1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item 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 SThe 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.Item Computational prediction of mirnas in banana (musa spp.) and evaluation of their role in virus infection(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Kokila Sajeev, Anurag Mathew; Sony, K BThe study entitled "Computational prediction of miRNAs in banana (Musa spp.) and evaluation of their role in virus infection" was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram and Central Tuber Crops Research Institute (ICAR-CTCRI), Sreekariyam, Thiruvananthapuram during 2016–2018. The objective of the study was to predict miRNAs in banana using bioinformatics tools and to validate and analyze their expression during BBrMV infection. Computational miRNA prediction tool NovoMIR was used for the prediction of miRNAs in banana genome. Analysis was performed with all the gene coding or nucleotide sequences of banana and 85 pre-miRNAs were predicted from 11 chromosomes. Most of the pre-miRNAs ranged from 140–180 nt. G+C% content of pre-miRNAs ranged from 24-77% and A+U% content ranged from 23-76%. MFE of pre-miRNAs were found by using an online application RNAfold web server. MFE of pre-miRNAs ranged from -20 Kcal/mol to -194.4 Kcal/mol. AMFE of pre-miRNAs ranged from -18.9 Kcal/mol to -85.4 Kcal/mol. All the predicted pre-miRNAs by NOVOMIR had MFE ≤ -20 Kcal/mol. MFEI of pre-miRNAs ranged from 0.85 to 1.82. BLAST analysis of 85 pre-miRNAs against annotated mature miRNAs in miRBase resulted in 52 mature miRNAs. The targets for the 52 mature miRNAs were predicted using the web tool psRNATarget and were functionally annotated by Blast2Go analysis server. Targets were identified for 40 miRNAs in banana genome. A total of 124 targets were found, with each miRNA having more than one target. Validation of the predicted miRNAs were done in in vitro banana plants of variety Nendran. Three months old tissue culture plants were infected with BBrMV virus by using aphids. Twenty healthy aphids were released on BBrMV infected sucker for 30 min for acquisition feeding, after starving for 10 min. These aphids were further released on tissue culture plants for 12-14 h for infection. The infection process was repeated twice a day for 7 days. For experimental validation, five miRNAs and their target genes having role in biological process were selected and stem-loop/gene specific primers were designed. RNA was isolated from healthy and infected leaf samples and reverse transcribed to cDNA. Expression analysis using RT-qPCR showed the presence of all the five miRNAs in healthy and BBrMV infected leaf samples. Out of them, miR-3900-5p, miR-9112 and miR-5417 were found up-regulated, miR-6928-5p downregulated and miR-3900-5p remain unchanged in infected samples and there was a positive correlation with the expression of their corresponding target genes. In the present study, 52 mature miRNAs have been predicted using bioinformatics tools in banana genome. Targets for 40 miRNAs were identified in banana genome. The five miRNAs selected were validated along with their targets. Expression analysis showed regulation of four of them during virus infection, indicating the possibility of their role in stress response in banana. The remaining miRNAs predicted need validation.Item Characterization and validation of microsatelite markers for resistance to vascular streak dieback disease in cocoa (Theobroma cacao L.)(Centre for Plant Biotechnolgy and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Waghmare Sandesh Tulshiram; Deepu MathewCocoa is the third important plantation crop next to coffee and tea. The global production and consumption of cocoa is 27.00 lakh MT. Among the fungal diseases, Vascular Streak Dieback (VSD) caused by Ceratobasidium theobromae is the main constraint in cocoa growing countries, causing heavy losses in mature trees as well as seedlings. The VSD disease cannot be effectively controlled by chemicals and hence breeding for the development of resistant varieties is the best strategy to tackle the disease. In order to confirm the transfer of a desired gene into the offspring, conventional breeding methods rely on the field screening which will be highly influenced by the environmental factors. Marker assisted selection is an alternate where the tightly linked molecular markers will be employed to confirm the presence of the gene of interest in the selected plants. Five ISSR and one SSR markers linked to VSD resistance were identified at Kerala Agricultural University (Chandrakant, 2014). The present study was undertaken with the objective of validating the identified SSR and ISSR markers and to characterize the ISSR markers to identify the corresponding SSR markers. For validation and characterization, twenty VSD resistant hybrids and four susceptible clones were used. For molecular analyses, good quality genomic DNA was isolated from twenty four genotypes and ISSR markers UBC 811, UBC 815, UBC 826, UBC 857, UBC 866 and SSR marker mTcCIR 42 were screened. ISSR analysis had shown that all the primers are capable to differentiate resistant and susceptible genotypes. The SSR assay has also differentiated the resistant and susceptible genotypes. The distinct markers generated in resistant genotypes using UBC 811, UBC 826 and UBC 857 were eluted, cloned to pGEMT vector and sequenced. The nucleotide sequences were annotated using BLAST, ORF finder and SSR finder. The BLASTn of UBC811A and UBC811D nucleotide sequence have shown that this resistance locus lie in the chromosome V of Theobroma cacao genome. BLASTn of UBC826A, UBC826B and UBC857 has positioned these loci in chromosome III. ORF1 and ORF3 in UBC811D are shown to code for aflatoxin biosynthesis regulatory protein and NAD(P)H dehydrogenase quinine, respectively. ORF1 in UBC826B and ORF5 in UBC857-2 code for potassium transporter 27 (0sHAK-27) and structural polyprotein precursor of VP2, capsid protein VP2, respectively. All these proteins are identified to have definite roles in defence pathways. The frequency and distribution of SSR motifs, dimmers to decamers, in these ISSR markers and the corresponding primers were identified. The reported ISSR and SSR markers were validated and found to be successful in differentiating resistant and susceptible genotypes of cocoa; thereby these markers can be used in marker assisted breeding for VSD resistance.Item Development of recombinant coat protien for immunodetection of cucumber mosaic virus infecting banana(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2019) Alan C Antony; Vimi LouisBanana (Musa spp.) is infected by four well characterised plant viruses viz., Banana bunchy top virus, Cucumber mosaic virus (CMV), Banana bract mosaic virus and Banana streak virus. Among these, CMV causes devastating effect on tissue culture banana plants. The study entitled “Development of recombinant coat protein for immunodetection of Cucumber mosaic virus infecting banana” was carried out using existing facilities of Department of Biochemistry, Indian Institute of Science, Bangalore, Division of Plant Pathology, Banana Research Station, Kannara and Department of Plant Pathology, College of Horticulture, Vellanikkara, Thrissur during 2018- 2019. The present study was carried out to produce recombinant coat protein, which can be utilised later for producing high quality antiserum for the detection of CMV infecting banana. Cucumber mosaic virus infected samples were collected based on various characteristic symptoms and screened by direct antigen coating immunosorbent assay using commercially available CMV polyclonal antiserum. Isolate namely, KANC- 4, KANC- 2 and NDRNS- 4 showed maximum absorbance at 405 nm and hence selected for molecular detection using reverse transcriptase polymerase chain reaction with CMV- CP specific primer. The PCR product was purified and CMV- CP amplicon of NDRNS- 4 isolate was ligated to pGEM- T linear plasmid vector, which was later transformed into E. coli DH5α cells. Positive clones were selected according to blue-white screening. Cloning i.e., E.coli DH5α/pGEM- T/CMV- CP was confirmed through colony PCR using coat protein specific primer, restriction digestion of recombinant plasmids using EcoR1 enzyme followed by sequencing. The vectors viz. pRSET- C and pET28a were selected for the expression of CMV- CP gene in E. coli. Coat protein specific forward (5’GGG GCT AGC ATG GAC AAA TCT GAA TCA ACC3’) and reverse primers(5’CCC GGA TCC TTA CTC TCC ATG GCG TTT AG 3’) were designed along with recognition sites of restriction enzymes BamH1 and Nhe1.The annealing temperature of designed primer was standardised as 55°C using gradient PCR. The coat protein gene of CMV was amplified at 750 bp using designed primers and high fidelity Pfu DNA polymerase enzyme. Expression vectors as well as amplicon were subjected to ligation and the recombination in expression plasmids (pRSET- C/ CMV- CP and pET28a/CMV- CP) were confirmed through PCR and sequencing. The plasmid with maximum homology i.e., pRSET-C/CMV- CP was selected for further studies. The recombinant plasmid was transformed into E. coli BL21(DE3)pLysS cells for the expression of CMV- CP gene and the expression of 25 kDa recombinant CMV coat protein was confirmed in 12 per cent sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS- PAGE). Tris- NaCl buffer of pH 8.0 was selected for solubilising the recombinant protein using ExPASy - protein translation tool. The recombinant protein was further purified through Nitrilotriacetic acid column purification, in which the 6X histidine tagged recombinant protein was bound with agarose coated nickel beads. Buffers containing imidazole were used for the elution of histidine tagged recombinant protein, since imidazole competes with histidine for the binding site in nickel beads. Each fraction viz., cell pellet, supernatant, flow through, wash and elution were collected and later detected for protein using SDS- PAGE. Absence of 25 kDa protein in cell pellet indicated that the recombinant coat protein completely soluble in Tris- NaCl buffer (pH 8.0). Confirmation of recombinant coat protein was carried out through DAC- ELISA and western blotting using commercially available polyclonal CMV antiserum (1: 2000; NRCB, Trichy). The recombinant coat protein developed through this study could be utilised for large scale production of antiserum for immunodetection of CMV.Item Identification of graft transmissible resistant factors and development of si RNA mediated resistance in cassava against cassava mosaic geminivirus(Department of Plant Pathology, College of Agriculture, Vellayani, 2017) Asha B Nair; Umamaheswaran, KItem Triazole,strobilurin and its combination fungicides for the management of anthracnose and fruit rot of chilli(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Anjana, R S; Joy, MItem Evaluation of siRNA mediated banana bract mosaic virus (BBrMV) resistance in banana plants with ihpRNA construct for replicase gene(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Harshitha, C K; Soni, K BItem Integrated management of alternaria leaf spot of cabbage (Brassica oleracea var. capitata (L.))(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Madhu Kiran Gunda, V N S; Susha S TharaItem Molecular charecterization of taro bacilliform virus (TaBV)(Department of Plant Biotechnology, College of Agriculture, 2017) Aarathy, M B; Makeshkumar, TItem Characterization of PR proteins in selected calliclones of black pepper in relation to phytophthora foot rot disease(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Debashis Sahoo; Shylaja, M RBlack pepper (Piper nigrum L.), the king of spices is severely affected by Phytophthora foot rot disease caused by Phytophthora capsici. The disease results in severe crop loss, and valuable genotypes are lost every year due to this dreadful disease. The local cultivars and released varieties of black pepper are susceptible to the pathogen, but variation exists in genotypes in the degree of tolerance and mechanism of defense to the disease. The plants resist the pathogen infection by accumulating a number of defense related proteins in the intercellular spaces which are collectively known as pathogenesis related (PR) proteins. The study was aimed to characterize PR proteins in selected eleven calliclones of black pepper along with susceptible variety Panniyur-1 after challenge inoculation with Phytophthora capsici so as to get more insight on the defense mechanism of Phytophthora foot rot. Investigations on disease reaction of the selected calliclones and variety Panniyur-1 after challenge inoculation with Phytophthora capsici, β-1,3-glucanase activity and protein analysis by SDS-PAGE was carried out at 0, 24, 48 and 72 hours after inoculation. Protein profiling by 2D-gel electrophoresis and protein identification by MALDI-TOF MS / MS in the most tolerant and susceptible calliclone and in silico analysis for characterization of proteins were also attempted in the present study. In leaf symptom bioassay, variety Panniyur-1 showed susceptible reaction as compared to calliclones of Cheriakanyakkadan and Kalluvally. Based on β-1,3-glucanase activity and expression of 16.5 kDa band in SDS-PAGE, clone KLCC 89 was selected as the tolerant calliclone. 2D-gel electrophoresis was attempted in the selected tolerant calliclone, KLCC 89 and susceptible variety Panniyur-1. Proteome analysis by 2D-gel electrophoresis could locate 167 differentially expressed protein spots in KLCC 89, 24 hours after inoculation. Analysis by PDQUEST software could select four protein spots (Spot 1, Spot 2, Spot 3 and Spot 4) from 167 spots based on higher degree of differential expression. The selected spots were sent to Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram for protein identification by MALDI-TOF. Analysis by MALDI-TOF and MASCOT search could identify 15 hit peptides from the selected four protein spots. Analysis and functional characterization of the 15 hit peptides by BLAST2GO revealed the defense response of tolerant calliclone KLCC 89 against Phytophthora foot rot disease. The enhanced expression of plastocyanin protein, TRAF-like family proteins, RUBISCO dependent glycolate and glyoxylate metabolism, light dependent ROS production during photorespiration, F-box proteins, synthesis of antimicrobial metabolites and retrotransposition activity were observed in the tolerant calliclone KLCC 89 as defense related responses. Plastocyanin is involved in regulation of photosynthesis to meet the requirements of nutrient competition by the P. capsici whereas the TRAF-like family proteins is involved in regulation of programmed cell death. The increment in RUBISCO, regulates the glycolate and glyoxylate metabolism for H2O2 production. F-box proteins are involved in regulation of jasmonate regulated defense-related pathways. The study could characterize PR proteins in the selected calliclones and investigate in depth the Phytophthora capsici interaction in black pepper at proteome level. The future research should focus on validation of identified proteins in defense mechanism, characterization of novel protein in KLCC 89, in-depth investigations on retrotransposons in defense mechanism of Phytophthora foot rot tolerance in black pepper, metabolic engineering of the pathways triggering transcription of PR-genes and transgenic / cisgenic research for Phytophthora foot rot resistance.