Genomic Variation and Diversification in Begomovirus Genome in Implication to Host and Vector Adaptation

Begomoviruses (family Geminiviridae, genus Begomovirus) are DNA viruses transmitted in a circulative, persistent manner by the whitefly Bemisia tabaci (Gennadius). As revealed by their wide host range (more than 420 plant species), worldwide distribution, and effective vector transmission, begomoviruses are highly adaptive. Still, the genetic factors that facilitate their adaptation to a diverse array of hosts and vectors remain poorly understood. Mutations in the virus genome may confer a selective advantage for essential functions, such as transmission, replication, evading host responses, and movement within the host. Therefore, genetic variation is vital to virus evolution and, in response to selection pressure, is demonstrated as the emergence of new strains and species adapted to diverse hosts or with unique pathogenicity. The combination of variation and selection forms a genetic imprint on the genome. This review focuses on factors that contribute to the evolution of Begomovirus and their global spread, for which an unforeseen diversity and dispersal has been recognized and continues to expand.

Tobacco Leaf Curl Puer Virus: a Novel Monopartite Begomovirus Infecting Nicotiana Tabacum in China

Begomoviruses (family Geminiviridae) cause serious diseases in many economic crops and weeds globally. This study characterized a begomovirus isolated from a tobacco plant with leaf curl in Puer, Yunnan Province, China. Analysis of the viral genome obtained from a diseased Nicotiana tabacum plant showed a novel monopartite begomovirus. The DNA-A-like sequence (2741 nt) has the typical Old World monopartite begomovirus genome organization, sharing the highest nucleotide sequence identity (81.2%) with that of Tomato yellow leaf curl Vietnam virus (TYLCVV). According to the current International Committee on Taxonomy of Viruses (ICTV )taxonomic criteria, this is a new species of begomovirus for which the name “Tobacco leaf curl Puer virus” is proposed.

siRNA biogenesis and advances in topically applied dsRNA for controlling virus infections in tomato plants

A non-transgenic approach based on RNA interference was employed to induce protection against tomato mosaic virus (ToMV) infection in tomato plants. dsRNA molecules targeting the cp gene of ToMV were topically applied on plants prior to virus inoculation. Protection was dose-dependent and sequence-specific. While no protection was achieved when 0–16 µg dsRNA were used, maximum rates of resistance (60 and 63%) were observed in doses of 200 and 400 µg/plant, respectively. Similar rates were also obtained against potato virus Y when targeting its cp gene. The protection was quickly activated upon dsRNA application and lasted for up to 4 days. In contrast, no detectable antiviral response was triggered by the dsRNA from a begomovirus genome, suggesting the method is not effective against phloem-limited DNA viruses. Deep sequencing was performed to analyze the biogenesis of siRNA populations. Although long-dsRNA remained in the treated leaves for at least 10 days, its systemic movement was not observed. Conversely, dsRNA-derived siRNA populations (mainly 21- and 22-nt) were detected in non-treated leaves, which indicates endogenous processing and transport through the plant. Altogether, this study provides critical information for the development of novel tools against plant viruses; strengths and limitations inherent to the systems are discussed.

Tomato leaf curl Patna virus causing tomato leaf curl disease in Bangladesh

Tomato leaf curl virus (ToLCV) has appeared as a potential threat to the tomato production in the world. ToLCV, a member of the family Geminiviridae may contain either bipartite or monopartite genome. The genetic nature of a monopartite ToLCV isolate characterized from the tomato leaf curl diseased samples of Jamalpur district, Bangladesh (ToLCV-JB) has been reported. The products of rolling circle amplification (RCA) were digested, cloned and sequenced. Sequence analysis revealed the features of begomovirus genome organization in the ToLCV-JB isolate, containing six open reading frames. BLAST analysis showed 100% sequence similarity with tomato leaf curl Patna virus (EU862323.1) and more than 80% similarity with other reported monopartite begomoviruses. Hence, the virus isolate was registered as Tomato leaf Curl Patna virus-[Bangladesh:Jamalpur:2014] isolate ToLCV-JB (Genebank Accession: KU933675.1) according to the suggestion of NCBI. Recombination analysis also did not show any genetic exchange between ToLCV-JB and ToLCV-Patna virus. Moreover, they belong to the same cluster as observed in phylogenetic analysis. The present work suggests the possibility of cross-border spread of ToLCV-Patna viruses without mutation and this could pose a threat to tomato production in Bangladesh as well as in the Asian continent.

Detection and identification of Begomovirus infecting Cucurbitaceae and Solanaceae in Yogyakarta, Indonesia

Subiastuti AS, Hartono S, Daryono BS. 2019. Detection and Identification of Begomovirus infecting Cucurbitaceae and Solanaceae in Yogyakarta, Indonesia. Biodiversitas 20: 738-744. Begomovirus genome has high plasticity that led to evolve rapidly. Begomovirus is one of a remarkably successful group of emerging viruses as the results from combination of many factors. Planting systems in Indonesia which often overlapping two or more plant species in one land has high possibility for occurring mixed infection. It is also suggested has high contribution to increase Begomovirus diversity. The aim of this research is to do preliminary identification of Begomovirus infected-Solanaceae and Cucurbitaceae in Yogyakarta based core coat protein (CP) gene sequence. A total of 50 melon, 50 chili, 30 eggplants, and 30 watermelon samples which showed Begomovirus symptoms were observed from several fields in Yogyakarta and Purworejo, Indonesia during 2016. Almost 90% of infected samples for each plant were tested by PCR and showed positive for Begomovirus. Based on coat protein (CP) gene nucleotide sequence identity, Begomovirus infected Solanaceae in Indonesia has close relationship with Pepper yellow leaf curl Indonesia virus (PepYLCIV) and Tomato yellow leaf curl Kanchanaburi virus (TYLCVKaV), while in Cucurbitaceae has close relationship with Squash leaf curl China virus (SLCCV) Tomato leaf curl New Delhi virus (ToLCNDV). All collected isolates showed highest sequence identity with isolates from South-East Asia and China. However, further analysis that including full genome characterization is still needed to explain Begomovirus evolution in Indonesia.

First Report of Okra enation leaf curl virus and Associated Cotton leaf curl Multan betasatellite and Cotton leaf curl Multan alphasatellite Infecting Cotton in Pakistan: A New Member of the Cotton Leaf Curl Disease Complex

Cotton (Gossypium hirsutum L.) is an important and widely cultivated crop in Pakistan, upon which many rely for economic security. Cotton leaf curl disease (CLCuD) is caused by a complex comprising of more than eight species in the genus Begomovirus (family Geminiviridae) with associated betasatellite and alphasatellites. During 2011, characteristic symptoms of leaf curl disease were widespread (>40%), and the whitefly Bemisia tabaci (Genn.) vector of the leaf curl complex was abundant in commercial cotton fields in Burewala, Pakistan. Symptoms included vein thickening, upward or downward leaf curling, and foliar enations. To test for the presence of a begomovirus(es), total DNA was extracted from 100 mg of symptomatic leaf tissues from five different plants (isolates CLCuDBur1 to 5) using the CTAB method (1). Total DNA extracts were used for rolling circle amplification (RCA) using TempliPhi DNA Amplification Kit (GE Healthcare). Of the five field isolates, the RCA product for only one, CLCuDBur3, digested with HindIII, produced an apparently full-length ~2.7 kb fragment, suggesting that CLCuD-Bur3 represented a distinct isolate. The 2.7-kb fragment was cloned into the plasmid vector pGEM-3Zf+ (Promega, Madison, WI). To test for the presence of associated alphasatellites and betasatellites, the PCR primers, AlphaF/R and BetaF/R (2), were used to amplify the putative 1.4-kbp molecules. The resultant 1.4-kb PCR products were ligated into the pGEMT-Easy vector and cloned. Cloned inserts for each were subjected to DNA sequencing, bidirectionally. The cloned monopartite, helper begomovirus genome (HF567945), one betasatellite (HF567946), and one alphasatellite (HF567947) sequences were determined and found to be 2,742, 1,358, and 1,376 bases long, respectively. Pairwise sequence comparisons were carried out for each using the 10 most closely related species or strains (identified in GenBank using BLASTn) using MEGA5 software. The CLCuDBur3 genome sequence shared its highest identity (99.6%) with Okra enation leaf curl virus (OELCuV) (KC019308), so CLCuDBur3 is a variant of OELCuV, a begomovirus reported previously from Abelmoschus esculentus (L.) (okra) plants in India. The betasatellite and alphasatellite shared their highest nt identity at 96 and 98.7% with Cotton leaf curl Multan betasatellite (CLCuMB) (AM774311) and Cotton leaf curl Multan alphasatellite (CLCuMA), respectively (misnamed as CLCuBuA in GenBank) (FN658728). Additionally, the HindIII-digested RCA products were analyzed by Southern blot hybridization using a DIG-labeled DNA probe specific for the intergenic region of either Cotton leaf curl Burewala virus (CLCuBuV) or OELCuV. The OELCuV, but not the CLCuBuV, probe hybridized with HindIII digested RCA products (CLCuDBur3 genome), confirming the presence of OELCuV and the absence of CLCuBuV, the latter being the most prevalent begomovirus species infecting cotton in Pakistan. This is the first report of OELCuV infecting cotton plants in Pakistan, underscoring the discovery of yet another begomovirus member of the CLCuD complex. Further, the possible co-infection of cotton by OELCuV and other recognized species of the CLCuD complex could facilitate further diversification (potentially, through recombination) and lead to the emergence of new variants with the potential to cause damage to the cotton crop in Pakistan. References: (1) J. J. Doyle and J. L. Doyle. Focus. 12:13, 1990. (2) M. Zia-Ur-Rehman et al. Plant Dis. 97:1122, 2013.

Identification of Chili leaf curl virus Causing Leaf Curl Disease of Petunia in Oman

Petunias (Petunia × hybrida) are the most important ornamental plants in Oman. In 2012, petunias were observed in public parks and airport landscape in Dhofar region with symptoms of upward leaf curling, yellowing and vein clearing, and size reduction in leaves. Almost all plants in the surveyed landscape showed high infestation of Bemisia tabaci and symptoms that suggested infection with a begomovirus. Six symptomatic samples were collected from three different sites. All symptomatic samples were found PCR-positive with diagnostic primers for begomovirus (3) when DNA extracted from infected leaves was used as template. Nucleic acids extracted from the symptomatic leaves were used to amplify circular DNA molecules by rolling circle amplification method. The amplified concatameric products were digested with restriction enzyme PstI, which yielded a product ∼2.8 kb in size. The putative begomovirus fragment was cloned and sequenced in both orientations. Partial sequences of six clones were 99 to 100% similar and thus only two clones, PT-2 and PT-3, were fully sequenced. The whole genomes of both clones were 2,761 bp, and both were deposited in GenBank under accession numbers HF968755 and HF968756 for the isolates PT-2 and PT-3, respectively. Both sequences had six open reading frames; Rep, TrAP, REn, and C4 genes in complementary sense; and CP and V2 genes in virion-sense, typical of the begomovirus genome organization. Upon alignment, the two sequences showed 99.4% nucleotide identity with each other, thus representing isolates of a single begomovirus species. BlastN comparison showed PT-2 and PT-3 from petunia were 94 to 95% identical to the sequences of ChCLV from Oman (JN604490 to JN604500), which were obtained from other hosts. ClustalV multiple sequence alignment showed that isolates PT-2 and PT-3 shared maximum sequence identity of 93.3 and 92.8%, respectively, with an isolate of ChLCV-OM (JN604495). According to ICTV rules for begomoviruses, PT-3 should be considered to be a new strain of ChLCV-OM and PT-2 a variant of the already existing ChLCV-OM strain. We propose the name for this new strain as the “Petunia strain” of Chili leaf curl virus (ChLCV-Pet). Two infectious clones were constructed from the PT-2 and PT-3 sequences, clones as 1.75-genome sequences in a binary vector, suitable for agroinfection to confirm their infectivity. Both clones, PT-2 and PT-3, produced typical leaf curl disease symptoms upon inoculation on petunia 18 days post inoculation. The presence of the same virus in symptomatic field infected and inoculated petunia was confirmed by Southern blot using 650 bp DIG labeled probe prepared from CP region of PT-3 isolate. ChLCV-OM, a monopartite begomovirus, is widely associated with leaf curl disease of tomato and pepper in Oman, with its origin traced to the Indian subcontinent (2). Identification of a new strain of ChLCV from petunia provides evidence of an ongoing rapid evolution of begomoviruses in this region. Although petunia has been tested as an experimental host for some begomoviruses (1,4), this is the first report of petunia as natural host for ChLCV, a begomovirus previously reported in tomato and pepper in Oman. References: (1) Cui et al. J. Virol. 78:13966, 2004. (2) Khan et al. Virus Res. 177:87, 2013. (3) Khan et al. Plant Dis. 97:1396, 2013. (4) Urbino et al. Arch. Virol. 149:417, 2003.

First Report of Tomato rugose yellow leaf curl virus Infecting Tomato in Argentina

Tomato plants exhibiting typical symptoms of begomovirus infection, including leaf deformation, curling, and yellowing, were collected from cultivated fields in Lavalle Department, Corrientes, Argentina, in 2010. Although the number of affected plants was only 2% within a farm, the finding is of considerable importance since the white fly Bemisia tabaci is widely spread within the country, even in other southernmost areas such as the cinturón hortícola de Buenos Aires (horticultural belt around Buenos Aires). DNA isolated from infected tomato leaves collected from three symptomatic tomato plants was amplified by PCR with specific primers designed to amplify a region of component A and B of the Begomovirus genome (3). The amplified DNA fragment was sequenced and a new set of primers were designed based on the obtained sequences. A DNA fragment of about 1,300 bp was amplified and later the complete genome, which was 2,683 bp long. No fragments were obtained when template DNA was from non-infected leaf samples. The 2,683-bp fragment was annotated at the NCBI under Accession No. KC132844. Analysis by NCBI BLAST showed that it was highly homologous to DNA-A component of Begomovirus. Furthermore, the genome organization was typical of DNA-A component of bipartite New World begomovirus. The sequence had one open reading frame (ORF) on the viral-sense strand (AV1/CP) and four ORFs on the complementary-sense strand (AC1/Rep, AC2/TrAp, AC3/REn, and AC4). In order to confirm this finding, the viral genome was amplified by rolling circle amplification (RCA, TempliPhi 100 Amplification Kit, Amersham Biosciences) as described by the manufacturer instructions. The RCA full-length product was digested with XhoI generating a 2,700-bp DNA fragment, suggesting the presence of only one restriction site, in agreement with the bioinformatics analysis of the KC132844 sequence. This PCR product was used as template in PCR reactions with specific primers to DNA-A or DNA-B components. While the DNA-A primers generated the expected 1,300-bp fragment, those homologous to the DNA-B component did not generate amplifications. These results confirmed the identity of the DNA-A component of the isolate MT8. The full sequence of the DNA-A component was 94% homologous to the DNA-A sequence of the Uruguayan begomovirus Tomato Rugose Yellow Leaf Curl Virus-[U4.1] (JN381823.1). Therefore, considering our results and the criteria proposed by Fauquet (1), isolate MT8 is a new species of begomovirus described recently (2). This is the first report of TRYLCV in one of the main areas of tomato production in Argentina. This virus might be accompanying another begomovirus TYVSV that provoked yellow veins symptoms in tomato plants cultivated in the same area of Corrientes. These viruses appeared recently and concomitantly with the introduction of the white fly Bemisia spp. in the area, which is one of the main production areas of tomato and provides fresh tomatoes to the whole country, and in wintertime to the city of Buenos Aires, when the horticultural belt around Buenos Aires is not under production. References: (1) C. M. Fauquet et al. Arch Virol 153:783, 2008. (2) B. Márquez-Martín et al. Arch Virol 157:1137, 2012. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.