Chilli Leaf Curl Research Articles

Molecular Characterization of the Begomovirus Associated Satellites Infecting Spinacia Oleracea and Capsicum Annum Plants in Kohat, Pakistan

Begomovirus is a major and economically important genus of the Geminiviridae family. It comprises a wide range of viruses that infect a number of dicot plants including the horticulture crops, cereal crops, aromatic plants, vegetable crops, medicinal plants and weeds in various regions of the world. This study aims to investigate and correlate the various symptoms of begomovirus / satellites in different plants grown in the vicinity of Kohat, Pakistan. Furthermore, the characterization of the selected virus-associated satellites at the molecular level is also studied. Samples of suspected plants showing begomoviral infection were collected from the Kohat District. Genomic DNA was extracted from the infected plants and subjected to PCR using DNA-1/DNA-2 and Beta01/Beta02 for alpha satellites and beta satellites, respectively. The amplified PCR products were cloned and sequenced commercially. After sequencing, in silico sequence and phylogenetic analysis was also performed. Our study discovered that many plants in the Kohat District display begomovirus and satellite disease symptoms with mild to extreme disease severity. Disease incidence is especially high in okra. Beta satellites were isolated and sequenced from Spinacia oleracea and Capsicum annum plants and they showed more than 90% sequence similarity with chilli leaf curl and tomato leaf curl beta satellites. The existence of betasatellites in spinach and chilli plants was discovered for the first time in the Kohat region. Moreover, the distribution of these highly pathogenic variants of chilli leaf curl and tomato leaf curl betasatellites in the district Kohat has been reported previously.

Molecular Characterization of the Begomovirus Associated Satellites Infecting Spinacia Oleracea and Capsicum Annum Plants in Kohat, Pakistan

Begomovirus is a major and economically important genus of the Geminiviridae family. It comprises a wide range of viruses that infect a number of dicot plants including the horticulture crops, cereal crops, aromatic plants, vegetable crops, medicinal plants and weeds in various regions of the world. This study aims to investigate and correlate the various symptoms of begomovirus / satellites in different plants grown in the vicinity of Kohat, Pakistan. Furthermore, the characterization of the selected virus-associated satellites at the molecular level is also studied. Samples of suspected plants showing begomoviral infection were collected from the Kohat District. Genomic DNA was extracted from the infected plants and subjected to PCR using DNA-1/DNA-2 and Beta01/Beta02 for alpha satellites and beta satellites, respectively. The amplified PCR products were cloned and sequenced commercially. After sequencing, in silico sequence and phylogenetic analysis was also performed. Our study discovered that many plants in the Kohat District display begomovirus and satellite disease symptoms with mild to extreme disease severity. Disease incidence is especially high in okra. Beta satellites were isolated and sequenced from Spinacia oleracea and Capsicum annum plants and they showed more than 90% sequence similarity with chilli leaf curl and tomato leaf curl beta satellites. The existence of betasatellites in spinach and chilli plants was discovered for the first time in the Kohat region. Moreover, the distribution of these highly pathogenic variants of chilli leaf curl and tomato leaf curl betasatellites in the district Kohat has been reported previously.

Spatial distribution of chili leaf curl disease and its correlation with environmental factors in Southern Irrigated Plain Zone of Pakistan

Spatial distribution of chili leaf curl disease and its correlation with environmental factors in Southern Irrigated Plain Zone of Pakistan

Suppression of chili leaf curl virus (ChLCV) incidence in chili (Capsicum annuum L.) across Bangladesh via manipulated planting date and spacing

A 2-year factorial experiment was undertaken in RARS, Jessore in 2019 and 2020 following randomized complete block design with three replicates to evaluate the impact of planting date and spacing on ChLCV disease development and its vector whitefly abundance in chili (Capsicum annuum) modulating chili growth and yield attributes. The field experiments were found consistent between the years improving the reliability and generalizability of the data. The factors planting date and spacing separately produced statistical variation in ChLCV epidemiological parameters along with growth and yield attributing characters. Of the three chili planting dates, early planting (January 1) significantly suppressed ChLCD incidence, severity and its vector whitefly abundance over mid (February 1) and late planting (March 1) dates. Among four spacings, spacious plant spacing (50 × 60 cm) performed significantly better than other spacings in reducing ChLCD incidence and thereby expediting chili growth and yield. No statistical variation was documented between two narrow spacings of 50 × 30 cm and 50 × 40 cm in most of the studied parameters. Regression analyses depicted AUSiPC and AUDPC declined by 13.07 and 12.66 with each unit increase in plant spacing ensuring good fits of the models. A three-variables contour plot illustrated how vector whitefly abundance in chili stimulated ChLCD incidence and reduced chili yield while a 3-D wireframe pointed out that ChLCD incidence accelerated both AUSiPC and AUDPC. PCA analysis identified the major components of the studied parameters impacting disease incidence and chili yield. Cluster analyses well supported the PCA analysis and distinctly branched all the studied parameters into two, one associated with chili yield promoting characters and another with yield constraining ChLCD epidemiological attributes positioned in different nodes with variable similarity matrix. Molecular PCR analysis confirmed the presence of ssDNA (+) ChLCV in sampled plants.

Solanum pseudocapsicum: Wild source of resistance to Chilli leaf curl disease

The present investigation was undertaken to screen Solanum pseudocapsicum, against chilli leaf curl disease (ChiLCD) which is the most destructive virus disease in terms of incidence and yield loss in chilli peppers. The screening was done both under natural epiphytotic condition and through challenge inoculation using viruliferous whiteflies. The genus, which is perennial in nature and has a life span of about 10 years, was found free of infection under field screening even after four years of continuous exposure to high disease pressure. Under challenge inoculation, S. pseudocapsicum was found asymptomatic upon infection with ChiLCV and ToLCNDV, the two pre-dominant begomovirus prevalent in the study area. S. pseudocapsicum plants exposed to leaf curl disease under field condition was analysed for presence of ChiLCV through PCR and was found to show presence of virus indicating it to be a symptomless carrier of the disease. S. pseudocapsicum has proven to be a source of resistance against ChiLCD.

Biology and Interaction of the Natural Occurrence of Distinct Monopartite Begomoviruses Associated With Satellites in Capsicum annum From India.

Chili (Capsicum annuum L.) is an important vegetable and spice crop of tropical and sub-tropical regions. Chili plants showing upward leaf curling, leaf crinkling, and leaf yellowing symptoms, collected from Sikar district of Rajasthan, India, were found to be associated with begomovirus and satellite molecules. The presence of virus was confirmed by PCR using virus-specific primer. The full-length genomic DNA-A of three begomovirus (MM-1, CS-1 and RV-1) and two satellites (MM-2 and MM-3) were cloned which was identified from single symptomatic chili plant. The genome organization of isolated three viruses is similar to those of other Old World monopartite begomoviruses. The comparison of the sequences and closest phylogenetic relationships for the begomoviruses, betasatellite and alphasatellite DNAs revealed that MM-1 was designated as DNA-A of Chili leaf curl virus (ChiLCV), CS-1 is considered to be a new distinct species of Tomato leaf curl Gujrat virus (ToLCGV) whereas RV-1 as a new strain of Cotton leaf curl Multan virus (CLCuMuV). The DNA-A component of ChiLCV showed 8.6%, ToLCGV of 16.6% and CLCuMuV of 7.7% average evolutionary divergence, concomitantly, the betasatellite and alphasatellite molecule had 9.9% and 5.9% overall sequence divergence, respectively. Interestingly, most of the begomoviruses were found to be intra-species recombinants. The dN/dS ratio and Tajima D value of all viral DNA-A component and their associated betasatellite showed their selective control on evolutionary relationships. The nucleotide substitution rates were determined for the DNA-A genomes of ChiLCV (7.22 × 10–4 substitutions site–1 year–1), CLCuMuV (1.49 × 10–4 substitutions site–1 year–1), ToLCGV (7.47 × 10–4 substitutions site–1 year–1), the genome of associated ChiLCB (4.20 × 10–4 substitutions site–1 year–1) and CLCuMuA (1.49 × 10–4 substitutions site–1 year–1). Agro-inoculation studies indicate that the presence of DNA betasatellite induce severe symptoms in N. benthamiana and chili, suggesting prerequisite association for typical disease development.

Differential expression profiling of defense related genes for Leaf Curl Virus (ChiLCV) in resistant and susceptible genotypes of Chiili

The present investigation was undertaken to study the expression of eight defense related genes in leaf curl resistant line DLS-Sel-10 and susceptible line Phule Mukta after different days post inoculation (dpi) with viruliferous white flies to understand their role in resistance to chilli leaf curl virus (ChiLCV). The expression level of Ca PPO, Ca AsPer, Ca ATP/ADP and CaTopoII was observed to be higher in resistant genotype DLS-Sel-10 than the susceptible Phule Mukta at all the time points studied. Expression of CaNBS-LRR increased up to 12 dpi while that of Ca Thionin, and Ca SKP1 increased up to 24 dpi in the resistant line, thereafter it started declining. The CaSPI expression did not show any specific pattern in both the test plants. The heat map clustered all the genes under study into two major clusters based on their expression profiles, one comprising CaAsPer, Ca-Thionin, CaATP/ADP transporter, CaPPO and CaTopoII while other group comprised CaSKPI, Ca NBS and CaSPI. The challenge inoculation of the test genotypes also revealed that viral titre increased at a much slower rate in DLS-Sel-10 than Phule Mukta, suggesting thereby that DLS-Sel-10 is resisting the accumulation of ChiLCV and has a more active defense machinery than Phule Mukta.

Molecular diagnosis of begomovirus associated with Chilli leaf curl disease in Jeddah, Saudi Arabia.

Chilli (Capsicum annum L.) is well known as ‘wonder spice’. This is a very valuable cash crop grown as a vegetable globally. Chilli leaf curl disease is a major threat and global concern for the cultivation of Chilli by farmers and growers. In this work, the molecular diagnosis, genetic diversity, phylogenetic relationship, and begomovirus association with Chilli leaf curl disease have been discussed. The infected leaves were randomly harvested from the Chilli field, at Jeddah, Saudi Arabia. A group of begomovirus vector, whiteflies were also observed on the Chilli crop and infected weeds growing in the neighboring field. The begomovirus was confirmed by coat protein gene specific primer, dot blot hybridization, sequencing and sequence analysis. The full coat protein gene was found to have 774 nucleotides. The nucleotide sequences analysis shared the highest identity with Tomato yellow leaf curl virus reported earlier infecting tomato from Saudi Arabia, and the lowest identity was observed with Tomato yellow leaf curl virus Oman isolate. The overall sequence identity ranged from more than ninety percent among the analyzed sequences. The phylogenetic relationship analysis formed the major three clusters and showed the closed clustering with Tomato yellow leaf curl virus isolates. The natural spread of the Tomato yellow leaf curl virus on the Chilli crop from other crops poses an important and serious threat to Chili cultivation in the Kingdom of Saudi Arabia. Based on the literature review and current evidence, this is the first report of leaf curl disease of Chilli from Saudi Arabia.

Molecular and biological characterization of Chilli leaf curl virus and associated betasatellite infecting Cucurbita maxima in Oman.

During a survey in February 2016, leaf curl disease symptoms were witnessed in Cucurbita maxima plants in Al-Batina commercial farm in Oman. Symptoms exhibited were characteristic of begomovirus infection as leaf curling, yellowing, and color breaking followed by mosaic pattern. The transmission electron microscopy confirmed the presence of typical twinned geminate typical of Geminate virus particles. Rolling circle amplification (RCA) was employed to characterize the unknown causal agent of C. maxima disease. In molecular identification RCA produced nearly 2.8 and 1.4kb DNA molecules corresponding to begomovirus and satellite molecules, cloned and sequenced them. In Blast, species demarcation tool and phylogenetic analysis revealed the begomovirus and satellite isolates were determined as Chilli leaf curl virus (ChLCV) and tomato leaf curl betasatellite (ToLCB). In biological analysis by agrobacterium mediated inoculation, ChLCV displayed upward leaf curling and vein swelling symptoms in Nicotiana benthamiana plants; however, in presence of ToLCB enhanced downward leaf curling and crumpling symptoms were revealed. This study provides the first evidence that ChLCV and ToLCB caused leaf curl disease of C. maxima in Oman.

Cotton leaf curl Kokhran virus in association with Chili leaf curl betasatellite infecting mungbean (Vigna radiata.) and black gram (Vigna mungo.) in Pakistan

In 2016, begomovirus-like symptoms were observed in mungbean (Vigna radiata) and black gram (Vigna mungo) in Pakistan. Symptomatic and asymptomatic leaf samples were collected and total genomic DNA was extracted. Full length begomovirus and betasatellite genomes were PCR amplified and cloned in plasmid pTZ/57RT cloning vector. The cloned molecules were subjected to Sanger sequencing. Based on Sequence Demarcation Tool, the begomovirus clone SR-3 identified in mungbean showed 94.50% highest identity with cotton leaf curl Kokhran virus-Kokhran (CLCuKoV-Ko), while the clone SR-5 isolated from black gram showed 99.7% highest identity with CLCuKoV-Ko. In phylogenetic tree analysis the identified isolates made a lower branch tree with CLCuKoV-Ko. The betasatellite clone SR-79 isolated from black gram showed 97% similarity with chili leaf curl betasatellite (ChLCB) and the betasatellite clone SR-93 isolated from mungbean showed 97.7% similarity with ChLCB. Southern blot analysis also confirmed the presence of CLCuKoV-Ko. This is the first report of CLCuKoV-Ko associated with ChLCB infecting both mungbean and black gram in Pakistan. This presents the new combination of disease complex and increase in host range of this virus species, which shows a real threat to other crops especially legume crops in Pakistan.

Molecular mapping of dominant gene responsible for leaf curl virus resistance in chilli pepper (Capsicum annuum L.).

A resistant source (S-343) having monogenic dominant resistance to chilli leaf curl virus disease (ChiLCVD) has been identified at Punjab Agricultural University (PAU), Ludhiana. The F2 mapping population of 204 plants was derived from the cross MS-341 (susceptible) × S-343 (resistant) to identify the linked marker with the disease-resistant gene. Out of the 685 single-sequence repeats (SSRs) used, only 160 primers showed parental polymorphism. These 160 polymorphic primers were used for bulk segregant analysis and only eight SSR primers were able to differentiate the resistant and susceptible bulks. The linkage analysis revealed that the two markers CA 516044 and PAU-LC-343-1 were found linked with the disease-resistant gene covering a total distance of 15.7 centimorgan (cM). The two primers CA 516044 and PAU-LC-343-1 were found located on chromosome 6 of the pepper genome at a genetic distance of 6.8cM and 8.9cM, respectively, from the resistant gene. The validation of linked markers was performed using 26 resistant and susceptible genotypes developed at PAU, Ludhiana by former researchers. The validation of the primers revealed that there was a correlation between phenotypic and genotypic data of the used genotypes, and these markers can be used for the marker-assistedbreeding procedures for transferring ChiLCVD resistance until the gene-based markers will be developed. The markers described in this study are the first-ever molecular markers identified as linked to the ChiLCVD-resistant gene.

First Report of Duranta leaf curl virus Infecting Ficus virens Showing Leaf Curl Symptoms in Pakistan

Ficus virens (family: Moraceae) is a deciduous tree found in the Indian subcontinent, some other Southeast Asian countries, and northern Australia. It is valued for its latex, wood, and medicinal and edible products. In December 2018, during a routine survey to record begomoviruses other than cotton infecting viruses in Pakistan, curling of young leaves, which is a typical symptom of infection by viruses of the genus Begomovirus, was observed in F. virens trees in a park in Lahore, central Punjab, Pakistan. The total DNA of three trees was extracted using the Viral Gene-spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea) and used as a template in PCR amplification with begomovirus-specific primers: Beg-F (5′-CCGTGCTGCTGCCCCCATTGTCCGCGTCAC-3′) and Beg-R (5′- CTGCCACAACCATGGATTCACGCACAGGG-3′). The amplified 1-kb DNA was cloned into the pGEM-3Zf (+) vector (Promega, Madison, WI) and sequenced by a commercial sequencing service (Macrogen, Seoul, Korea). NCBI BLAST results revealed a nucleotide (nt) sequence identity of 95, 93, and 92% with chilli leaf curl India virus, rose leaf curl virus, and papaya leaf crumple virus, respectively. Based on the sequence obtained, new primers, DCP-F (5′-AGCACATTGGTAAAGTCATGTGTG-3′) and DCP-R (5′-GGATATCATGTCTGGACTCAAATG-3′), were designed to obtain full-length DNA-A of the begomovirus. An approximately 2.8-kb product was amplified and cloned. The resulting sequence (GenBank accession no. MN537564) showed 97.43% nt identity with duranta leaf curl virus (DLCV; KT948069), a Begomovirus species reported in the ornamental shrub Duranta erecta in Pakistan by Iram et al. (2005) and by Anwar et al. (2017). In the former case, only DNA-A of DLCV was found without any associated molecules such as DNA-B or DNA β, whereas, in the latter case, tomato leaf curl New Delhi virus DNA-B was found in association with the DNA-A of DLCV, like in many other Old World begomoviruses. The attempt to amplify DNA-B through PCR using newly designed primers NDB-F (5′-AAACACAGGAGGGATCGGAG-3′) and NDB-R (5′-CACAGATTTCCTTACGCGTATATT-3′) based on the sequence KT948071 was unsuccessful. Similarly, efforts to detect alphasatellite and betasatellite molecules in infected F. virens using universal primers (Bull et al. 2003; Briddon et al. 2002) were unsuccessful. Furthermore, Southern blot hybridization was carried out using [³²P]-radiolabeled PCR product of 1 kb obtained with Beg-F/R primers as a probe. This produced dsDNA bands of different conformations as well as a ssDNA band, which confirms the presence of DLCV in F. virens. Members of the family Geminiviridae, to which begomoviruses belong, mainly infect herbaceous hosts, but they have also been reported in a few woody hosts, such as citrus chlorotic dwarf associated virus in citrus (Loconsole et al. 2012), grapevine red blotch virus in grape (Al Rwahnih et al. 2013), jatropha mosaic virus in Jatropha multifida (Polston et al. 2014), and apple geminivirus 1 in apple tree (Liang et al. 2015). To our knowledge, this is the first report of DLCV infecting F. virens in Pakistan. The results obtained here indicate that F. virens may serve as an alternative host of DLCV and a possible source of infection to the commonly grown D. erecta ornamental garden hedge.

In silico analysis of chili encoded miRNAs targeting Chili leaf curl begomovirus and its associated satellite

Chilli leaf curl virus (ChiLCV), a member of Geminiviridae family (Genus: Begomovirus), is one of the most destructive plant virus. Micro (mi) RNAs, are the endogenous non-coding small RNAs that play significant roles in plant growth and stress resistance by degrading targeted mRNA or repressing mRNA translation. Computational methods have identified numerous miRNAs in many plant species, whereas there is no report of chilli miRNAs targeting essential genes of ChiLCV genome and associated satellites. In this study, we have predicted chilli encoded miRNAs that could be used for silencing against chilli leaf curl virus (Accession no. MF737343) infection. We predicted several potential mir miRNAs, exhibited high complementarities with V1 (CP) and C1 (Rep) genes of ChiLCV. Other overlapping genes V2, C2 and C3 were also targeted by mir-miRNAs.

Genetic analysis for resistance to leaf curl disease in Chilli Peppers (Capsicum annuum L.) under specific situations

Leaf curl is a serious viral disease of chilli caused by a group of bigomoviruses dominated by chilli leaf curl virus (ChiLcv). With the aim to study the mode of inheritance of the ChiLcv disease resistance, a resistant genotype DLS-Sel.10 was crossed with a susceptible genotype Phule Mukta and F1, F2, B1 and B2 generations were developed. The parents along with the segregating generations were screened under natural conditions as well as challenged inoculation with viruliferous whiteflies carrying predominant ChiLcv.PCR amplification of viral genome-specific marker confirmed the presence of virus in all the tested plants however, only susceptible plants produced symptoms. The F1 plants showed susceptibility under both natural and challenged inoculation conditions indicating that the resistance is of recessive nature. On Chi-square test, it was found that susceptible and resistant plants of the two F2populations segregated in a ratio of 3 susceptible : 1 resistant plants and the B2 population derived from DLS-Sel.10/Phule Mukta//DLS-Sel.10 segregated in a ratio of 1 resistant : 1 susceptible plant suggesting that the Chilcv is goverened by a single recessive gene. The findings of this study will help in breeding for ChiLcv resistance in Chilli.

Screening chilli genotypes for whitefly (<em>Bemisia tabaci </em>Genn.) resistance: A vector for chilli leaf curl virus

Whiteflies (Bemisia tabaci) are one of the most important insect-pests of chilli worldwide mainly due to their role in transmission of Begomoviruses like Chilli Leaf Curl Virus (ChL CV). In this study, selected chilli genotypes (N = 125) including wild and cultivated species were evaluated against whitefly using free choice assays and parameters like number of whiteflies settled and nymphal population (both early and late stage) were considered to identify less preferred genotypes. The genotypes were also evaluated for plant traits namely trichome density, leaf colour and biochemical content (phenols and flavonoids). The results showed that the whitefly population and nymphal density were significantly different among the genotypes evaluated. Whitefly settlers and their nymphal density were positively correlated with the leaf non-glandular trichomes. The chilli genotypes viz., IHR 4283, IHR 4329, IHR 4300, IHR 4321, IHR 4338 recorded the less numbers of whiteflies as well as nymphs and grouped as the least preferred genotypes. On the contrary, the genotypes IHR 4586 A-1, IHR 4588 and IHR 4330 recorded the highest numbers of whiteflies. The role of plant traits in whitefly resistance in chilli was discussed in detail.

Host range studies of <em>Chilli veinal mottle virus </em>(Chi VMV) in chilli (<em>Capsicum annuum </em>L.)

Chilli (Capsicum annuum L.), being the most important remunerative vegetable and is susceptible to a wide range of viruses which are the major constraints in its production resulting to heavy crop losses. Among these, after Chilli leaf curl virus, Chilli veinal mottle virus (Chi VMV) is the major prevalent virus. Host range studies under glasshouse conditions revealed that Chi VMV is transmitted mechanically. Among 41 host plants tested, nine different plant species (Datura metel, Capsicum annuum, Physalis floridana, Solanum nigrum, Lycopersicon esculentum, Amaranthus spp, Nicotiana tabaccum cv. White Burley, Nicotiana tabaccum cv. Samsun and Capsicum frutescense.) induced characteristic systemic mottling symptoms within 7 to 14 days of inoculation. The rest of the hosts remained asymptomatic.

Identification of resistant sources against chilli leaf curl virus disease through field and molecular screening in chilli

A study was conducted to discover resistant sources in 109 F6 families of a Recombinant Inbreed Lines (RIL) population against Chilli Leaf Curl Virus (ChiLCV) disease, a serious menace to chilli crop in India. The Population was characterized for yield and disease related traits that revealed presence of significant variability in the population. Fruit yield followed by number of fruits per plant, fruit weight and PDI (at 120 days after transplanting) were key traits contributing significant portion of this variation as explained by principal component analysis. Twenty virus-free plants were identified under natural field condition at 60 and 120 days after transplanting along with molecular screenings using universal primers. These potent plants having ChiLCV resistance and superior plant and fruit morphology will be utilized in breeding programme for development of resistant varieties/hybrids against chilli leaf curl virus disease.

Evaluation of the effects of an eco-friendly crop protection system on management of whitefly-vectored chilli leaf curl virus disease in Sri Lanka

The present study was conducted to evaluate an eco-friendly crop protection system having less reliance on pesticides with an existing crop protection system adopted by farmers depending on frequent use of pesticides for management of whitefly-transmitted virus diseases of chilli. Further, quantification of defense-related enzymes in chilli leaf tissues and calculation of field use environmental impact quotient of pesticide use were done for the crop grown under the two systems. Field experiments were conducted at five locations, namely Kilinochchi, Mahailluppallama, Kundasale, Peradeniya and Rahangala using chilli cv. MI Green for two consecutive growth seasons to evaluate the two crop protection systems. Percentage incidence of chilli plants showing virus-like symptoms varied significantly by location, growth stage and the interaction effect between location x crop protection system in both seasons. Chilli leaf curl complex infestation and leaf deformation were the most prominent in the chilli plants showing virus-like symptoms. In general, there was no significant variation (P = 0.05) between the two crop protection systems on the activity of four defense enzymes tested (i.e. peroxidase, β-1,3-glucanase, chitinase and phenylalanine ammonia lyase) in chilli tissues. The eco-friendly system had lower environmental impact of pesticides compared to the existing crop protection system practiced by farmers in chilli fields. Multi-location trials for two growing seasons confirmed that the eco-friendly package tested is equally-capable as the existing crop protection system for the management of virus diseases in chilli in Sri Lanka with a lower environmental impact.

Molecular and biological characterization of Chilli leaf curl virus and associated Tomato leaf curl betasatellite infecting tobacco in Oman

BackgroundIn Oman tobacco (Nicotiana tabacum; family Solanaceae) is a minor crop, which is produced only for local consumption. In 2015, tobacco plants exhibiting severe downward leaf curling, leaf thickening, vein swelling, yellowing and stunting were identified in fields of tobacco in Suhar Al-Batina region, Oman. These symptoms are suggestive of begomovirus (genus Begomovirus, family Geminiviridae) infection.MethodsCircular DNA molecules were amplified from total DNA extracted from tobacco plants by rolling circle amplification (RCA). Viral genomes were cloned from RCA products by restriction digestion and betasatellites were cloned by PCR amplification from RCA product, using universal primers. The sequences of full-length clones were obtained by Sanger sequencing and primer walking. Constructs for the infectivity of virus and betasatellite were produced and introduced into plants by Agrobacterium-mediated inoculation.ResultsThe full-length sequences of 3 begomovirus and 3 betasatellite clones, isolated from 3 plants, were obtained. Analysis of the full-length sequences determined showed the virus to be a variant of Chilli leaf curl virus (ChiLCV) and the betasatellite to be a variant of Tomato leaf curl betasatellite (ToLCB). Both the virus and the betasatellite isolated from tobacco show the greatest levels of sequence identity to isolates of ChiLCV and ToLCB identified in other hosts in Oman. Additionally clones of ChiLCV and ToLCB were shown, by Agrobacterium-mediated inoculation, to be infectious to 3 Nicotiana species, including N. tabacum. In N. benthamiana the betasatellite was shown to change the upward leaf rolling symptoms to a severe downward leaf curl, as is typical for many monopartite begomoviruses with betasatellites.ConclusionsThe leaf curl disease of tobacco in Oman was shown to be caused by ChiLCV and ToLCB. This is the first identification of ChiLCV with ToLCB infecting tobacco. The study shows that, despite the low diversity of begomoviruses and betasatellites in Oman, the extant viruses/betasatellites are able to fill the niches that present themselves.

Disease incidence and molecular indexing of viruses infecting King chilli (Capsicum chinense Jacq) in North East India

A survey was conducted in King chilli growing areas of four Northeast states of India namely, Assam, Nagaland, Manipur and Meghalaya for virus disease incidence particularly Cucumber mosaic virus (CMV), Chilli veinal mottle virus (ChiVMV) and Chilli leaf curl virus (ChiLCV). The virus disease incidence based on symptomatology ranged from 15 to 65% with highest incidence recorded in Golaghat district of Assam and lowest in the Ukhrul district of Manipur. The polymerase chain reaction based detection using coat protein gene specific primers for the three viruses revealed the presence of CMV and ChiVMV in all the four states whereas ChiLCV was recorded only in one location from Assam. In two of the samples collected for CMV and ChiVMV, mixed infection of the two viruses was confirmed. Based on molecular method based detection, the incidence of CMV infection in King chilli among all the samples tested for the virus was 32.5% whereas it was 25.97% for ChiVMV.