Indian Journal of Agricultural Research

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Indian Journal of Agricultural Research, volume 57 issue 6 (december 2023) : 836-840

Survey and Molecular Identification of Begomovirus Associated with Chilli Leaf Curl Disease in Kurnool District of Andhra Pradesh

B.H. Chaithanya1,*, T. Srinivas2, B.V. Bhaskara Reddy3
1Regional Agricultural Research Station, Acharya N.G. Ranga Agricultural University, Nandyal-518 502, Kurnool, Andhra Pradesh, India.
2Krishi Vigyan Kendra, Acharya N.G. Ranga Agricultural University, Banavasi, Yemmiganur-518 323, Kurnool, Andhra Pradesh, India.
3Regional Agricultural Research Station, Acharya N.G. Ranga Agricultural University, Tirupati-517 502, Chittoor, Andhra Pradesh, India.
Cite article:- Chaithanya B.H., Srinivas T., Reddy Bhaskara B.V. (2023). Survey and Molecular Identification of Begomovirus Associated with Chilli Leaf Curl Disease in Kurnool District of Andhra Pradesh . Indian Journal of Agricultural Research. 57(6): 836-840. doi: 10.18805/IJARe.A-6028.
Background: Chilli is an important spice and vegetable crop grown in tropical and sub-tropical climate. Recently, Chilli leaf curl disease has become a serious threat to chilli growing farmers. 

Methods: The present study was aimed to know prevalence of leaf curl disease in chilli. For this, roving field survey was carried out in 63 randomly selected chilli fields covering 21 villages in seven mandals of Kurnool district in Andhra Pradesh during 2019-20. Attempts were also made on molecular diagnosis of begomovirus associated with chilli leaf curl disease.

Result: The average mean per cent incidence of chilli leaf curl virus in Kurnool district was 50.76% ranging from 25.0% to 82.2%. During the field survey, the highest mean leaf curl incidence was noticed in Peddakadubur mandal (71.1%) followed by Gonegandla (59.96%) and Nandavaram (52.27%) mandals, while the lowest leaf curl incidence was observed at Adoni (37.83%) mandal in Kurnool district. The amplified coat protein gene was sequenced and obtained 770bp sequence which was deposited in Gen Bank. The obtained sequence shared maximum identity of 99.7% with chilli leaf curl virus Guntur isolate (MN417112). As per species demarcation and nomenclature criteria of begomovirus, the virus associated with chilli leaf curl disease in Kurnool district shared > 94% sequence identity with Chilli leaf curl virus. Hence it was identified as chilli leaf curl virus (ChiLCV).
Chilli (Capsicum annuum L.) is an important spice crop grown mainly in tropical and sub-tropical climate. India is the largest consumer, producer and exporter of the chilli in the world accounting for 13.76 million tonnes of production annually and it is cultivated in an area of 7.75 lakh hectares with a production of 14.92 lakh tonnes during 2014-15 (Geetha and Selvarani, 2017). Major chilli growing states in India are Andhra Pradesh, Assam, Gujarat, Maharashtra, Nagaland, Orissa, Rajasthan, Tamil Nadu and West Bengal. Various fungal, bacterial and viral diseases worsen the quality and quantity of chilli production. Chilli leaf curl disease is one of the major limiting factor in chilli production transmitted by whitefly (Bemisia tabaci) (Gennadius) (Hemiptera: Aleyrodidae) which can cause significant reductions in yield and quality of chilli (Kumar et al. 2006). Epidemics of ChiLCV can lead to cent per cent yield loss which results severe economic consequences (Senanayake et al., 2006). Since 2017-18, severe incidence of leaf curl disease was reported in Kurnool district of Andhra Pradesh.

In order to find out occurrence of viral disease and to assess the levels of resistance/tolerance and susceptibility of chilli cultivars grown in farmer’s fields at Kurnool district, roving field survey was conducted in Kurnool district of Andhra Pradesh. The attempts were also made to identify the associated begomovirus species with the leaf curl disease of chilli in Kurnool district of Andhra Pradesh.
Survey
 
Roving field survey was under taken in major chilli growing mandals of Kurnool district andhra Pradesh from July to October months during 2019-20. Totally, seven mandals (Adoni, Aspari, Gonegandla, Kodumur, Nandavaram, Peddakadubur and Yemmiganur) were selected and from each Mandal three villages were selected. A minimum of three fields were selected randomly in each village for determining the disease status. Incidence of the disease, variability of disease symptoms and crop variety were recorded. The per cent disease incidence of chilli viral disease complex was recorded. The per cent disease incidence was calculated using the following formula.
 
Detection of virus
 
Total DNA was isolated from diseased chilli leaf samples using cetyl trimethylammonium bromide (CTAB) method. The presence of chilli leaf curl virus was confirmed by PCR amplification with coat protein specific primers 5′-AGAATTATGTCCAAGCGACCA-3′ and 5′-AAGCGTTGG GGATACACAAA-3′ (Sinha et al. 2013). The PCR amplification  was carried out in a thermal cycler. The PCR reaction mixture contained 2 μl DNA sample, 1 μl of each primers (Conc. 10 mM), 0.5 μl dNTPs (10 mM), 2 μl Mgcl2 (25 mM), 2.5 μl 10 x reaction buffer, 0.2 μl Taq polymerase and 15.8 μl sterile HPLC-H2O which gave final volume of 25 μl PCR reaction. The conditions for PCR reaction were, an initial denaturation at 94°C for 5.00 minute followed by 34 cycles of denaturation at 94°C for 30sec., annealing at 55°C for 30sec and extension for 1minute at 72°C. Then final extension at 72°C for 7 minute was included. Amplified PCR product were separated by electrophoresis on 1.5% agarose gel and documented by Gel documentation system. The amplified PCR product of 750 bp was purified using PCR Purification Kit and sequenced. Database search with study sequence was done in BLAST at NCBI website. The multiple sequence alignments were performed using Clustal W programme in BioEdit software and phylogenetic analysis was done with Mega 7 Using neighbour joining method with 1000 bootstrap replicates.
Survey, symptomatology and disease incidence
 
The major disease symptoms observed in most of the fields surveyed were severe upward leaf curling with puckering, crinkling of leaves and reduced intermodal length. In few locations, other characteristic symptoms i.e reduced leaf size, leaf distortion, blistering, stunting and smaller fruits were also observed (Table 1 and Fig 1). The characteristic field symptoms of chilli leaf curl virus were upward leaf curling, puckering, reduced leaf size with no or less number of fruits (Senanayake et al. 2007).

Table 1: Types of viral symptoms observed in different mandals of Kurnool district.



Fig 1: Different symptoms of chilli leaf curl disease in surveyed fields.



The results of field survey indicated the presence of leaf curl disease in all seven mandals of Kurnool district that were surveyed. The data collected during filed survey was presented in Table 2. Maximum leaf curl incidence of 82.2 per cent was recorded in Peddakadubur village and minimum incidence of 25.0 per cent was recorded in Nagarur village of Aspari  mandal. The mandal wise mean incidence of leaf curl disease revealed that maximum leaf curl incidence was observed in Peddakadubur mandal (71.1%) followed by Gonegandla (59.96%), Nandavaram (52.27%), Yemmiganur (48.03%),  Aspari  (44.63%), Kodumur (41.50%)  and with minimum leaf curl incidence in Adoni (37.83%) mandal of Kurnool district. Similarly, 40-70% of percent disease incidence of leaf curl was recorded in Kurnool district of Andhra Pradesh (Bagavatha devi et al. 2019).

Table 2: Incidence of leaf curl virus infecting chilli in Kurnool district of Andhra Pradesh.



The variation of symptomatology of plant in different mandals indicated the presence of mixed infections of begomoviruses and infestation of sucking pests like thrips and mites. During the survey it was observed that severely affected plants produced no fruits or with significantly reduced fruits. The reasons for higher leaf curl disease incidence were continuous cultivation of chilli crop over the years (Monocropping), seasonal weather conditions which influence the sucking pest population, more number of sprayings with neonicotinoid insecticides during initial stages of crop which increases the resurgence of white fly population (vector) and non-adoption of integrated management practices. The sucking pests (whiteflies and thrips) were invariably present in all surveyed fields. Leaf curl due to sucking pest is reversible and can be managed through vector control, but leaf curl due to virus is irreversible and cannot be controlled (Dore et al. 2017).

Information regarding chilli hybrids was collected from chilli growers during survey. It was observed that, majority of the farmers in the surveyed fields cultivated Teja, Syngenta and Deluxe hybrids (Devanur deluxe, Kohinoor deluxe, Nuziveedu deluxe and Super deluxe). Among all these, Devanur deluxe and Super deluxe were the major chilli hybrids grown in the surveyed fields. The chilli hybrids were compared based on percent disease incidence recorded during survey and found that Teja and Syngenta cultivars recorded less PDI (25.00-45.2%) of leaf curl disease compared to deluxe cultivars (30.6-82.2%) which might be due to broader leaf size of deluxe hybrids which favours sucking pest infestation, apart from tolerance levels are compared to Teja chilli hybrid.
 
Detection of virus and characterization of virus associated with chilli leaf curl disease
 
The genomic DNA was isolated from leaf samples with characteristic symptoms of begomo virus infection from Kodumur mandal were subjected to polymerase chain reaction (PCR) using ChiLCV coat protein specific primers 5′-AGAATTATGTCCAAGCGACCA-3′ and 5′-AAGCGTTG GGGATACACAAA-3′. PCR amplification of 775 bp was observed with all isolated DNA samples. The amplified product of 775 bp was subjected to PCR purification and sequenced. The nucleotide sequence of coat protein gene (772 bp) of study isolate was deposited in GenBank and obtained accession no (MZ691516). Whenever full length sequence of begomovirus is not available, the sequence of coat protein gene serves as an effective marker in begomovirus virus identification (Rybicki, 1994).

Different begomovirus sequences from NCBI site were used in comparative analysis. A BLAST search of GenBank revealed close sequence similarity with the Chilli leaf curl virus. The complete nucleotide sequence of coat protein gene of study isolate with other begomovirus from NCBI database revealed that it has 99.7% nucleotide similarity with chilli leaf curl virus-Guntur isolate followed by ChiLCV-Raichur isolate (Table 3) and least sequence similarity with Croton yellow vein mosaic virus (79%). In phylogeny analysis, tree forms three clusters consisting of ChiLCV and ChiLCMV (one cluster),ToLCV  and ToLCNDV (Fig 2).

Table 3: The details of begomoviruses used for sequence analysis and per cent nucleotide similarity of study isolate with other begomoviruses.



Fig 2: Phylogenetic tree based upon alignments of the coat protein gene of Begomoviruses. The tree was constructed using MEGA 7.0 at 1000 boot strap values (·) marked indicates the sequence accession obtained from Kodumur mandal of Kurnool district andhra Pradesh.



In India, Khan et al (2006) reported the association of Tomato leaf curl New Delhi virus (ToLCNDV) with chilli leaf curl disease in Lucknow. Later in 2007, the association of chilli leaf curl virus with chilli leaf curl disease was reported in Jodhpur (Senenayake et al., 2007). In the present study we confirmed the association of ChiLCV with leaf curl disease complex in Kurnool district of Andhra Pradesh.
In chilli field survey, it was observed that 25.00 to 82.20% percent disease incidence of leaf curl disease recorded in seven mandals of Kurnool district andhra Pradesh. Maximum leaf curl incidence was reported in Peddakadubur mandal (82.2%) and minimum disease incidence was recorded in Aspari mandal (25.00%). In molecular characterization of coat protein gene of study viral isolate is identified as Chilli leaf curl virus (ChLCV).
The authors declare that they have no conflicts of interest.

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