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 Molecular cloning and characterisation of coat protein gene of banana bract mosaic virus(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Darshan Gowda, M R; Anita Cherian, KBanana (Musa spp.), identified as ‘tropical treasure’ is grown extensively in the tropical and sub tropical regions of the world. Diseases, especially those caused by viruses are major constraints for the profitable cultivation of banana. Among the viral diseases, banana bract mosaic is one of the most important, which leads to an yield reduction ranging from 52 to 75 per cent. This disease is caused by Banana bract mosaic virus (BBrMV) which is a member of genus Potyvirus and family Potyviridae. In case of any viral disease, early diagnosis is very important since symptomless hosts carry the viral inoculum. Development of molecular clones of viral genome has immense application in the field of disease diagnostics and management. Hence, the present study was carried out with the objective to develop molecular clones of coat protein (CP) gene of BBrMV and to characterize it.The infected suckers were collected from Banana Research Station (BRS), Kannara and maintained in the insect proof net house of Department of Plant Pathology. The symptoms developed on different plant parts under natural field conditions were documented which included longitudinal, irregular, reddish streaks of varying sizes on the base of pseudostem, mosaic pattern on bracts, fan like orientation of leaves, spindle shaped lesions on leaves, reduced bunch size and malformed fingers.The serodiagnostic technique namely, Direct Antigen Coating-Enzyme linked immuno sorbent assay (DAC-ELISA) was validated by determining the antibody titre with different dilutions of primary antibody viz., 1:100, 1:200, 1:300, 1:500 and it was found that BBrMV could be best detected at 1:200 dilution along with 1:500 dilution of secondary antibody. Later, the presence of virus particles in the samples were confirmed by DAC-ELISA using the standardized combination of primary and secondary antibody dilutions. Dot Immuno Binding Assay (DIBA) was validated to detect BBrMV and showed positive reaction for infected leaf sample which was detected by the development of purple coloured spots on nitrocellulose membrane.The genome of BBrMV is RNA and hence, molecular detection of virus was standardized by Reverse Transcription- PCR (RT-PCR). Total RNA was isolated by two different protocols using different reagents. Among the two methods, the one with Ambion Purelink RNA Reagent was the most appropriate for RNA isolation from banana since it provided highest quality and quantity of RNA compared to the protocol with TRIzol reagent. The isolated RNA was converted into complementary DNA (cDNA) using First Strand cDNA synthesis kit. RTPCR amplification of coat protein gene was standardized using gene specific reported primer (B1/B2) and designed primer (BCPF1/R1) which yielded amplicons of approximate size, 605 bp and 850 bp respectively. The molecular cloning of CP gene was done in Escherichia coli (DH5- alpha). The presence of gene insert in transformed colonies were confirmed by colony PCR using plasmid specific primer (T7 and SP6) which yielded amplicons of expected band size of 1150 bp. The amplified colony PCR products were sequenced to obtain CP gene sequence of BBrMV. The characterization of cloned CP gene of BBrMV was carried out by in silico analysis. The blast analysis revealed that the CP gene sequence of the virus showed maximum homology of 99 per cent to KER2 isolate from Kasargod, Kerala (Accession no. KF385491). The sequence exhibited significant nucleotide identity (99 to 96 per cent) and amino acid identity (95 to 83 per cent) with other nucleotide and protein sequences of BBrMV available in the database of Genbank. The phylogenetic analysis by the alignment of CP gene sequences of selected 22 isolates also revealed that the present isolate was more similar to KER2 isolate and the Indian isolates did not show any relationship based on geographical origin.The recombinant clones developed in the present study could be applied in serodiagnosis and genetic engineering. This could be also used as disease diagnostic probes for more sensitive molecular diagnostic techniques like Nucleic acid spot hybridization.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 Etiology and management of mosaic disease in ginger (Zingiber officinale Roscoe)(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Ananthu, N; Umamaheswaran, KItem 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 Molecular characterization of virus causing infectious chlorosis disease of banana(Department of Plant Pathology, College of Horticulture Vellanikkara, 2017) Ahamed Mujtaba, V; Anita Cherian, KThe experiment entitled “Nutrient management in strawberry (Fragaria x ananassa Duch.)” was undertaken at Regional Agricultural Research Station, Ambalavayal, Wayanad during the year 2016-17. Performance of strawberry variety Winter Dawn was evaluated under nine treatments and a control in the open field viz., FYM 10 t ha-1 + NPK 50:20:50 kg ha-1 (T1); FYM 10 t ha-1 + NPK 75:30:75 kg ha-1 (T2 ); FYM 10 t ha-1 + NPK 100:40:100 kg ha-1 (T3); FYM 20 t ha-1 + NPK 50:30:100 kg ha-1 (T4); FYM 20 t ha-1 + NPK 75:40:50 kg ha-1 (T5); FYM 20 t ha-1 + NPK 100:20:75 kg ha-1 (T6); FYM 30 t ha-1 + NPK 50:40:75 kg ha-1 (T7); FYM 30 t ha-1 + NPK 75:20:100 kg ha-1 (T8); FYM 30 t ha-1 + NPK 100:30:50 kg ha-1 (T9) and an absolute control (T10), without any nutrient application. All the treatments were on par and superior over the control (T10) in case of plant height. In case of plant spread, T2, T3, T5, T6, T7, T8 and T9 were on par and superior over the control while T1 and T4 were on par with each other but differs with other treatments. All the treatments except T2 were on par and superior over the control with respect to number of leaves per plant. Application of treatments had no significant effect on days to first flowering. In case of number of flowers and clusters per plant, T1, T2, T3, T5, T6, T7, T8 and T9 were on par and superior over the control while T4 was on par with the control (T10). Days to first harvest was minimum in T6, T7, T8 and T9 which were on par while all other treatments were on par with the control (T10).In case of number of fruits and yield per plant, T7 (FYM 30 t ha-1 + NPK 50:40:75 kg ha-1) and T8 (FYM 30 t ha-1 + NPK 75:20:100 kg ha-1) were on par and superior over other treatments including T1, T2, T3, T4, T5, T6 and T9 which were on par and superior over the control. Average fruit weight recorded under T3, T5, T6, T7, T8 and T9 were on par which was followed by T2 on par with T4 and T1. Days to final harvest was not found to be influenced by the application of different treatments. Biochemical characters of fruits viz., TSS, acidity and TSS/acidity ratio were not having any significant effect due to the application of treatments. In case of total sugars, T3, T7, T8 and T9 were having the highest content and were on par which was followed by T5 on par with T1, T2, T4, T6 and T10. The overall sensory score was highest in T7 followed by T8. Application of different treatments had no significant effect on the shelf life of strawberry fruits. N, P, K and Ca content in the plant were not significantly affected by any treatment while Mg content was found to be on par in all treatments and superior over the control. Soil analysis after the harvest of the crop revealed that the values for soil EC, available P, K, Mg and S were found to be elevated while soil pH, organic carbon and available Ca content were found to be at lower levels than the initial values before planting. It was concluded that among different nutrient combinations evaluated, T7 (FYM 30 t ha-1 + NPK 50:40:75 kg ha-1) with a BC ratio of 3.06 can be recommended for further optimization and refinement.