PG Thesis

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Subject: search.filters.subject.Chilli leaf curl virus

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    Identification of sources of resistance and effective non chemical methods for the management for major viral disease of chilli in Kerala
    (Department of Plant Pathology, College of Agriculture Vellanikkara, 2023) Sujisha, C S; Sumiya, K V
    The present study was undertaken in an endeavor to gain some understanding regarding the viral diseases affecting chilli and devising suitable management strategies to contain them. A purposive sampling survey conducted involving 3 districts of Kerala, namely Palakkad, Thrissur and Malappuram covering over 15 locations and 44 samples revealed symptoms descriptive of viral infection like curling, puckering, cupping and yellowing of leaves, dwarfing of plants and low fruit set in affected plants. To identify the etiological agent(s) associated with the symptoms, transmission studies were carried out. Whitefly transmission and graft transmission gave positive results whereas none of the samples that were sap transmitted exhibited symptoms indicating the presence of a geminivirus. Detection methods like Transmission Electron Microscopy (TEM) and Enzyme Linked Immunosorbant Assay (ELISA) were also carried out. Geminate particles were observed but no mosaic causing virus was detected in EM. ELISA performed on samples with mosaic like symptoms for the presence of potyvirus and CMV did not detect the presence of suspected virus in it. Hence, it was concluded that leaf curl disease was the only major disease affecting the crop in the area. Characterization of the virus (s) responsible for the leaf curl disease was attempted. DNA was isolated from seven representative samples after which PCR (Polymerase Chain Reaction) was performed with two sets of primers, degenerate (DENG) primer and a specific primer. The PCR amplified products of four representative isolates were sequenced and analysed for homology with sequences available in NCBI using BLAST software. Three of the isolates showed maximum nucleotide identity to the Pepper leaf curl Bangladesh virus isolate India/Coimbatore/Chilli/2008 segment DNA-A, complete sequence (HM007096.1) and the other isolate showed maximum identity with Chilli leaf curl virus-India isolate Raichur segment DNA-A, complete sequence (MK161454.1). A phylogenetic tree was constructed combining different geminiviruses reported from different crops to study the sequence similarity existing amongst them. The amino acid sequence of the coat protein gene of the isolate PKD-1 was predicted using the ExPASy translation tool available online. With the aim of identifying source (s) of resistance against chilli leaf curl disease in Kerala, two sets of screening experiments were carried out. In the initial field screening of 30 accessions, Violet Mulak, a local accession remained symptomless whereas Arka Lohit (IIHR) and Phule Jyothi (MPKVV) remained highly resistant. The molecular confirmation of the incidence of Chilli leaf curl virus (ChiLCV) in the field was done by performing PCR using primers specific to the CP gene region of the concerned virus. A pot experiment was set up to once again score the disease reaction of 48 accessions including those used in field screening along with a few more accessions. In this experiment, Phule Jyothi remained symptomless and Violet mulak turned out highly resistant whereas Arka Lohit remained HR. Twentysix random infected accessions were confirmed for the presence of ChiLCV in them. The three accessions that were adjudged highly resistant following the conclusions of the abovesaid two screening experiments were subjected to graft transmission. Fifty days old field exposed plants of the resistant sources were used as rootstocks in wedge grafting with scions from known susceptible variety. At 60 days of grafting, rootstocks and scions failed to amplify the primer indicating the absence of virus in them. From all these experiments, what could be inferred is that these accessions possessed true resistance against the virus up to 50 days after transplanting in the field. A management study to evaluate the efficacy of certain plant extracts and biocontrol agents in containing chilli leaf curl disease was undertaken the results of which indicate spraying the leaf extract of Azadirachta indica (10 %) or Mirabilis jalapa (10 %) as the best treatment. The only biocontrol agent with an effect on par with the best treatments was Bacillus subtilis applied as seed treatment (10g kg-1 seed) and foliar spray (20g l-1). All the four plant extracts did have a reducing effect on the disease severity with the least mean recorded in A. indica (10 %).
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    Nutrient based management of chilli leaf curl virus in Chilli (Capsicum annuum L.)
    (Department of Plant Pathology, College of Agriculture, Vellayani, 2019) Shilpa Sankar; Radhika, N S
    The study entitled “Nutrient based management of Chilli leaf curl virus in Chilli (Capsicum annuum L.) was conducted at Department of Plant Pathology, College of Agriculture, Vellayani from 2017 to 2019. The main objectives were to serologically and molecularly characterize the virus causing leaf curl in chilli and to study the role of nutrient application in the management of the disease. The present investigation was carried out in four experiments viz., collection of Begomovirus infecting chilli from different cultivated areas, symptomatology, serological diagnosis and molecular characterization of the virus causing chilli leaf curl and nutrient based management of chilli leaf curl. The survey was undertaken from December 2018 to March 2019 to investigate the disease incidence in major chilli growing areas of Palakkad (Vadakarapathy and Kozhinjanpara) and Thiruvananthapuram districts (College of Agriculture, Vellayani) of Kerala. In Thiruvananthapuram, the disease incidence ranged from 69.33 to 80 per cent whereas Palakkad recorded maximum incidence of 73.33 per cent in Vadakarapathy village and Kozhinjanpara village recorded the disease incidence of 55.71 to 71.70 per cent. The common symptoms of the disease were upward curling and puckering of leaves, and stunting of whole plant. Other symptoms viz., reduced leaf size, yellowing, petiole elongation, crinkling and mottling of leaves with few or no fruits were also observed. Serological diagnosis of the disease was carried out using triple antibody sandwich – enzyme linked immunosorbent assay (TAS-ELISA) using polyclonal antisera specific to Begomovirus, Sri Lankan cassava mosaic virus (SLCMV). All the samples collected from College of Agriculture, Vellayani were detected with the virus whereas none of the samples from Palakkad district were positive to the virus. Molecular characterization of the virus using universal primers specific to coat protein of Begomovirus viz., AV-AC and DENG through Polymerase chain reaction (PCR) revealed that the DNA isolated from samples of Vellayani could yield an amplicon size of ̴ 550 bp (AV-AC) and ̴ 500 bp (DENG); thus confirming the presence of the virus. But no PCR amplification was observed in any of the samples from Palakkad district. Blast analysis with the sequence of coat protein of Vellayani revealed that the virus had 96.63 per cent similarity with Chilli leaf curl Vellanad virus. Nutrient status of healthy and diseased leaves revealed that the per cent of major nutrients viz., nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg); and micronutrients viz., manganese (Mn), zinc (Zn), copper (Cu) and boron (B) in the infected leaves was low compared to the healthy leaves but in sufficient level. Whereas, the per cent of sulphur (S) and iron (Fe) in infected leaves was high and in toxic level. Comparatively higher levels of all the major and micro nutrients were recorded in the soils collected from the infected fields than the soils from the healthy field. A pot culture study was conducted at Department of Plant Pathology in completely randomized design consisting of ten treatments and three replications with chilli variety Vellayani Athulya from May 2019 to August 2019. Cent per cent disease incidence was recorded in all chilli plants before imposing the treatments. Soil samples were taken and analysed for major and micro nutrients at pre-treatment stage. Urea, rajphos, murate of potash, lime and magnesium sulphate (MgSO4) were used as the source of major nutrients like N, P, K, Ca and Mg, respectively. Whereas, manganese sulphate (MnSO4), zinc sulphate (ZnSO4), copper sulphate (CuSO4), borax and potassium silicate were used as source for micronutrients such as Mn, Zn, Cu, B and Si, respectively. After imposing the treatments, the highest coefficient of infection (75.0) was recorded in untreated infected plants whereas, the plants supplemented with nutrients as per package of practices (POP) + B @ 10 kg ha-1, POP + (ZnSO4) @ 20 kg ha-1 and, basal application of 1/2 N + full P + 1/2 K followed by 0.5 per cent foliar application of NPK 19:19:19 at fortnightly recorded the lowest coefficient of infection (25.0). TAS-ELISA conducted with all the experimental plants confirmed the presence of virus. Lower virus titre value was observed in the plants supplemented with nutrients as per POP recorded maximum level of major nutrients. Among the treatments highest fruit yield of 48.85 g plant1 was obtained from plants supplemented with nutrients as per POP + B @ 10 kg ha-1 was found to be most effective in reducing the coefficient of infection with better yield. Thus, the present study revealed that the serological diagnosis of the disease carried out using TAS-ELISA with antisera SLCMV and molecular characterization of the virus using primers AV-AC and DENG through PCR confirmed the presence of virus in Vellayani. The BLAST analysis of Chilli leaf curl Vellayani isolate thus showed 96.63 per cent similarity with Chilli leaf curl Vellanad virus. It was also indicated that Chilli leaf curl virus in chilli could be managed by the application of nutrients as per package of practices recommendation along with boron as borax @ 10 kg ha-1 which need to be validated under field conditions.
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    Molecular cloning and characterization of virus causing leaf curl disease of capsicum spp.
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2019) Niranjana Menon, C; Anita Cherian, K
    Chilli is one of the most important crops cultivated across the globe, as vegetable, spice and for industrial purposes. According to the statistics of National Horticulture Board (2017), the crop covers an area of 1860 ha in the state of Kerala with an average production of 12470 tonnes. During the last decade, the threats posed by the emerging begomoviruses infecting solanaceous crops have affected the economic cultivation of chilli. Chilli leaf curl disease caused by Chilli leaf curl virus belonging to the genus Begomovirus and family Geminiviridae is a serious constraint to chilli production in India which causes upto 100 per cent yield loss especially when infected at an early stage of the crop. Considering the importance of the disease, the present study was undertaken with the objective to study the incidence and symptomatology of chilli leaf curl disease and to characterize and clone the coat protein gene of the Chilli leaf curl virus isolates. The project initiated with purposive sampling surveys conducted in eleven different locations of Thrissur district, Kerala to document the incidence and symptomatology of leaf curl disease on chilli plants. The disease incidence recorded during the survey ranged from 43.30 to 85.00 per cent under open field conditions and from 45.75 to 79.40 per cent under protected conditions while the disease severity ranged from 43.60 to 81.54 per cent and from 49.40 to 87.50 per cent, respectively under open field conditions and protected conditions. The symptomatology of chilli leaf curl disease on different parts of the plant such as leaves, internodes, fruits and the whole plant under natural conditions was documented during the survey. The symptoms observed on the leaves of infected chilli plants under natural conditions include upward curling, crinkling, puckering, vein banding, interveinal chlorosis, size reduction of leaf lamina and leaf malformation. The fruits produced by the infected plants showed size reduction and deformation. The infected plants were stunted and bushy in appearance. The transmission of the virus by insect vector, Bemisia tabaci and grafting was studied and the symptoms were documented. The newly emerged leaves after artificial inoculation expressed symptoms such as curling, puckering and crinkling along with stunting of plant growth. Molecular characterization of the four virus isolates collected from various locations of Thrissur district viz., VKA1 VKA2, KAR1 and KOD4 and two isolates viz., VLNY1 and PKD1 collected from Vellayani, Thiruvanathapuram district and from Vithinasseri, Palakkad district, respectively were undertaken. The total genomic DNA from virus infected chilli leaf samples was isolated and subjected to PCR amplification of viral coat protein gene to confirm the presence of virus infection. PCR amplification of the isolated DNA was carried out using two Begomovirus specific degenerate (universal) primers, namely, AV494 / AC1048 (Wyatt and Brown, 1996) and Deng 540 / 541 (Deng et al., 1994). The amplicons of size 550 bp were obtained and were sequenced. The partial coat protein gene of size 550bp were also cloned into the vector pTZ57R/T and transformed into DH5α strain of Escherichia coli and the true recombinants with desirable insert were confirmed by colony PCR. The sequence data obtained in the study were subjected to in silico analysis to assess the diversity of the isolates. The nucleotide BLAST (BLASTn) analysis revealed more than 90 per cent sequence identity with Chilli leaf curl Vellanad virus isolate (Accession No. NC038442.1) from Vellanad region of Thiruvanathapuram district, Kerala. The translated nucleotide - protein BLAST (BLASTx) analysis of the viral sequences revealed more than 96 per cent sequence identity with Chilli leaf curl Vellanad virus coat protein sequence (accession no. YP_009506391.1). The coat protein sequences of all the six isolates were translated into corresponding amino acid sequence by ExPASy Translate tool and were used for further analysis and interpretation. The phylogenetic analysis revealed that, the isolates VKA2, KAR1 and KOD4 had very distinct sequence alignment when compared to other Chilli leaf curl virus isolates from India. The results indicated that, the three isolates viz., VKA2, KAR1 and KOD4 could be new strains of Chilli leaf curl virus infecting chilli. Three, possibly new strains of Chilli leaf curl virus infecting chilli have been identified and hence the study highlights the need for monitoring the emergence of new strains of plant viruses especially begomoviruses infecting solanaceous crops grown in Kerala. As this disease is one of the most important challenges to chilli cultivation, the information generated from the study could also be applied for the timely detection and effective disease management