) shared a sequence identity of more than 93.5% with several already known Chilli leaf curl virus (ChiLCV) isolates infecting different crops and were classified as a new variants of ChiLCV infecting different crops in India (

Table 2. Maximum percentage nucleotide sequence identity of DNA-A component of begomoviruses isolated from papaya with other known begomovirus isolates and assigning acronyms to the isolates.

Sl. No.	Begomovirus Isolate	Accession No.	Maximum NSI * (%)	Begomovirus	Name Assigned Based on NSI	Virus Acronyms
1.	PLC-1	OL630090	88.6	ToLCKV-[IN:GJ:AHM:Tom:16]-MH5770301	Papaya leaf curl Bagalkote virus [India:Karnataka:Belagavi:Papaya:2021]	PaLCuBKV-[IN:Kar:Bel:Pap:21]
2.	PLC-2	OL630091	96.3	ChiLCV-DU[IN:ND:DU:Pap:09]-HM140364	Chilli leaf curl virus [India:Karnataka:Belagavi:Papaya:2021]	ChiLCV-[IN:Kar:Bel:Pap:21]
3.	PLC-3	OL677445	96.5	ChiLCV-[IN:Kar:Rai:Chi:17]-MK161454	Chilli leaf curl virus [India:Karnataka:Belagavi:Papaya:2021]	ChiLCV-[IN:Kar:Bel:Pap:21]
4.	PLC-4	OL677443	85.4	PaLCuV-[IN:Kar:Madi:Cal:18]-MK087120	Papaya leaf curl Bagalkote virus [India:Karnataka:Bagalkote:Papaya:2021]	PaLCuBKV-[IN:Kar:Bgk:Pap:21]
5.	PLC-7	OL677444	88.6	BYVBV-[IN:OD:Bhu:Okra:03]-FJ589571	Papaya leaf curl Bagalkote virus [India:Karnataka:Bagalkote:Papaya:2021]	PaLCuBKV-[IN:Kar:Bgk:Pap:21]
6.	PLC-9	OL677446	98.5	ChiLCV-[IN:Kar:Rai:Chi:17]-MK161454	Chilli leaf curl virus [India:Karnataka:Bagalkote:Papaya:2021]	ChiLCV-[IN:Kar:Bgk:Pap:21]
7.	PLC-11	OL677447	98.5	ChiLCV-[IN:Kar:Rai:Chi:17]-MK161454	Chilli leaf curl virus [India:Karnataka:Bagalkote:Papaya:2021]	ChiLCV-[IN:Kar:Bgk:Pap:21]
8.	PLC-18	OL677448	98.0	ChiLCV-DU[IN:ND:DU:Pap:09]-HM140364	Chilli leaf curl virus [India:Karnataka:Kalaburagi:Papaya:2021]	ChiLCV-[IN:Kar:Kal:Pap:21]
9.	PLC-22	OL677449	93.5	ChiLCV-DU[IN:ND:DU:Pap:09]-HM140364	Chilli leaf curl virus [India:Karnataka:Kalaburagi:Papaya:2021]	ChiLCV-[IN:Kar:Kal:Pap:21]
10.	PLC-25	OL677450	92.4	PaLCuV-[IN:Kar:Madi:Cal:18]-MK087120	Papaya leaf curl virus [India:Karnataka:Kalaburagi:Papaya:2021]	PaLCuV-[IN:Kar:Kal:Pap:21]
11.	PLC-28	OL677451	93.5	CYVMV-[IN:ND:Cro:08]-JN817516	Croton yellow vein mosaic virus [India:Karnataka:Kalaburagi:Papaya:2021]	CYVMV-[IN:Kar:Kal:Pap:21]
12.	PLC-31	OL677452	93.0	PaLCuV-[IN:Kar:Madi:Cal:18]-MK087120	Papaya leaf curl virus [India:Karnataka:Vijayapura:Papaya:2021]	PaLCuV-[IN:Kar:Vij:Pap:21]
13.	PLC-32	OL677453	87.2	ToLCV-[In:Kar:Ben:Chr:17]-MG758145	Papaya leaf curl Haveri virus [India:Karnataka:Haveri:Papaya:2021]	PaLCuHV-[IN:Kar:Hav:Pap:21]

* NSI: Nucleotide Sequence Identity.

and Table S4). Isolate PLC-28 showed nt identity of 93.5% with Croton yellow vein mosaic virus (CYVMV) infecting croton, and the other two isolates (PLC-25 and PLC-31) shared an nt identity of more than 92% with Papaya leaf curl virus (PaLCuV) infecting calendula.

As per the current species/strain demarcation criteria [38], six isolates (PLC-2, PLC-3, PLC-9, PLC-11 PLC-18 and PLC-22) were strains of ChiLCV, one isolate (PLC-28) was a strain of CYVMV and two isolates (PLC-25 and PLC-31) were strains of PaLCuV. The remaining four isolates (PLC-1, PLC-4, PLC-7 and PLC-32) shared a nt identity of less than 91% with already known begomoviruses infecting different crops and have maximum nt identity with Tomato leaf curl Karnataka virus (ToLCKV) (MH5770301, 88.6% identity), Papaya leaf curl virus (PaLCuV) (MK087120, 85.4% identity), Bhendi yellow vein Bhubhaneswar virus (BYVBV) (FJ589571, 88.6% identity) and Tomato leaf curl virus (ToLCV) (MG758145, 87.2% identity) (Table 2 and Table S4). Among the four isolates, three (PLC-1, PLC-4 and PLC-7) shared more than 91% nt identity among them and less than 91% nt identity with all other reported begomovirus isolates, and hence were considered as isolates of novel begomovirus, which is also supported SDT analysis (Figure 3). Therefore, we propose the name Papaya leaf curl Bagalkote virus [India:Karnataka:Bagalkote:Papaya:2021]. One isolate (PLC-32) was distinct from all other begomovirus isolates, including the isolates in the current study, and was considered as a novel begomovirus, for which were propose the name Papaya leaf curl Haveri virus-[India:Karnataka:Haveri:Papaya:2021] (Table 2).

Comparison of overall nt identity of the intergenic region (IR) revealed that six isolates (PLC-2, 3, 9, 11, 18 and 22) showed a maximum nt identity, with several ChiLCV isolates infecting different crops. Additionally, two isolates (PLC-4 and PLC-31) shared maximum nt identity with PaLCuV isolates and another one isolate (PLC-1) shared more identity with ToLCKV isolates. However, isolates PLC-7 and PLC-25 shared maximum nt identity with BhVBhV and PLC-28, and PLC-32 shared maximum nt identity with CYVMV and PaLCrV, respectively (Table 2 and Table S4).

The IR nt sequence length among 13 begomovirus isolates in the current study ranged from 296 to 305, which is a similar range to that of other begomoviruses (ChiLCV, PaLCuV, BhVBhV, CYVMV, and PaLCrV) infecting different crops. Within the IR, incomplete two direct repeat iteron sequences (GGTACT nt positions 2628–2633 and 2662–2667) were identified in the IR of PLC-1, PLC-4, PLC-11 and PLC-18, whereas an inverted repeat of Rep binding sites was observed in PLC-3 (GGTACT nt positions 2627–2632 and 2662–2667) and PLC-9 (GGTACT nt position 2630–2635 and 2665–2670). Similarly, Rep binding sites in PLC-7 (GGTACT nt positions 2633–2638 and 2668–2673), PLC-2 and PLC-25 (GGTACT nt positions 2633–2638 and 2668–2673), PLC-22 (GGTACT nt positions 2628–2633 and 2663–2668) PLC-28 and PLC-31 (GGTACT nt positions 2625–2630 and 2661–2666) and PLC-32 (GGTACT nt positions 2605–2610 and 2639–2644) were predicted in the adjacent region of TATA box [39,40]. Iterons shared significant homology with iterons identified in DNA-A’s of the begomoviruses reported so far.

Comparisons of the predicted amino acid sequences (ORFs) of the 13 leaf curl isolates of papaya were made with other known begomoviruses (Table S5). ORF AV2, CP (AV1), Rep (AC1), TrAP (AC2) and ORF REn (AC3) of the leaf curl isolates (PLC-2, 3, 9, 11, 18, 22 and 32) showed maximum aa sequence identity, with several isolates of ChiLCV. ORF AV2 and CP (AV1) of leaf curl isolates (PLC-1, 4, 7, 25, 28 and 31) have showed maximum aa sequence identity, with several PaLCuV isolates infecting different crops. In the case of ORF Rep (AC1), two isolates each (PLC-28 and PLC-31) and (PLC-1 and PLC-4) showed a maximum aa sequence identity with CYVMV and ToLCV isolates, respectively. Similarly, ORF TrAP (AC2) of four papaya leaf curl isolates (PaLCuV-1, 7, 28 and 31) showed maximum aa sequence identity with several isolates of PaLCuV infecting different crops. However, ORF REn (AC3) of six papaya leaf curl isolates (PLC-1, 4, 7, 25, 28 and 31) has showed maximum aa sequence identity with several isolates of ToLCV infecting tomato and other crops. Similarly, ORFAC4 of nine papaya leaf curl isolates (PLC-4, 7, 3, 9, 11, 18, 22, 25 and 31) showed a maximum aa sequence identity with several isolates of PaLCuV. Isolates PLC-1, 2 and 28 showed maximum aa sequence identity with ToLCV, ChiLCV and CYVMV isolates infecting different crops, respectively (Table S5).

3.5. Phylogenetic Analysis

A phylogenetic tree was generated by comparing 13 papaya leaf curl isolates from the present study with 66 different begomoviruses available in the GenBank database. As expected from the pair-wise similarity analyses, phylogenetic analysis revealed grouping of all newly characterized sequences into two major clusters along with previously identified species (Figure 4). Cluster I contains six papaya leaf curl isolates (PLC-2, PLC-3, PLC-9, PLC-11, PLC-18 and PLC-22), closely clustered with the ChiLCV infecting different crops in India. Cluster II contains three papaya leaf curl isolates (PLC-4, PLC-25 and PLC-31), clustered with PaLCuV isolate infecting calendula in India. However, four papaya leaf curl isolates (PLC-1, PLC -7, PLC-28 and PLC-32) formed a distinct cluster with known monopartite begomoviruses, i.e., ToLCKV, BYVBV, CYVMV and ToLCV, reported from different parts of India.

3.6. Neighbor–Net and Recombination Breaking Point Analysis

Complete nt sequence of 13 papaya leaf curl isolates infecting papaya were subjected to neighbor–net analysis. Results revealed genetic variation patterns with high degrees of phylogenetic conflict, indicating that the viral sequences have recombinant origin and that there is possibility of them being the derived from begomoviruses (ChiLCV, PaLCuV, CYVMV and BYVBV isolates) infecting different crops. (Figure 5).

Computational tests were performed for detecting the recombination events using Recombination Detection Program (RDP), GENECOV, Bootscan, Max Chi, Chimera, Si Scan and 3Seq based upon statistical likelihood (p values) of strand swapping among parental genotypes (irrespective of inter and intragenic exchange or the number of crossovers/recombination breakpoints) using RDP5 package. An analysis provided evidence for the presence of past recombination events. Recombination events detected by at least three methods were considered to exclude unreliable signals and it was apparent from this analysis that these novel papaya-infecting begomoviruses exhibited multiple detectable recombination events with major and minor parent evidence that are most closely related to begomoviruses. Based on this information, we subjected the sequences to breakpoint analysis with the RDP program to determine the potential recombination sites present in the 13 papaya leaf curl isolates (Table S6 and Figure 6). The isolates PLC-2, PLC-18 and PLC-22 were detected with one and PLC-3, PLC-9 and PLC-11 isolates detected with two recombination events. The common recombination event for these six isolates was detected between PaLCuV (KX353622) (major parent) and PaLCrV (HE580236) (minor parent) (p < 1.875 × 10⁻³) for all recombination methods integrated with RDP) infecting papaya could be considered as a major event for the emergence of this virus and suggests that all papaya leaf curl isolates might have derived from recombination and derived from above parents.

Similarly, recombination events were also detected in PLC-25 (5 events) and PLC-31 (6 events) isolates. In these two recombination events were common, which are between ToLCKV (HM007094) (major parent) and CYVMV (FN645926) (minor parent) (p < 2.651 × 10⁻⁵) and CYVMV (FN645901) (major parent) and ChiLCV (OL677445) (minor parent) (p < 9.177 × 10⁻⁴). These could be considered major events for the emergence of this virus.

The isolate PLC-28 has recorded five recombination events. However, an event between PaLCuV (MH807203) as major parent and ChiLCV (HM140364) as minor parent was detected by all methods with a p value < 2.996 × 10⁻³. In the case of PLC-32, two events out of four, between ToLCV(MG758145) (major parent) and ChiLCV (OL630091) (minor parent) (p < 7.920 × 10⁻¹⁰) and PaLCuV (KX302713) and ChiLCV (OL677448) (p < 6.621 × 10⁻¹²) were detected by all methods and could be considered as major events for the emergence of this isolate. The isolate PLC-1 has been recorded four recombination events. In that event between CYVMV (FN645901) (major parent) and ChiLCV (OL677445) (minor parent) with p value < 9.177 × 10⁻⁴ was detected by all methods, whereas isolates PLC-4 and PLC-7 have showed six and three potential recombination events, respectively. In these two isolates, a single predicted event having PaLCuBKV (OL630090) as the major parent and BhVBhV (FJ589571) as the minor parent was recorded by all methods integrated with RDP with a p value < 7.023 × 10⁻⁵.

4. Discussion

Papaya-infecting begomovirus and its distribution is mainly restricted to Asian countries, and recently it was also reported in North American countries [6,17]. Papaya leaf curl disease was first recorded in Tamil Nadu by Thomas and Krishnaswami [24]. Later, viral infection of papaya was recorded from Pakistan [9] and India [25]. Although begomovirus prevalence on papaya is seen in different countries, their incidence and diversity are high in the states of India. This might be due to the large papaya production areas, the growing of susceptible varieties, and fast evolution of begomoviruses leading to emergence of new species or strains through recombination, and the occurrence of different whitefly cryptic species [41,42,43,44,45,46,47,48,49,50,51].

A sampling survey, conducted in the present study, focused on the major papaya-growing regions of Karnataka State, India and 32 samples were collected from 15 locations covering five districts (Bagalkote, Belagavi, Haveri, Kalaburagi and Vijayapura). Symptoms recorded at different locations were almost identical, with notable characteristic symptomatic expression seen on leaves with severe leaf curl. A literature survey also showed that similar type of symptoms, such as downward leaf curling, vein thickening and clearing, rubbery, fragile, distorted petioles curling, deformation and yellowing of young leaves, were well documented on papaya [10,15,25,52].

Preliminary analyses of 32 samples collected from the 15 locations representing five districts of Karnataka, India, conducted by PCR using degenerative primers, indicated that the papaya plants were infected with begomoviruses. Based on partial genome sequence information, 13 representative isolates were considered for complete genome sequencing in order to establish the begomovirus species diversity on papaya in the surveyed area. The genome sequence revealed its organization in all 13 isolates which were typical of begomoviruses originating from the Old World, coding for six ORFs [53,54,55]. Genome sequences revealed, that among the 13 isolates, 9 were showing nucleotide identity above the threshold percentage given for begomoviruses species demarcation [38] with already reported viruses, and four virus isolates were found to be distinct and had a nucleotide identity of less the 91%. Based on the criteria for distinguishing strains of the begomoviruses [56] among the 9 isolates showing >91% sequence identity, five isolates (PLC-2, PLC-3, PLC-9, PLC-11 and PLC-18) were considered as variants of Chilli leaf curl virus isolates infecting different crops, two isolates (PLC-25 and PLC-31) were considered as variants of PaLCuV infecting calendula, one isolate (PLC-22) was considered as a variant of ChiLCV infecting papaya, and another one isolate (PLC-28) was considered as a variant of Croton yellow vein mosaic virus (CYVMV) infecting croton. Based on the ICTV species demarcation criteria (nucleotide identity of less than 91% for DNA-A component), four isolates (PLC-1, PLC-4, PLC-7 and PLC-32) were considered as distinct. Among these, three shared more than 91% nt identity among themselves and were considered as variants of novel begomoviruses, for which we propose the name Papaya leaf curl Bagalkote virus [India:Karnataka:Bagalkote:Papaya:2021] (PLC-1, PLC-4 and PLC-7). Further, one isolate, PLC-32 was distinct from all the reported begomoviruses as well the isolates in the study and considered as another novel begmovirus, for which the name Papaya leaf curl Haveri virus [India:Karnataka:Haveri:Papaya:2021] (PLC-32) is proposed. These novel begomoviruses identified in papaya, adding to the known complexity of begomoviruses associated with this crop in southern India.

PaLCuV not only infects papaya, but it also infects Rhynchosia capitata [18], Soyabean [18], Gossypium sp. [19], aster [20], grain amaranth [21], ornamental shrub Pedilanthus tithymaloides [22] and Aster alpines [23]. Besides this, sequences of PaLCuV genome from different crops are available in the GenBank database such as sunhemp (GQ200448 and GQ200446), cluster bean (LT009399), Glycine max (JN807765) and Nicotiana glutinosa (HM143914), Physalis peruviana (LN845920), and Cape gooseberry (LT009400) from Indian subcontinent. Similarly, begomoviruses infecting other crops can also infect papaya and the results of the current study provided evidence for the cross-taxonomic movement of begomoviruses. Further, results also revealed that nearly 50% of infected papaya samples collected for the study were found to be associated Chilli leaf curl virus.

Analysis of the intergenic region (IR) sequences of the genome of 13 papaya-infecting begomovirus isolates revealed that two iteron sequences were identified in the genome component in all the isolates. These iterons are recognition sites for sequence-specific binding of the cognate Rep protein [57]. Depending on the viral species, any modifications with respect to the sequence, size or number of iterons, or even a point mutation in these motifs, can adversely affect Rep binding, both in vivo and in vitro [40,58].

Recombination is one of the major forces in increasing plant virus variability and adaptation to new hosts, often leading to the emergence of new variants and resistance-breaking virus strains. Recombination can also increase the fitness of plant viruses by repairing defective viral genomes or efficiently removing deleterious mutations that result from error-prone replication. The frequency of recombination is affected by several factors, including the viral replication proteins and various features of the viral templates involved. Host genes also affect viral recombination, suggesting a complex interaction between a given virus and its host during viral adaptation and evolution [59].

Recombination analysis of papaya isolates genomes suggests it might have played a part in the origin of begomovirus isolates causing papaya leaf curl. Different recombination methods were used for analysis in the current study to provide evidence for past recombination events, and such evidence was found in all 13 papaya-infecting begomovirus isolates with the major and minor parents. The evidence further suggests that most of the recombination breakpoints were detected in the Rep, CP and REn genes at sites that had previously been reported to be recombination hotspots within begomovirus genomes by scientists [60,61]. Most of the genome fragments present within these novel begomovirus sequences probably originated from begomovirus species known to infect papaya and other hosts. These results showed that the homologous recombination events occurred at an intra-specific level; these are much more often in DNA viruses with genetic crossing over [62]. Similar intra-specific recombination was reported by Venkataravanappa et al. [50]. Mixed infections, pseudo-recombination, recombination and synergism are the major factors for the evolution of novel species, resulting in diversification and evolution of begomoviruses, and resulting in turn in epidemics outbreaks [42,63,64,65]. Recombination is one of the main mechanisms that has contributed to the diversification and evolution of begomoviruses [66]. The identification of two begomovirus novel species among the 13 isolates sequenced indicated the evolutionary mechanism in aiding the emergence of new begomoviruses that are capable of infecting papaya and it was in line with the evidence found out in nucleotide and phylogenetic analyses. Current study has brought for the first-time information on the diversity and distribution of the begomoviruses infecting papaya in regions of Karnataka, India. Results of the current investigation revealed that there are presently five major papaya-infecting begomoviruses (PaLCuBKV, ChiLCV, PaLCuV, CYVMV and PaLCuHV) in the sampled regions of Karnataka, India.

5. Conclusions

The survey and characterization of begomoviruses associated with papaya leaf curl disease we performed here provided a picture of papaya associated begomovirus diversity in the major papaya production region of Karnataka, India. The survey, symptomatology, characterization and their phylogenetic relationship indicate that leaf curl disease in papaya is associated with an array of existing (ChiLCV, PaLCuV and CYVMV) and novel (PaLCuBKV and PaLCuHV) begomviruses in major papaya-growing areas of Karnataka, India. Documentation of different viruses and their strains of viruses present in a particular area provide important information for strategies to contain the leaf curl disease of papaya.