Chilli Leaf Curl Disease Research Articles

Characterization of a new begomovirus and betasatellite associated with chilli leaf curl disease in India.

Chilli leaf curl disease (ChiLCD) is a serious problem and a major limitation to chilli (Capsicum spp.) cultivation in India. Leaves of a chilli plant showing leaf curl symptoms were collected from the Gonda district in Uttar Pradesh, India, in April, 2013. Full-length genomes of a begomovirus and an associated betasatellite were amplified, cloned and sequenced. The size of the begomovirus genome and the betasatellite were 2760 bp and 1374 bp, respectively. The nucleotide sequence of the begomovirus genome shared maximum identity (89%) with pepper leaf curl Bangladesh virus-India isolate Chhapra (PepLCBV, JN663853), below the threshold for species demarcation. Sequence analysis showed that the begomovirus is a potential recombinant between viruses related to PepLCBV and chilli leaf curl virus (ChiLCV). The name chilli leaf curl Gonda virus (ChiLCGV) is being proposed. The betasatellite associated with ChiLCGV was identified as tomato leaf curl Bangladesh betasatellite (ToLCBDB). Agroinoculation of the viral genome along with betasatellite induced severe leaf curl symptoms in Nicotiana benthamiana. ChiLCGV and ToLCBDB characterized in this study represent a new begomovirus-betasatellite complex infecting Capsicum.

A new monopartite Begomovirus associated with betasatellite molecule causing leaf curl disease of chilli in India.

30Apr 2016 A new monopartite Begomovirus associated with betasatellite molecule causing leaf curl disease of chilli in India. Abhinav Kumar , Vartika Sinha , Zainul A. Khan , Neera B. Sarin , Seema Singh and Archana Tiwari. School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi-110067, India. Dept. of Biosciences, Jamia Milia Islamia, New Delhi-110025, India. University of Kashmir Hazratbal, Srinagar, Jammu and Kashmir- 190006, India School of Sciences, Noida International University, G.B. Nagar, Greater Noida Plot No.1, Sector-17-A, India

Identification and molecular characterization of a new recombinant begomovirus and associated betasatellite DNA infecting Capsicum annuum in India.

Capsicum annuum (Chilli) is a perennial herbaceous plant that is cultivated as an annual crop throughout the world, including India. Chilli leaf curl disease (ChiLCD) is a major biotic constraint, causing major losses in chilli production. During 2014, leaf samples of chilli plants displaying leaf curl disease were collected from the Ahmedabad district of Gujarat, India. These samples were used to isolate, clone and sequence viral genomic DNA and an associated betasatellite DNA molecule. Sequence analysis showed 90.4 % nucleotide sequence identity to the previously reported chilli leaf curl virus-[India:Guntur:2009] (ChiLCV-[IN:Gun:09]. As per ICTV nomenclature rules, ChiLCV-Ahm represents a new species of begomovirus, and we therefore propose the name chilli leaf curl Ahmedabad virus-[India:Ahmedabad:2014] (ChiLCAV-[IN:Ahm:14]). The associated betasatellite DNA showed a maximum of 93.5 % nucleotide sequence identity to a previously reported tomato leaf curl Bangladesh betasatellite and may be named tomato leaf curl Bangladesh betasatellite-[India:Ahmedabad:Chilli:2014].

Complexity of begomovirus and betasatellite populations associated with chilli leaf curl disease in India.

Chilli, which encompasses several species in the genus Capsicum, is widely consumed throughout the world. In the Indian subcontinent, production of chilli is constrained due to chilli leaf curl disease (ChiLCD) caused by begomoviruses. Despite the considerable economic consequences of ChiLCD on chilli cultivation in India, there have been scant studies of the genetic diversity and structure of the begomoviruses that cause this disease. Here we report on a comprehensive survey across major chilli-growing regions in India. Analysis of samples collected in the survey indicates that ChiLCD-infected plants are associated with a complex of begomoviruses (including one previously unreported species) with a diverse group of betasatellites found in crops and weeds. The associated betasatellites neither enhanced the accumulation of the begomovirus components nor reduced the incubation period in Nicotiana benthamiana. The ChiLCD-associated begomoviruses induced mild symptoms on Capsicum spp., but both the level of helper virus that accumulated and the severity of symptoms were increased in the presence of cognate betasatellites. Interestingly, most of the begomoviruses were found to be intra-species recombinants. The betasatellites possess high nucleotide variability, and recombination among them was also evident. The nucleotide substitution rates were determined for the AV1 gene of begomoviruses (2.60 × 10- 3 substitutions site- 1 year- 1) and the βC1 gene of betasatellites [chilli leaf curl betasatellite (ChiLCB), 2.57 × 10- 4 substitution site- 1 year- 1; tomato leaf curl Bangladesh betasatellite (ToLCBDB), 5.22 × 10- 4 substitution site- 1 year- 1]. This study underscores the current understanding of Indian ChiLCD-associated begomoviruses and also demonstrates the crucial role of betasatellites in severe disease development in Capsicum spp.

RNAi mediated broad-spectrum transgenic resistance in Nicotiana benthamiana to chilli-infecting begomoviruses.

Two RNAi constructs were designed targeting chilli-infecting begomoviruses and associated betasatellites. Broad-spectrum resistance was achieved against multiple begomoviruses associated with leaf curl disease of chillies in India. Chilli leaf curl disease (ChiLCD) caused by begomoviruses (family: Geminiviridae) has emerged as one of the most devastating viral diseases of chilli, especially in the Indian sub-continent. The severity of disease incidence is expanding at an alarming rate due to the emergence of new begomoviruses with greater ability to infect this crop in almost all the major chilli producing regions of India. In this study, we applied the RNA interference (RNAi) based strategies to control infection of chilli-infecting begomoviruses (CIBs). For this, we have generated transgenic Nicotiana benthamiana plants harboring two different intron hairpin RNAi constructs [designated as TR1 (AC1/AC2) and TR2 (AC1/AC2/βC1)] using conserved regions of viral genome and associated betasatellite. During our study, we observed that, two lines harboring TR1 construct (13-1 and 2-4) and one line harboring TR2 construct (5-1) have shown resistance to the most predominant Indian CIBs like Chilli leaf curl virus-Pakistan isolate Varanasi, Tomato leaf curl New Delhi virus-isolate chilli, and a newly identified begomovirus species, Chilli leaf curl Vellanad virus. Resistant lines accumulated transgene-specific siRNAs, confirming RNAi-mediated resistance against these viruses. Furthermore, these resistant lines also displayed delayed symptom appearance and milder symptoms, as compared to virus-inoculated non-transgenic plants. Average viral DNA accumulation in the resistant lines was reduced up to 90% as compared to non-transgenic plants. Thus, our study demonstrated the application of RNAi-mediated approach in providing resistance against diverse monopartite and bipartite begomoviruses associated with ChiLCD.

Evidence of Association of aTomato Leaf Curl New Delhi Viruswith Chilli Leaf Curl Disease in Western Uttar Pradesh, India

Chilli Leaf Curl Disease (ChLCD) is an important limiting factor affecting chilli production on Indian subcontinent and is caused by begomoviruses. During November’ 2011, very high disease incidence (up to 100% of plants) was observed in chilli fields near Meerut district of Western Uttar Pradesh, India with Symptoms such as upward leaf curling, crowding and yellowing of leaves and stunting of whole plants. In this paper we report the evidence of a Tomato Leaf Curl New Delhi Virus (ToLCNDV) and associated betasatellite responsible for the leaf curl disease in Chilli.

A new begomovirus–betasatellite complex is associated with chilli leaf curl disease in Sri Lanka

Leaf curl disease of chilli (LCDC) is a major constraint in production of chilli in the Indian subcontinent. The objective of this study was to identify the begomovirus species occurring in chilli in Sri Lanka, where the LCDC was initially recorded in 1938. The virus samples were collected from the North Central Province, the major chilli growing region in Sri Lanka with a history of epidemic prevalence of LCDC. The virus could be readily transmitted by Bemisia tabaci to chilli, tomato and tobacco, where vein clearing followed by leaf curl developed. The genome analysis of two isolates obtained from two distantly located fields showing 100% LCDC, revealed that the DNA-A genome (2754 nucleotides) shared 89.5% sequence identity with each other and 68.80-84.40% sequence identity with the other begomoviruses occurring in the Indian subcontinent. The closest identity (84.40%) of the virus isolates was with Tomato leaf curl Sri Lanka virus (ToLCLKV). The results support that a new begomovirus species is affecting chilli in Sri Lanka and the name Chilli leaf curl Sri Lanka virus (ChiLCSLV) is proposed. Recombination analysis indicated that ChiLCSLV was a recombinant virus potentially originated from the begomoviruses prevailing in southern India and Sri Lanka. The genome of betasatellite associated with the two isolates consisted of 1366 and 1371 nucleotides and shared 95.2% sequence identity with each other and 41.50-73.70% sequence identity with the other betasatellite species. The results suggest that a new begomovirus betasatellite, Chilli leaf curl Sri Lanka betasatellite is associated with LCDC in Sri Lanka. This study demonstrates a new species of begomovirus and betasatellite complex is occurring in chilli in Sri Lanka and further shows that diverse begomovirus species are affecting chilli production in the Indian subcontinent.

Development of PCR-based diagnostic probe to detect begomoviruses infecting chilli in the hot arid region of Rajasthan

A survey was conducted in major chilli-growing hot arid regions of Rajasthan, namely, Bikaner, Nagur, Jodhpur and Jalore districts, during November 2009. Among the four districts surveyed for chilli leaf curl disease (ChLCD), maximum disease incidence was recorded in Jodhpur (98%) followed by Jalore district (88%). The number of whiteflies was also counted in top, middle and bottom leaf of chilli plants grown in these areas. The average number of whiteflies per plant ranged from 0.0 to 4.0. Higher number of whiteflies (4.0) was recorded in Jodhpur and lowest (1.8) in Jalore district. On the basis of conserved region in the genome of begomoviruses infecting chilli, a set of primers was designed to amplify all begomoviruses infecting chilli by PCR; ChCPF 5'-ATTAGGGCTAAGAATTATGTC-3' and ChCPR 5'-AAATTCCAATCTTTATTAATT-3'. These primers were validated by cloning and sequencing (HM004433) of PCR-amplified products and detection from infected chilli leaf samples. These primers were utilised while screening chilli cultivars against begomovirus infection in the asymptomatic plants. Our investigation suggests that the leaf curl disease of chilli is widespread in the hot arid regions of Rajasthan and is caused by a begomovirus associated with a satellite DNA β. The PCR primers designed in this study could be highly useful in chilli breeding programmes.

Virus–vector Relationships, Host Range, Detection and Sequence Comparison of Chilli leaf curl virus Associated with an Epidemic of Leaf Curl Disease of Chilli in Jodhpur, India

AbstractAn epidemic of chilli leaf curl disease was recorded in 2004 in Jodhpur, a major chilli‐growing area in Rajasthan, India. Several isolates were efficiently transmitted by the whitefly (Bemisia tabaci), all of which induced severe leaf curl symptoms in chilli. A single whitefly was capable of transmitting the virus, and eight or more whiteflies per plant resulted in 100% transmission. The minimum acquisition access period (AAP) and inoculation access period (IAP) were 180 and 60 min, respectively. The virus persisted in whiteflies for up to 5 days postacquisition. Of 25 species tested, the virus infected only five (Capsicum annuum, Carica papaya, Solanum lycopersicum, Nicotiana tabacum and N. benthamiana). The virus was identified as Chilli leaf curl virus (ChiLCV), which shared the closest sequence identity (96.1%) with an isolate of ChiLCV from potato in Pakistan and showed sequence diversity up to 12.3% among the ChiLCV isolates reported from India and Pakistan. A betasatellite was identified, which resembled most closely (97.3%) that of Tomato leaf curl Bangladesh betasatellite previously reported from chilli and tomato leaf curl in India. The betasatellite was very different from that reported from chilli leaf curl in Pakistan, indicating that different betasatellites are associated with chilli leaf curl in India and Pakistan. We describe here for the first time the virus–vector relationships and host range of ChiLCV.

A new monopartite begomovirus species, Chilli leaf curl Vellanad virus, and associated betasatellites infecting chilli in the Vellanad region of Kerala, India

Capsicum annuum (chilli) is one of the most important commercial crops cultivated in the Indian sub-continent for vegetable, spice and industrial purposes. Leaf curl disease of chilli has become a serious problem in the Vellanad district …

Pepper leaf curl Lahore virus requires the DNA B component of Tomato leaf curl New Delhi virus to cause leaf curl symptoms.

BackgroundBegomoviruses are whitefly-transmitted geminiviruses with genomes that consist of either two components (known as DNA A and DNA B) or a single component (homologous to the DNA A component of bipartite begomoviruses). Monopartite begomoviruses are often associated with a symptom-modulating DNA satellite (collectively known as betasatellites). Both bipartite and monopartite begomoviruses with associated satellites have previously been identified in chillies showing leaf curl symptoms in Pakistan.ResultsA chilli plant (Capsicum annum) with chilli leaf curl disease symptoms was found to contain a begomovirus, a betasatellite and the DNA B component of Tomato leaf curl New Delhi virus (ToLCNDV). The begomovirus consisted of 2747 nucleotides and had the highest sequence identity (99%) with Pepper leaf curl Lahore virus (PepLCLV-[PK: Lah:04], acc. no. AM404179). Agrobacterium-mediated inoculation of the clone to Nicotiana benthamiana, induced very mild symptoms and low levels of viral DNA, detected in systemically infected leaves by PCR. No symptoms were induced in Nicotiana tabacum or chillies either in the presence or absence of a betasatellite. However, inoculation of PepLCLV with the DNA B component of ToLCNDV induced leaf curl symptoms in N. benthamiana, N. tabacum and chillies and viral DNA accumulated to higher levels in comparison to plants infected with just PepLCLV.ConclusionsBased on our previous efforts aimed at understanding of diversity of begomoviruses associated with chillies, we propose that PepLCLV was recently mobilized into chillies upon its interaction with DNA B of ToLCNDV. Interestingly, the putative rep-binding iterons found on PepLCLV (GGGGAC) differ at two base positions from those of ToLCNDV (GGTGTC). This is the first experimental demonstration of the infectivity for a bipartite begomovirus causing chilli leaf curl disease in chillies from Pakistan and suggests that component capture is contributing to the emerging complexity of begomovirus diseases in the region.

A Single Species of Betasatellite is Prevalent in Chilli across North Central Pakistan and Shows Phylogeographic Segregation

Chilli leaf curl disease is an important constraint to production of chilli peppers (Capsicum annum L.) across the Indian subcontinent and is caused by begomoviruses in association with betasatellites. Betasatellites are symptom modulating single-stranded DNA satellites associated with the majority of monopartite begomoviruses and are responsible for diseases of many crops including chilli peppers. Here, we have studied the diversity of a betasatellite of chillies in the Punjab and North Western Frontier Province of Pakistan. Sequence analysis of thirteen full-length clones showed that a single species of satellite, Chilli leaf curl betasatellite (ChLCB), is prevalent in chilli throughout the surveyed region. ChLCB has only been identified in Pakistan and infecting chilli. It thus probably represents a chilli adapted satellite. The ChLCBs fall into two groups, the first occurs in the less intensively cultivated north and shows a high level of sequence diversity (relative to the second group), while the second occurs in the southern cotton-growing areas and shows little sequence diversity. Between these two areas there is a region of overlap where both ChLCB types occur. The phylogeographic segregation of ChLCB we attribute to geographic isolation (in the north), leading to divergence and intensive cultivation (in the south), leading to homogenization. However, the lack of diversity (only a single species) bodes well for the prospects of achieving resistance to the disease by either conventional or pathogen-derived strategies.

Infectivity of the cloned components of a begomovirus: DNA beta complex causing chilli leaf curl disease in India

The full-length genome of a begomovirus and its cognate DNA-beta satellite component associated with chilli leaf curl disease (ChLCD), originating from Varanasi, India, were cloned. Sequence analysis revealed that the viral genome (EF190217) is 2,750 bp and the DNA-beta satellite (EF190215) is 1,361 bp in length. Agroinoculation with partial tandem repeats of the viral genome along with the satellite induced symptoms typical of ChLCD in chilli and Nicotiana benthamiana. However, symptom expression was delayed and milder when the viral genome was agroinoculated alone in these hosts. Sequence comparisons revealed that the genome had the highest sequence identity (95%) with that of chilli leaf curl virus-PK[PK:Mul:98]. The DNA-beta satellite shared maximum sequence identity (88%) with a DNA-beta satellite associated with tomato leaf curl disease from Rajasthan (ToLCBDB-[IN:Raj:03]). These results demonstrate that ChLCD is caused by a complex consisting of the monopartite chilli leaf curl virus and a DNA-beta satellite component. This is the first experimental demonstration of Koch's postulates using cloned DNA molecules associated with chilli leaf curl disease.

First report of Chilli leaf curl virus affecting chilli in India

) is an important spice crop cultivated through-out India. Leaf curl disease of Chilli has emerged as a serious problem inthe Jodhpur district, the major chilli growing area of Rajasthan state. Dur-ing December 2004, very high disease incidence (up to 100% of plants)was observed in farmers’ fields in Narwa and Tinwari villages. The char-acteristic field symptoms were upward curling, puckering and reduced sizeof leaves. Severely affected plants were stunted and produced no fruit. Thevirus from field samples from Narwa village was transmitted by whitefly(Bemisia tabaci) to 50–100% of chilli test plants, which produced veinclearing, curling and stunting symptoms. Electron microscopic examina-tion of field samples revealed few, typical geminate particles. The presenceof a begomovirus was confirmed by PCR using the degenerate primersAVF28 5′-GCCCACATYGTC TTYCCNGT-3 ′ and AV29R 5′-GGCTTYCTRT ACATRGG-3 ′, which gave a ca.1·0 kb product. Cloning and sequencingof the PCR product yielded a 995 bp sequence (Acc. No. DQ445255). ABLAST search of GenBank revealed close similarity of the sequence withthe intergenic region and part of the replication initiator protein, AV1 andAV2 genes of Chilli leaf curl virus-[Pakistan: Multan] (ChiLCuV-[Pk:Mul];AF336806) reported from Pakistan (Shih et al., 2003).In India, Tomato leaf curl New Delhi virus (ToLCNDV) was recentlyshown to be associated with chilli leaf curl disease occurring in Lucknow(Khan et al., 2006). Two more Begomovirus species, Cotton leaf curlMultan virus (CLCuMV) (Hussain et al ., 2004) and Pepper yellow leaf curlIndonesia virus (PepYLCIDV) (Tsai et al ., 2006), have been reported to beassociated with chilli leaf curl in Pakistan and Indonesia respectively.Sequence analysis showed that the virus isolated from Jodhpur wasdistantly related (59·1-67·9 % identity) to CLCuMV, PepYLCIDV andToLCNDV. However, it shared 96·5% identity with ChiLCuV-[Pak:Mul].Given the close sequence identity with ChiLCuV-[Pk:Mul], the virusisolated from Jodhpur is considered to be ChiLCuV. This is the firstreport of ChiLCuV affecting chilli in India and the recent report of Khanet al. (2006) show that chilli leaf curl in India is caused by more than onebegomovirus.ReferencesHussain M, Mansoor S, Iram S, Zafar Y, Briddon RW, 2004. First report of

Identification of host plant resistance to pepper leaf curl virus in chilli (Capsicum species)

Three hundred and seven genotypes belonging to four cultivated and one wild species of Capsicum were screened against pepper leaf curl virus (PepLCV) causing devastating leaf curl disease of chilli (Capsicum annuum). Initial screening was done under field conditions based on coefficient of infection (CI), disease reaction to each genotype was assigned. Subsequently, selfed progenies of eight symptom-less and highly resistant lines were challenged by viruliferous white fly under glasshouse conditions, out of which only three genotypes, viz. GKC-29, BS-35 and EC-497636 showed no symptom. Using scion and root stalk of susceptible genotype (Pusa Jwala), these three putative symptom-less genotypes were further challenged by grafting and alternate grafting. The resistant reactions of GKC-29, BS-35, EC-497636 were confirmed because even after 50 days of successful grafting/alternate grafting, no viral symptom appeared on all the grafted plants of these genotypes. When subjected to PCR amplification with degenerate primers deigned to detect gemnivirus like PepLCV, the three symptom-less genotypes did not show any amplification, suggesting that the resistant reaction in three identified symptom-less resistant sources was because of the absence of viral genome and they are not symptom-less carrier.