Alternanthera yellow vein virus (AYVV); a betasatellite independent begomovirus infecting Sonchus palustris in Pakistan

Alternanthera yellow vein virus (AYVV); um begomovírus independente de betassatélites infectando Sonchus palustris L. no Paquistão

Abstract

Satellites associated begomoviruses are the most diverse group of plant viruses in tropical and subtropical regions. In Pakistan, during field surveys in 2019-2020, Sonchus palustris (a weed plant) was observed showing begomovirus symptoms i.e., vein yellowing and mosaic patterns on leaves. Rolling circle amplification from total isolated DNA of symptomatic leaves was performed to amplify circular viral genomes. Subsequent cloning and sequencing showed that a new strain of Alternanthera yellow vein virus (AlYVV) is associated with vein yellowing disease of S. palustris. The identity percentage analysis through BLAST search and SDT analysis showed that the new strain is 94-98% identical to AlYVV isolates reported from Pakistan, India and China. In phylogenetic tree, it clustered with AlYVV-[PK:E prostrata:15-KX710155], AlYVV-[PK:E prostrata:13]-KX906697] and AlYVV-[PK:E prostrata:11]-KX906694] previously reported from Pakistan. There was no detectable level of betasatellite or any other satellite molecule in the samples studied here. Phylogenetic analysis of Rep and CP genes of AlYVV with corresponding genes of closely related viruses circulating in Southeast Asia showed intra-specific recombination involving both complementary and virion sense region of virus. Relaxed clock and Bayesian Skyline Plot analysis based on CP gene sequences indicated slight higher substitution rates (4.75 x 10^-3 substitutions/nucleotide/year). In the Indian subcontinent satellite-associated monopartite begomoviruses predominately infect crops and non-crop plants. But AlYVV is found infecting mostly non-crop plants independent of satellite molecules. We hypothesize here that AlYVV evolved as a true monopartite begomovirus in the Indian sub-continent and could be a great threat to introduced crops under suitable conditions. Such studies are crucial to understand probable future epidemics of begomoviruses in the region.

Keywords:
begomovirus; phylogeny; recombination; Bayesian analysis; mutation

Resumo

Os begomovírus associados aos satélites são o grupo mais diversificado de vírus de plantas encontrado em regiões tropicais e subtropicais. No Paquistão, durante pesquisas de campo entre 2019 e 2020, a espécie Sonchus palustris L. (uma planta daninha) foi observada apresentando sintomas de begomovírus, ou seja, amarelecimento das veias e padrões de mosaico nas folhas. A Amplificação em Círculo Rolante (ACR) a partir de DNA isolado total de folhas sintomáticas foi realizada para amplificar genomas virais circulares. A clonagem e sequenciamento subsequentes mostraram que uma nova cepa de Alternanthera yellow vein virus (AlYVV) está associada à doença do amarelecimento das veias de S. palustris. A análise da porcentagem de identidade por meio de pesquisa BLAST e análise SDT mostrou que a nova cepa é 94-98% idêntica aos isolados de AlYVV relatados no Paquistão, Índia e China. Na árvore filogenética, essa cepa se agrupou com AlYVV-[PK:E prostrata:15-KX710155], AlYVV-[PK:E prostrata:13]-KX906697] e AlYVV-[PK:E prostrata:11]-KX906694] relatada anteriormente de Paquistão. Não houve nível detectável de betassatélite ou qualquer outra molécula satélite nas amostras estudadas aqui. A análise filogenética de genes Rep e CP de AlYVV com genes correspondentes de vírus intimamente relacionados que estão circulando no Sudeste Asiático mostrou recombinação intraespecífica envolvendo a região complementar e de sentido viral do vírus. Relógio molecular relaxado e análise de Bayesian Skyline Plot (BSP) com base nas sequências do gene CP indicaram taxas de substituição ligeiramente mais altas (4,75 x 10-3 substituições/nucleotídeo/ano). No subcontinente indiano, os begomovírus monopartidos associados aos satélites infectam predominantemente culturas e plantas não cultivadas. Mas o AlYVV é encontrado infectando principalmente plantas não cultivadas, independentemente de moléculas satélites. Desenvolveu-se a hipótese de que o AlYVV evoluiu como um verdadeiro begomovírus monopartido no subcontinente indiano e pode ser uma grande ameaça às culturas introduzidas em condições adequadas. Tais estudos são cruciais para entender prováveis e ​​futuras epidemias de begomovírus na região.

Palavras-chave:
begomovírus; filogenia; recombinação; análise Bayesiana; mutação

1. Introduction

Whitefly-transmitted begomoviruses of Geminiviridae family are emerging as major pathogens on food and fiber crops in the Indian subcontinent (Mansoor et al., 2006). These viruses have ~2.6kb to 5.2kb circular single-stranded genomes and are transmitted by whitefly (Zerbini et al., 2017). The emergence of more diverse begomovirus species with broader host range could be the result of multiple infections, recombination, component capture/exchange and emergence of different biotypes of whiteflies (Mansoor et al., 2006; Czosnek et al., 2017). The natural sources of resistance are limited and often prone to breakdown due to emergence of highly virulent strains (Amrao et al., 2010).

Weeds serve as alternative host for crop-infecting begomoviruses, when main cropping season is not there. There are many reports of begomoviruses and associated satellites i.e., alphasatellite and betasatellite (Briddon and Stanley, 2006) infecting weeds, like Sonchus arvensis, Eclipta prostrata and Alternanthera (Mubin et al., 2009; Guo and Xhou, 2005; Mubin et al., 2010; Ghulam et al., 2017).

The genomes of begomoviruses consist of either two genomic components of approx. 2.8kb, known as DNA A and DNA B i.e., New World (NW) begomoviruses or of a single component homologous to the DNA A component of the bipartite viruses i.e., Old World (OW) monopartite begomoviruses (Briddon, 2003; Mansoor et al., 2006). Betasatellites are pathogenicity determinant molecules while alphasatellites are diverse in nature and do not play a significant role in the development of disease symptoms. Although Alpha-Rep protein was found to be a suppressors of gene silencing, but its downstream pathways are not understood (Nawaz-ul-Rehman et al., 2010). In the Old World, alphasatellites and betasatellites molecules are associated mostly with monopartite begomoviruses (Mansoor et al., 2001; Briddon and Stanley, 2006). We know more about begomoviruses infecting field crops as compared to overall begomovirus diversity in agriculture ecosystem. Based on available data of begomoviruses diversity, it can be hypothesized that a large number of unknown begomoviruses reside within non-cultivated plant species (Haider et al., 2007; Iram et al., 2005; Murtaza et al., 2018; Mubin et al., 2009, 2012).

The Sonchus palustris belonging to family Asteraceae is an annual herb found in subtropical and tropical regions of the world including Pakistan. S. palustris can grow and thrive in the varied geographical conditions, mainly found around the wet crop fields and moist places. Objective of this study is to uncover the component viruses, possibility of variation in the disease complex and understand the phylogeny of begomoviruses infecting S. palustris.

During a survey conducted in 2019-20, natural occurrence of vein yellowing disease was observed on S. palustris. We analyzed symptomatic plants for the possible presence of components of begomovirus disease complex using rolling circle amplification-based protocol, which has improved our ability to identify circular DNA viruses in large numbers. As expected, vein-yellowing disease was associated with AlYVV while no betasatellites or alphasatellites were detected from the plants analyzed. Phylogenetic analysis of AlYVV and coat protein and replication associated genes was also analyzed. This data will help in understanding the diversity of begomoviruses in weed hosts may aid in devising control strategies against begomoviruses.

2. Materials and Methods

2.1. Virus sources

Ten Sonchus palustris symptomatic plants showing vein yellowing were collected from farmer’s fields in Faisalabad, Punjab. Two types of leaf samples; green leaves with no visible symptoms as control (Figure 1A) and leaves showing vein yellowing (Figure 1B), were collected. Young leaves were collected, labeled and transported on ice to lab and stored at -80°C. Total DNA was extracted from leaf samples by CTAB method described by Doyle and Doyle (1990).

Figure 1
Sonchus Palustris plant and cloning of begomovirus: (A) Asymptomatic plant showing vein yellowing symptoms; (B) Symptomatic plant; (C) Amplification using Phi29 DNA polymerase, restriction and cloning of viral molecules; (lane 1-2) Amplified RCA product, (lane 3-4) restriction of RCA product resulted in 2.8kb fragment using EcoRI, (lane 6-7) Confirmation of cloning of virus 2.8kb in pTZ57R vector using EcoRI and Hindlll, lane 5 shows 1Kb marker. Size of pTZ57R vector is also 2.88 Kb.

2.2. Cloning and sequencing of viral molecules

Total DNA extracted from infected leaves of S. palustris was subjected to rolling circle amplification (RCA) using ɸ29 DNA polymerase (Blanco et al., 1989). The RCA product of S. palustris DNA was restricted using different restriction enzymes i.e., SalI, BglII, HindIII, KpnI and EcoRI. The 2.8kb sized molecules (equal to the size of begomovirus) were generated by restriction digestion with EcoRI enzyme. The restricted product (2.8kb) was gel eluted and cloned into the pre-digested pTZ57R vector at EcoRI restriction site (Fermentas) and two clones out of 23 clones were completely sequenced to generate clone names pVirol901-2.8 and pVirol902-2.8.

2.3. Sequence analysis and recombination detection

Sequences were assembled and analyzed by the Lasergene DNA analysis package (v8; DNAStar Inc., Madison, WI, USA). Phylogenetic trees were generated, first by aligning the molecules using CLUSTAL-W, followed by Neighbor joint method of phylogenetic tree construction in MEGA7 program (Kumar et al., 2016). Sequences of AlYVV isolated from S. palustris are submitted to GenBank (waiting for accession numbers). The other viral sequences were downloaded from GenBank and virus abbreviations are used as described by Zerbini et al. (2017). After the sequence confirmation, detailed recombination analysis was conducted for viral molecules through RDP-4 (Martin et al., 2015). Prior to recombination analysis the sequences were aligned by CLUSTAL-W in MEGA7 DNA analysis software (Kumar et al., 2016) followed by recombination detection through RDP-4 program (Martin et al., 2015).

2.4. SDT and phylogenetic analysis

As a requirement of begomovirus taxonomy (Zerbini et al., 2017), further confirmation of virus was performed using Multiple sequence comparison by log-expectation (MUSCLE) alignment in sequence demarcation tool (SDT) (Muhire et al., 2014). Separate files for coat protein genes and replication-associated genes of all AlYVV isolates in the GenBank and in this study were also generated manually for Bayesian evolutionary analysis by sampling trees (BEAST) analysis (Drummond and Rambaut, 2007). For these two datasets (Rep and CP) nexus files were generated after aligning sequences in MEGA-7 as described above. The general time reverse (GTR+E) substitution was chosen as the best-fit model and the nucleotide dataset was partitioned into 3 sets (codon positions 1, 2 and 3). Coalescent Bayesian Skyline was chosen in the tree panel as a prior and each dataset was run for a chain length of 4x10_⁷ to ensure an adequate sample size in the MCMC panel of the BEAUTi module in BEAST (Drummond and Rambaut, 2007).

3. Results and Discussion

To avoid future epidemics and design control strategies for plant viral diseases, identification of initial viral inoculum is important. Begomoviruses infecting important crop plants, are one of the rapidly emerging groups of plant viruses, which can be attributed to various factors, including increased insect vector populations, presence of alternative hosts and/or rapid manifestation of insect vectors (Mansoor et al., 2006). These begomoviruses probably hibernate on alternate hosts when the main crop is not present in the field. A higher diversity of economically important viruses is found in weeds (Maliano et al., 2021). But unfortunately, these reservoirs are often neglected; partly because many weed species display non-significant viral symptoms and partly because cost and effort required acquiring sequence data from significant number of weed plants is huge. Pakistan is home for begomoviruses and there is a high disease incidence as well as high diversity of viruses infecting crops and non-crops like Zinnia elegans, Solanum nigrum, Ageratum conyzoides (Haider et al., 2007), Duranta erecta (Iram et al., 2005), Chili pepper, Tomato (Hussain et al., 2004; Shih et al., 2003; Mansoor et al., 1997) Croton bonplandianus (Amin et al., 2002) Okra, Watermelon and Radish (Mansoor et al., 2001; Mansoor et al., 2000a, b) Vigna aconitifolia (Qazi et al., 2006) Mungbean (Hameed and Robinson, 2004), Eclipta prostrata (Murtaza et al., 2018) papaya (Nadeem et al., 1997) Digera arvensis, Sonchus arvensis and Xanthium strumarium (Mubin et al., 2009; 2012; Mubin et al., 2009). Old World begomoviruses infecting economically important field crops are believed to be originated from the weeds/non-crop plants usually grow in or around the fields of these crops (Ndunguru et al., 2005; Nawaz-ul-Rehman et al., 2012). Resistance breaking begomoviruses, Cotton Leaf Curl Burewala Virus (CLCuBuV), which is a recombinant molecule of Cotton Leaf Curl Multan Virus (CLCuMuV) and Cotton leaf Curl Khokhran Virus (CLCuKV) (Amrao et al., 2010), was isolated from a weed known as X. strumarium L. found inside and around the cotton fields (Mubin et al., 2012).

3.1. Symptomatology and characterization of begomoviruses

Sonchus palustris is a perennial weed and found around water channels and in crop fields. In 2019-2020 during a field survey, symptomatic leaves of S. palustris, a common weed, showing vein yellowing were collected from different farmers fields growing cotton and vegetables in Punjab (Figure 1B). Vein yellowing was so conspicuous that the infected plants showed clear distinction from the non-infected ones (Figure 1A). Rolling circle amplification (RCA) (Blanco et al., 1989) was used to amplify all circular ssDNA molecules from isolated DNA of infected plant samples. Restriction with EcoRI enzyme yielded 2.8kb fragment i.e., size of begomovirus was cloned in pTZ57R (Figure 1C). The restriction digestion with EcoRI and HindIII enzyme confirmed the 2.8kb sized bands in the gel (Figure 1C). Sequencing of 2.8kb sized bands confirmed the presence of begomovirus. We were unable to amplify DNA B, betasatellites or alphasatellites from all samples with universal primers (Briddon et al., 2002, 2004; Rojas et al., 1993) using the dilution of same RCA dilution as a template in PCR reaction. The absence of betasatellites confirmed our previous results (Murtaza et al., 2018) showing that betasatellite might not be associated with AlYVV in S. palustris. For the first time AlYVV was amplified from Alternanthera plants in Hainan province of China during 2004 as new species of begomoviruses (Guo and Xhou, 2005). Since then AlYVV has been reported from E. prostrata and several other weeds in different Asian countries (Mubin et al., 2010; Murtaza et al., 2018). AlYVV seems to be prevalent in Pakistan, India and China and none of the hosts in these countries have shown to be associated with DNA-B component, suggesting it as a monopartite begomovirus. In China, no satellites have been reported to be associated with AlYVV, from E. prostrata or Alternanthera plants (Guo and Xhou, 2005), (Huang et al., 2006) (Table 1). Similarly in Vietnam, there are two reports of AlYVV infecting Z. elegance and E. prostrate (Ha et al., 2008). In case of Z. elegance AlYVV is associated with Alternanthera yellow vein betasatellite (AlYVB) but infectivity analysis showing trans-replication was not performed while in case of E. prostrata no satellite was found (Ha et al., 2008). In Pakistan, AlYVV has been found to be associated with satellites, like Ageratum yellow vein betasatellite (AYVB) and potato leaf curl alphasatellite (PotLCuA) in Sonchus arvensis (Mubin et al., 2010). Later on, AlYVV was found to be associated with new species of alphasatellite, Alternanthera yellow vein alphasatellite (EcYVA), but no betasatellite, in Eclipta prostrata (Murtaza et al., 2018). Infectivity analysis showed that when AlYVV was co-inoculated with Cotton leaf curl Multan betasatellite (CLCuMB) and betasatellite was not transreplicated by AlYVV. This gives an indication that for AlYVV to interact with betasatellite there is a need of suitable hosts. Or in other words certain host plants do not support betasatellites replication. AlYVV is among the few begomoviruses that are found in both China and Pakistan, as virus distribution is usually restricted by the Himalayan Mountain range which serves as a natural geographical barrier. In contrast, the viruses found in India, Pakistan and Bangladesh are often related due to their geographical proximity with no natural barriers. This is the first report of a begomovirus found in China, Vietnam, and Pakistan. The trade by land route may have disseminated the virus in China, Pakistan, and India.

Table 1
Geographical distribution and host range of Alternanthera yellow vein virus (AlYVV) and associated satellites.

Serial No	Virus Accession/Year	Host name	Country	Associated satellites	Reference
1	AlYVV-AM050736/2005	Alternanthera philoxeroide	China	No	(Guo and Xhou, 2005)
2	AlYVV- EF544601/2005	Eclipta prostrata	China	No	Unpublished
3	AlYVV-DQ375456/2006	Eclipta prostrata	China	No	(Guo and Xhou, 2005)
4	AlYVV- AJ965540/2006	Ludwigia hyssopifolia	China	No	(Huang et al., 2006)
5	AlYVV- DQ641704/2006	Eclipta prostrata	Vietnam	No	(Ha et al., 2008)
6	AlYVV- DQ641703/2006	Zinnia elegans	Vietnam	AlYVB	(Ha et al., 2008)
7	AlYVV- EF544604/2006	Eclipta prostrata	China	No	Unpublished
8	AlYVV- EF544603/2006	Eclipta prostrata	China	No	Unpublished
9	AlYVV-EF544602/2006	Eclipta prostrata	China	No	Unpublished
10	AlYVV-EU286798/2007	Eclipta prostrata	China	No	Unpublished
11	AlYVV- EU286797/2007	Eclipta prostrata	China	No	Unpublished
12	AlYVV-FJ712190/2008	Alternanthera philoxeroide	China	No	Unpublished
13	AlYVV- FJ015062/2008	Eclipta prostrata	China	No	Unpublished
14	AlYVV- FN432361/2009	Sonchus arvensis	Pakistan	AYVB and CLCuMuB	(Mubin et al., 2010)
15	AlYVV- LN795903/2013	Rumex nepalensis	India	No	Unpublished
16	AlYVV-KT717678/2013	Picrorhiza kurrooa	India	No	Unpublished
17	AlYVV-KX885031/2015	Eclipta prostrata	China	No	Unpublished
18	AlYVV-KX710155/2015	Eclipta prostrata	Pakistan	No	Unpublished
19	AlYVV-KX906694/2013	Eclipta prostrata	Pakistan	TYLCCNAEcl and ECYVA	(Murtaza et al., 2017)
20	AlYVV-MG686552/2017	Synedrella sp	India	No	Unpublished
21	AlYVV-LC316183/2012	Eclipta prostrata	India	No	Unpublished
22	AlYVV-LC316182/2012	Eclipta prostrata	India	No	Unpublished
23	AlYVV-KX906695/2013	Eclipta prostrata	Pakistan	TYLCCNAEcl and ECYVA	(Murtaza et al., 2018)
24	AlYVV-KX906696/2013	Eclipta prostrata	Pakistan	TYLCCNAEcl and ECYVA	(Murtaza et al., 2018)
25	AlYVV-KX906697/2013	Eclipta prostrata	Pakistan	TYLCCNAEcl and ECYVA	(Murtaza et al., 2018)

3.2. Sequence and phylogenetic analysis of AlYVV

Total of 23 full-length begomoviruses were cloned from 10 different samples of S. palustris. Partial sequencing showed that all molecules show maximum sequence homology to AlYVV (data not shown). Two molecules were completely sequenced. Begomoviruses isolated from S. palustris showed highest sequence homology with AlYVV though sampling was done from different areas. Total of 25 full-length sequences of ALYVV are present in the GenBank so far and mostly sequences were isolated from weed plants rather crops. The phylogenetic tree was constructed using 25 sequences of AlYVV (Table 1) along with AlYVV-[PK:S Palustris:20]-1 and AlYVV-[PK:S Palustris:20]-2 isolated from S. palustris and closely related begomoviruses. Sequence comparisons showed the begomovirus to be closely related to isolates of AlYVV (99.3 to 99.7% nucleotide sequence identity to 25 AlYVV sequences available in the databases) with the highest to isolates from Pakistan i.e., AlYVV-[PK:E prostrata:13]-KX906697, AlYVV-[PK:E prostrata:15]-KX710155] and AlYVV-[PK:E prostrata:11]-KX906694 from Pakistan (Figure 2). This indicates that the virus isolated from S. palustris is an isolate of AlYVV for which we propose the isolate descriptor for two AlYVV sequences as AlYVV-[PK:S Palustris:20]-1 and AlYVV-[PK:S Palustris:20]-2. The homology difference was observed as a point mutation throughout the genome as compared to recombination. In the phylogenetic tree (Figure 3) it was evident that sequences of the AlYVV had a high level of sequence similarity with the viruses isolated from Pakistan and clustered with AlYVV-[PK:E prostrata:13]-KX906697, AlYVV-[PK:E prostrata:15]-KX710155] and AlYVV-[PK:E prostrata:11]-KX906694. The virus cloned from S. palustris has the typical genome organization of monopartite begomoviruses (or DNA-A component of bipartite begomoviruses) with two ORFs in the virion-sense (encoding the V2 protein and coat protein [CP]) and four in the complementary sense (encoding the replication associated protein [Rep], the transcriptional activator protein [TrAP], the replication enhancer protein [REn] and the C4 protein). A phylogenetic tree, based on an alignment of the complete nucleotide sequence of the begomovirus isolated from S. palustris with selected begomovirus genome (or DNA A component) sequences is shown in Figure 3. This shows the sequence isolated from S. palustris to segregate with isolates of AlYVV, confirming it as an isolate of this species. Tree showed that this conservation in the sequence of AlYVV was not only consistent with respect to the geographical positions rather it was also consistent over the period of time. The sequence of the virus isolated from Pakistan in 2019-2020 had 98% to 100% similarity level with each other. Similarly, the full length AlYVV isolated from China in 2006 had least difference in the nucleotide sequence and had a higher level of homology with each other and these viruses were seemed to be the strains of each other without having considerable difference over a period of 8 to 10 years. Higher level of sequence similarity also revealed that apparently no recombination of AlYVV occurred with any other begomovirus. Sequence analysis of AlYVV reported so far from different countries revealed that the full-length viral molecules (~ 2.8kb) were found to be highly conserved and level of sequence homology was 92 to 98%. The homology difference could be observed as a point mutation throughout the genome as compared to recombination.

Figure 2
Color-coded matrix of pairwise nucleotide identity inferred from alignment of full-length AlYVV present in data bank. The matrix uses a discontinuous range of three shades of color (red, green and blue) differentiating two cut-off values representing the strain (93-94%, brown red) and the species (90-91%, yellow green) demarcation thresholds of begomoviruses. Identities were calculated with SDT v. 1.2.

Figure 3
Phylogenetic relationships and intra specific recombination patterns among different AlYVV molecules. The maximum-likelihood phylogenetic tree contains 27 known complete genomes of AlYVV from databank and two complete genomes determined in this study (indicated in black boxes). The tree was rooted on ToLCNDV (AB613826) as an out-group. The schematic representation of recombination events detected by RDP4. Arrows and blocks at the bottom correspond respectively to open reading frames (ORFs) and intergenic regions: pre-coat protein (AV2), coat protein (CP), replication-associated proteins (Rep and REn), transcriptional protein (TrAP), and AC4 region. AlYVV from different countries were colored differently. The colors of blocks represent the different ALYVV species and strains. Numbers at nodes indicate bootstrap confidence scores (1000 replicates).

3.3. Estimation of nucleotide substitution rates

The mean nucleotide substitution rates for the CP gene of AlYVV were determined using recombination free datasets with the relaxed clock and Bayesian Skyline Plot (BSP) method. For each dataset, the sequences were partitioned into the 3 codons positions. The mean substitute rates for CP gene of ALYVV was considerably higher (4.75 x 10^-3) substitutions/nucleotide/year, respectively) than those for the CLCuKoV CP (2.706 x10^-4) (Nawaz-ul-Rehman et al., 2012). This high substitution rate in CP gene is closer to substitution rate estimated for the bipartite begomovirus, East African cassava mosaic virus CP (1.37x10^-3 subst./nt/year) and Tomato yellow leaf curl virus CP (4.6x10^-4 subst./nt/year) (Nawaz-ul-Rehman et al., 2012). This may indicate that these viruses face the same evolutionary pressure, despite infecting different hosts in different parts of the world. To further estimate the selection pressure, we used the 3-position clock model in BSP analysis. Codon positions 1, 2 and 3 showed normal behavior for AlYVV-CP (0.965, 0.783 and 1.253).

3.4. SDT and recombination analysis

Sequences were aligned with begomovirus sequences available in databases using MUSCLE and pairwise identity scores were calculated with SDT (species demarcation tool). Viral sequence isolated in this study i.e., AlYVV-[PK:S Palustris:20]-1 shared 98% and 100% nucleotide identity, with isolates from Pakistan i.e., AlYVV-[CN:E prostrata:07]-EU286797, AlYVV-[PK:S arvensis:09]-FN432361 and AlYVV-[CN:A philoxeroide:05]-AM050736 (Figure 2). Nucleotide sequence homology of two begomoviruses with the most closely related begomoviruses was above the 97%, threshold for species demarcation, thus confirming that the begomoviruses found infecting S. palustris in Pakistan are isolates not new species. To find out any recombination, phylogenies of full-length AlYVV (Figure 3) AlYVV-Rep (Fig, 4A) and AlYVV-CP genes (Figure 4B) were studied. Interestingly trees have two major clades. Clade I contains AlYVV and clade II encompasses closely related viruses like LuYVVNV, RaLCV and AEV. This shows no interspecific recombination. But when phylogenetic trees generated for Rep and CP genes were analyzed along with full-length AlYVV tree, it showed interesting results. Clade I in CP tree and AlYVV tree distribution is uniform but in Rep tree differs in distribution considerably from CP and AlYVV trees. Not surprisingly the phylogenetic tree generated for Rep gene sequences shows AlYVV-[CN:A philoxeroide:05]-AM050736 segregates from rest of the ALYVV sequences. While for CP tree, AlYVV-[CN:A philoxeroide:05]-AM050736 forms group with AlYVV-[PK:E prostrata:13]-KX906695 and AlYVV-[VN:Hue:Z elegance:06]-DQ641703. Similarly for CP tree, AlYVV-[CN:E prostrata:05]-EF544601 and AlYVV-[CN:E prostrata:05]-EF544602 make a monophyletic group while for Rep these two group together with AlYVV-[CN:E prostrata:07]-EU286797 and other AlYVV from China. Indeed, the phylogenetic trees of CP and full-length molecules are alike. Where, the isolates of Indian sub-continent and Chinese origin cluster separately, indicating their geographical origin. However, the Phylogenetic tree for Rep gene shows segregation of Chinese isolates with the Indian sub-continent. The isolate AlYVV-[PK:S arvensis:09]-FN432361, which segregates with Chinese isolates in the full-length sequences tree is an indication of its recent introduction to the Indian subcontinent. However, its Rep gene is positioned along with the AlYVV molecules isolated in this study. The segregation of Chinese isolates with the Indian sub-continents isolates is a clear indication of recombination within the AlYVV isolates. Together these finding suggest that there might be no inter specific recombination in AlYVV but there is a definite intraspecific recombination on virion sense and complementary sense strand in AlYVV. AlYVV-[PK:S arvensis:09]-FN432361 shows recombination at complementary sense strand as Rep portion of it shows sequence homology with AlYVV-[CN:A Philoxeroides:08]-FJ712190 with p-value of P-value 2.441x10^-2. In CP tree and in Rep tree distribution of AlYVV-[CN:A Philoxeroides:08]-FJ712190 and AlYVV-[PK:S arvensis:09]-FN432361 supports this hypothesis. In full-length AlYVV tree, AlYVV-[CN:E prostrata:05]-EF544601 and AlYVV-[CN:E prostrata:05]-EF544602 cluster with AlYVV-[CN:E prostrata:08]-FJ015062 and AlYVV-[VN:E prostrata:06]-DQ641704 just like in Rep tree but in CP tree these two cluster with AlYVV-[IN:P kurrooa:13]-KT717678, AlYVV-[IN:Synedrella:17]-MG686552 and AlYVV-[IN:R nepalensis:13]-LN795903. This shows intraspecific recombination at virion sense strand with p-value of 1.328x10^-2. In contrast recombination analysis for CLCuBuV showed a distinct recombination pattern. The analysis showed CLCuBuV to consist of the virion-sense sequences of CLCuKoV and the complementary-sense sequences of CLCuMuV, so showing interspecific recombination (Nawaz-ul-Rehman et al., 2012). We need more sequencing data and experiments to understand recombination patterns in different begomoviruses.

Figure 4
Comparison of the phylogenetic trees of the Rep-gene (A) and CP-gene (B) of AlYVV for recombination analysis. AlYVV from different countries were colored differently. Lines show assortment of Rep gene and CP genes in different clusters showing intraspecific recombination. Rep and CP gene from AlYVV determined in this study are indicated in black boxes. Clade I contains AlYVV and clade II encompasses closely related viruses like LuYVVNV, RaLCV and AEV. The trees were arbitrarily rooted on the sequences of the Tomato leaf curl New Delhi virus (AB613826). Numbers at nodes indicate bootstrap confidence scores (1000 replicates).

4. Conclusions

Usually in Old World, begomoviruses are associated with betasatellites but in this study no betasatellite was found. AlYVV was found to be associated with satellites in some hosts but not proven through infectivity analysis. There are a number of reports showing multiple begomovirus infections in some hosts therefore it might be possible that these reported satellites were associated with some unamplified begomovirus. In present study, AlYVV was found to be recombination free across the infected samples collected from Punjab, which again is an interesting observation. The aim of this study was to understand the begomovirus complex responsible for the disease in S. palustris and subsequently phylogenetic and mutational analysis of AlYVV prevailing in the region. Findings of these studies were very interesting and novel as S. palustris proved to be a new reservoir for begomoviruses. So, there is always a chance that whitefly feed on different weed hosts and mix up these components. Crop plants showed specificity to specific viruses and components and thus only specific viruses and their components propagated in crop plants. Weeds act as reservoir of these viral components and recombination vessels where all the components are present together with greater chance of recombination. The host range of AlYVV is increasing which can be attributed to various factors, including increased insect vector populations, mutation, inter-specific recombination and/or rapid manifestation of insect vectors. In future such viruses could jump from weeds to main crops and cause economic loss.

Acknowledgements

We acknowledge all members of virology lab for help and support to perform experiments for this work. Higher education commission of Pakistan (HEC) under the grant numbers 1682 and 6427 funds the research in Virology lab. This work was partly funded by Researchers Supporting Project number (RSP-2021/26), King Saud University, Riyadh, Saudi Arabia.

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Publication Dates

• Publication in this collection
  13 June 2022
• Date of issue
  2022

History

• Received
  19 Mar 2022
• Accepted
  13 May 2022