Introduction

Cotton (Gossypium hirsutum) is an economically significant crop in Pakistan and its protection is a major concern. The most important threat for cotton plants is cotton leaf curl disease (CLCuD). Over the last decade, it has been estimated that about 7.1 million bales of cotton were lost due to CLCuD (Mahmood-ur-Rahman et al. 2012; Zubair et al. 2017). The agents responsible for CLCuD are begomoviruses, which require single-stranded DNA betasatellite molecules that are pathogenicity determinants and help in development of typical disease symptoms in cotton (Amin et al. 2011). Some betasatellite-begomovirus complexes are also found to be associated with alphasatellites (Nawaz-ul-Rehman and Fauquet 2009; Anwar et al. 2020). Alphasatellites are also single-stranded DNA molecules associated with geminivirus and nanoviruses. Geminialphasatellites are 1.4 Kb in size and are associated with begomoviruses and mastreviruses from family Geminiviridae (Hamza et al. 2018). The begomovirus disease complexes are rapidly evolving by swapping different satellite components leading to recombination and pseudorecombination which appears to be important for evading host resistance.

In the 1990s, CLCuD triggered a main loss of cotton in cotton growing districts of the Punjab province; however, there was a little effect in northern districts of the Sindh province while most cotton growing districts of southern Sindh remained unaffected. Although it has been reported that different begomoviruses affect different crops in Sindh, but there was no report in cotton till late nineties of the last century (Sanz et al. 2000). The begomoviruses and their associated whitefly vectors in Sindh were different from those which were reported in the Punjab province (Simón et al. 2003). During 1997–1998, CLCuD was reported to affect the cotton crop in the Sindh province as well, but the damage was not significant compared to that of Punjab (Mansoor et al. 1998). However, from 2003–2004, significant losses of cotton yield have been observed in Sindh, which has been proven to be associated with a new species of monopartite begomovirus which was reported by (Amrao et al. 2010) as cotton leaf curl Shahdadpur virus (CLCuShV).

A study on begomovirus diversity in cotton in two major cotton-growing provinces of Pakistan; Punjab and Sindh provinces reported as the dominant complexes the cotton leaf curl Kokhran virus Burewala strain (CLCuKoV-Bur) with the cotton leaf curl Multan betasatellite (CLCuMB) and a bipartite begomovirus tomato leaf curl New Dehli virus (Zaidi et al. 2016). Association of the cotton leaf curl Sindh alphasatellite and the cotton leaf curl Shahdadpur alphasatellite with the begomovirus-betasatellite complex have been reported (Amrao et al. 2010). Cotton leaf curl disease causing complex is changed after some time to evade the host resistance. Here we reported for the first time the association of tomato leaf curl Pakistan alphasatellite (ToLCPKA) and cotton leaf curl Multan alphasatellite (CLCuMA) with CLCuKoV-Bur in the Sindh province of Pakistan.

Materials and Methods

In 2018, cotton plants showing the typical CLCuD symptoms (leaf yellowing, curling and reddening, vein-thickening and foliar discoloration) were collected from different fields of Taluka Sakrand (Sindh, Pakistan) using sterile disposable gloves for each sample to avoid the cross contaimination. The collected samples were packed in clean polyethene bags and labelled with location and date of collection. Those samples are transported in ice-filled cooler to the molecular virology lab. Genomic DNA was extracted from a leaf sample by using the modified CTAB method as described by Akram et al. 2017 (Doyle and Doyle 1990; Akram et al. 2017) and visualised with 1% (w / v) agarose gel. The DNA then served as template in polymerase chain reaction (PCR) amplifications with the begomovirus specific primers uniNDVA3 (5’-GAG CTC GTG CAG TTG TCC CCA TTG CCC GCG TCA C-3’) and uniNDVA4 (5’-GAG CTC CAT AGG GGC TGT CGA AGT TG-3’), and the alphasatellite specific primers DNA101 (5’-CTG CAG ATA ATG TAG CTT ACC AG-3’) and DNA102 (5’-CTG CAG ATC CTC CAC GTG TAT AG-3’) (Bull et al. 2003). The amplicons were cloned in a TA plasmid vector and sequenced. Amplified PCR products were purified and sent for commercially available services of Sanger sequencing from Macrogen Inc. Soule, S. Korea. Received sequences of different amplicons of begomoviruses and alphasatellites were cleaned and assembled into a single consensus viral sequence using Lasergene (DNA-Star Inc., Madison, WI, USA) and submitted to the GenBank database. Potential genes in the begomovirus sequences and the associated betasatellite sequences were determined using the online NCBI tool ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/). Closely related sequences were retrieved from public databases using BLAST (Zhang et al. 2000) and multiple sequence alignments were performed using Clustal W, as implemented by MEGA 7.0 (Kumar et al. 2016). Pairwise sequence comparisons were done using MegAlign, as implemented by Lasergene and Sequence Demarcation Tool (SDT) (Muhire et al. 2014). Phylogenetic analysis was conducted by constructing a phylogenetic tree using the neighbor-joining algorithm as implemented by MEGA 7.0 with 1,000 bootstrap replicates. Abbreviations of begomovirus and their satellites names are shown as previously (Varsani et al. 2017).

Results

The complete nucleotide sequence of the begomovirus clone (IA6) (2759bp) (PK: Sin: CCRI: Gossy: 18 CLCuBuV_MW166879) confirmed its identity as a CLCuKoV-Bur strain. The IA6 clone had 99.7% sequence identity with CLCuKoV-Bur isolates (accession numbers FR750321 and LN713477) from Punjab Rahim Yar Khan and Sahiwal. The pairwise distance matrix (Fig. 1) of the IA6 clone with similar sequences from public databases demonstrated 99.7–99% identity with CLCuKoV-Bur strains. Phylogenetic analysis of the IA6 clone (PK: Sin: CCRI: Gossy: 18 CLCuBuV_MW166879) (Fig. 2) confirmed its homology with different CLCuKoV-Bur isolates from various regions of India and Pakistan.

From the same cotton sample, three alphasatellites were cloned (IA31, IA32 and IA33) consisting of two species. The sequence analysis of IA31 (1385 bp) (PK: Sind: Gossy: 18_CLCuMA_MW166934) and IA32 (1376 bp) (PK: Sind: Gossy: 18_CLCuMA_MW166935) revealed these as CLCuMA while IA33 was revealed as ToLCPKA. Specifically, the IA31 (accession number MW166934) and IA32 (accession number MW166935) clones (Fig. 3) showed 96.7–95.6% homology with CLCuMA sequences (accession numbers LT840038, LT840037, LT840024, LT840044), while the pairwise sequence alignment (Fig. 4) indicated that IA33 (1384 bp) (accession number MW166936) has 92.7% identity with ToLCPKA (HE966421) reported from the

Fig. 2: Phylogenetic analysis of the begomoviruse clone IA6. (PK:Sin:CCRI:Gossy:18 CLCuBuV-MW166879). Based on the alignment of the CLCuKoV-Bur sequences, a neighbor-joining phylogenetic tree was constructed, with closely related begomovirus sequences. Isolates in green were obtained in the present study. Sequence accession number of each isolate is listed together with the strain descriptors (in square brackets), which include the country, location, host, and year of sampling. The tree was rooted using the sequence of tomato mottle virus (ToMoV) as an outgroup

Fig. 1: Pairwise distance matrix of begomovirus sequences alinged by CLUSTAL W. The percent identity of the begomovirus IA6 clone (PK:Sin:CCRI:Gossy: CLCuBuV MW166879 ) with similar sequences from public databases is shown

Jhang district of Punjab. Phylogenetic analysis (Fig. 2) also showed that IA33 (PK: Sind: Gossy: 18_ToLCuPKA_MW166936) is grouping with different TLCuPKA isolates.

Discussion

Betasatellite associated monopartite begomovirus disease complex can significantly impact the cotton yield in Pakistan and Indian regions and was reported previously to occur most oftenly as multiple infection of distinct begomovirus species (Zaidi et al. 2016). This disease complex can readily advance by capturing additional new components, recombining and genetically modifying the existing components to overcome host resistance and to increase its host range (Sattar et al. 2013; Zubair et al. 2017). There are reports of more than a few monopartite begomoviruses involved in CLCuD in cotton in Pakistan. In the 1990s, minimum four different species of begomovirus, including CLCuKoV and CLCuMuV, CLCuAlV and PaLCuV were involved in CLCuD, either as single or mixed infections. In contrast, only one betasatellite molecule namely, CLCuMB, has been found to be part of the cotton leaf curl begomovirus complex (Briddon et al. 2004; Nawaz-ul-Rehman et al. 2017). Due to the emergence of the new recombinant CLCuKoV-Bur strain, the conventional resistant cotton varieties also become susceptible to CLCuD (Yasmeen et al. 2016).

Our current study focused on the diversity of begomoviruses and associated satellites in the Sindh province of Pakistan. Diversity studies in a cotton sample collected from Taluka Sakrand (Sindh province) revealed that it contained CLCuKoV-Bur associated with two alphasatellites, which were previously reported in Punjab Pakistan (Akhtar et al. 2010). Although the role of alphasatellites in disease etiology is not clear, it has been reported that it is an important component of begomovirus disease complex and a target of the host defense (Nawaz-ul-Rehman et al. 2010). The begomovirus clone (IA6) from cotton had 99.7% identity with CLCuKoV-Bur isolated from Rahim Yar Khan and Sahiwal in the Punjab province. From the same cotton sample, two types of alphasatellites were isolated, namely CLCuMA and ToLCPKA, demonstrating the fact that multiple alphasatellites can simultaneously be associated with a begomovirus inside a single host. In 2016, a study also reported the presence of three alphasatellites with a begomovirus in a single cotton plant in Punjab Pakistan (Siddiqui et al. 2016). Another recent study carried out in north western states of India revealed that analyses of cotton

Fig. 4: Pairwise distance matrix of alphasatellite sequences aligned by CLUSTAL W using sequence demarcation tool. The % identity of alphasatellite clones IA31(PK:Sind:Gossy:18 CLCuMA MW166934), IA32 (PK:Sind:Gossy:18 CLCuMA MW166935) and IA33 (PK:Sind:Gossy:18 ToLCuPKA MW166936) with similar sequences from public databases is shown

Fig. 3: Phylogenetic analysis of the alphasatellite clones IA31 (PK:Sind:Gossy:18_CLCuMA; MW166934), IA32 (PK:Sind:Gossy:18_CLCuMA; MW166935) and IA33 (PK:Sind:Gossy:18_ToLCuPKA; MW166936). A Neighbor-Joining phylogenetic tree was constructed based on alignments of the sequences of Cotton leaf curl Multan alphasatellite (CLCuMA), Tomato leaf curl alphasatellite (ToLCuA), Tomato leaf curl Pakistan defective alphasatellite (ToLCuPKDeA), Cotton leaf curl Shahdadpur alphasatellite (CLCuShA), Tomato leaf cur Pakistan alphasatellite (ToLCPKA) and Cotton leaf curl virus alphasatellite (CLCuVA). The tree was rooted using the sequence of cotton leaf curl Multan betasatellite (CLCUMB) as an outgroup

field symptomatic samples for three consecutive years, two different begomovirus species Cotton leaf curl Multan virus (with three different strains named as Cotton leaf curl Multan virus-Rajasthan, Cotton leaf curl Multan virus-Pakistan, Cotton leaf curl Multan virus-Faisalabad) and Cotton leaf Kokhran virus-Burewala strain were identified. CLCuMV-Raj strain; the most abundantly found begomovirus strain is this study was associated with Cotton leaf curl Multan betasatellite (CLCuMB) and Gossypium darwinii symptomless alphasatellite (GDarSLA), and Croton yellow vein mosaic alphasatellite (CrYVMoA). CLCuKoV-Bur was found second most abundant in cotton samples and associated satellites were CLCuMB and GDarSLA (Biswas et al. 2020). These studies support our findings that there is no species barrier in the association of satellites with helper begomoviruses as alphasatellites; tomato leaf curl Pakistan alphasatellite (ToLCPKA) was identified in cotton. The CLCuMA isolates from cotton have the highest similarity with those isolated from Malus pumila originating from Pakistan.

Conclusion

This study focused on characterizing the begomoviruses and associated satellites present in the Sindh province, Pakistan. Pakistan’s economy heavily depends on cotton and the major cause that decreased cotton yields is CLCuD. The causative agent of CLCuD was recognized as a begomovirus. Interestingly, begomoviruses possess the strategy of recombination and pseudorecombination, allowing them to overcome the plant defense mechanisms and control their machinery for propagating themselves. This is a serious problem as the virus can modify itself by recombination and evolve into a more virulent complex by capturing diverse components. In current study it is concluded that CLCuKoV-Bur associates with different alphasatellites to cope with host defense for bona fide infection. To the best of our knowledge this is the first report of ToLCuPKA associated with CLCuD in the Sindh region of Pakistan.

Acknowledgments

All authors are thankful to Higher Education Commission (HEC) Pakistan, for providing funds through project No. 5584/Punjab/NRPU/R&D/HEC/2016, which was awarded to Khadim Hussain.

Author Contributions

KH designed and directed the project. IA, IA and KH were involved in sample collection and performing the experimental work. IA, KR, SM and KH analyzed the data. KR, AA, KH, MT, SS and IKL wrote the manuscript. IKL and MT were involved in the revision of the final file of the manuscript.

Conflicts of Interest

Authors declare no conflict of interests and all authors read and approved the manuscript and agreed to submit it in IJAB for publication.

Ethics Approval

The present research does not involve any animal as experimental organism therefore approval from ethical committee was not needed.

Data Availability

The full genome sequences of clone IA6 (CLCuBuV PK: Sin: CCRI: Gossy: 18) is available under accession number MW166879 and alphasatellite clones IA31 and IA32 under accession numbers MW166934 and MW166935, respectively in Genbank database.

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