PhD Thesis
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Item Molecular diagnosis and management of Papaya ringspot virus causing papaya ringspot diseases(Department of Plant Pathology, College of Agriculture,Vellayani, 2024-04-17) Josiya Joy; KAU; Radhika, N SThe research work entitled “Molecular diagnosis and management of Papaya ringspot virus causing papaya ringspot disease” was undertaken in the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram, during 2019-24, with the objectives; molecular diagnosis and recombinant coat protein production of Papaya ringspot virus (PRSV), and evaluation of the efficacy of beneficial microorganisms and botanical inthe management of papaya ringspot disease (PRSD). Roving survey was carried out across five Agro-ecological units (AEUs) of Kerala. The disease incidence (DI) ranged from 50.25 per cent (Kayyur-Cheemeni) to 100 per cent (Kalliyoor, Venganoor, Balaramapuram, Pallichal, Kayamkulam, Mavelikkara, Velukkara, Irinjalakuda and Shoranur). Vulnerability index (VI) of the plants to PRSV in the surveyed locations ranged from 33.54 (Badiyadkka) to 98.22 (Kalliyoor). Serological and molecular detection confirmed the presence of PRSV in all the 20 symptomatic samples collected during survey. Phylogenetic tree constructed with the deduced amino acid sequences of CP gene of 11 Kerala PRSV isolates, revealed that Thiruvananthapuram isolates clustered together, indicating their relatedness during evolution. Mechanical inoculation on two months old Red Lady papaya seedlings under insect proof conditions was carried out to identify the most virulent PRSV isolate collected from different AEUs. At three months after inoculation (MAI), Kalliyoor isolate exhibited highest VI (96.63) followed by Alur (95.48) and Venganoor (95.22) isolates. The lowest VI was observed in Kayyur-Cheemeni isolate with 57.95 VI, followed by Cherpulassery (60.58). The recombinant coat protein of PRSV was induced in pLATE 31 expression vector with C terminal histidine tag, within BL21(DE3)pLysS expression host. PRSV recombinant coat protein was purified using Ni-NTA column chromatography. A single band was observed at 35 kDa in SDS-PAGE analysis and western blotting with PRSV antiserum, confirmed the presence of purified recombinant coat protein in the soluble fraction. Pot culture experiment was conducted to evaluate the management of PRSD using Piriformospora indica. The initial colonization of P. indica inside the papaya roots was observed five days after germination of the seeds grown in P. indica massmultiplied medium. Prophylactic colonization of P. indica exhibited lowest VI (23.10) and 68.27 per cent reduction in VI over control diseased plants at five months after PRSV inoculation (MAI). Double antibody sandwich - Enzyme linked immunosorbent assay (DAS-ELISA) at 5 MAI revealed lowest absorbance (0.23) indicating lowest virus titre in P. indica pre-colonized plants upon PRSV inoculation, compared to control diseased plants (1.23). The accumulation of reactive oxygen species (ROS) i.e., H2O2 and superoxides in the leaves were analyzed using DAB (diaminobenzidine) and NBT (nitro blue tetrazolium chloride) staining respectively. P. indica-colonized plants upon PRSV inoculation indicated a higher initial accumulation of ROS at three weeks after inoculation (3 WAI) and further reduction at 6, 9 and 12 WAI of PRSV, compared to control diseased plants. P. indica-colonized plants also exhibited enhanced antioxidant defence enzyme activity viz., catalase, peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, glutamate synthase and superoxide dismutase compared to control diseased plants. Amongst PRSV inoculated treatments, P. indica pre-colonized plants exhibited highest number of leaves (20.86), leaf area (365.14 cm2), plant height (108.29 cm), stem girth (7.26 cm), shoot biomass (423.43 g), root biomass (133.96 g) and chlorophyll content (2.84 mg g-1 of fw) at five months after transplanting (5 MAT). Effect of P. indica, Bougainvillea spectabilis leaf extract (10 %) and Pseudomonas fluorescens (2 %) were evaluated against natural incidence of PRSD under field conditions at Instructional farm, College of Agriculture, Vellayani and Coconut Research Station, Balaramapuram, Thiruvananthapuram. P. indica-colonized plants exhibited lowest VI (37.63), with highest reduction in VI over control (53.85 %) followed by B. spectabilis treated plants (37.49 %) and P. fluorescens treated plants (33.31 %) at 12 months after planting (12 MAP). In DAS-ELISA, lowest virus titre with absorbance of 0.438 was observed in P. indica-colonized plants at 12 MAP, compared to the highest virus titre in control plants (1.267). B. spectabilis treated plants (0.596) also exhibited reduction in virus titre followed by P. fluorescens treated plants (0.625) at 12 MAP. P. indica-colonized plants exhibited enhancement in growth parameters viz., number of leaves (24.00), leaf area (1127 cm2), stem girth (42.74 cm) and plant height (207.39 cm) at 12 MAP. P. indica-colonized plants also enhanced the yield by 44.68 per cent followed by B. spectabilis treated plants (24.13 %) and P. fluorescens treated plants (17.99 %). Moreover, fruits from P. indica-colonized plants expressed significantly superior quality parameters. Thus, findings from the present study could aid in the preliminary detection and management of the virus, thus mitigating the widespread infection caused by PRSV. The recombinant PRSV coat protein produced in this study could be used for the development of PRSV antiserum. Additionally, our research highlights the efficacy of eco-friendly management strategies for papaya ringspot disease, with P. indica-colonization @ 106 cfu g-1 and also with four foliar sprayings as well as soil drenching of B. spectabilis leaf extract (10 %) applied at fortnightly intervals, starting from one month after planting. More field trials are to be conducted to integrate this strategy in integrated disease management (IDM) package for the effective and sustainable management of PRSD in papaya.Item Fertigation studies in papaya (Carica papaya L.)(Department of Fruit Science, College of Agriculture,Vellayani, 2021) Karishma Sebastian; Bindu, BThe investigation entitled “Fertigation studies in papaya (Carica papaya L.)” was carried out in Instructional Farm, College of Agriculture, Vellayani during the period 2018 – 2020 with the objectives to standardize the nutrient level for yield improvement through fertigation and foliar nutrition in papaya variety Surya and to study the postharvest management practices for extending the shelf life of papaya fruits. The experiment was undertaken in two parts. In part I, standardization of nutrient level for fertigation and foliar nutrition was carried out in RBD with 14 treatments replicated thrice. A combination of four fertigation doses of 75 %, 100 %, 125 % and 150 % RDF of N (304.89, 406.52, 508.15 and 609.78 g urea plant⁻¹ year⁻¹ respectively based on soil test data in 76 fertigation) and K (426.25, 568.33, 710.42 and 852.50 g MOP plant⁻¹ year⁻¹ respectively based on soil test data in 76 fertigation) and three foliar sprays (1.0 % 19:19:19 at bimonthly interval starting from 4 MAP to 16 MAP, 0.5% ZnSO4 + 0.3% borax at 4 th, 8th, 12th and 16th MAP and water spray at bimonthly interval starting from 4 MAP to 16 MAP) were compared with soil application of recommended dose of NPK (187:170:341 g NPK plant-1 year-1 based on soil test data) (control 1) and 187:170:341 g NPK plant-1 year-1 based on soil test data as organic manures as combination of FYM, poultry manure and vermicompost in the ratio of 2:1:1 (control 2). In control 2, additional requirement of P and K were met through the application of rock phosphate and potassium sulphate respectively. Organic manure (15 kg FYM plant-1 ) was given uniformly to all treatments as basal. Lime and rock phosphate (500g and 850g respectively based on soil test data) was applied uniformly for all treatments as basal except controls. Urea and Muriate of Potash (MOP) were used as fertilizer sources for fertigation and applied weekly from 1 MAP to 20 MAP. Application of different levels of fertigation and different foliar sprays had significant effect on growth, yield and quality of papaya. Growth parameters viz., plant height, stem girth and number of leaves were significantly higher in plants receiving 100 % RD of N and K through fertigation and foliar sprays of 1.0 % 19:19:19 (T4) at bimonthly interval starting from 6 MAP to 16 MAP. T4 also recorded highest leaf area index at 6, 12, 18 MAP and at final harvest, flowering at the shortest height, highest number of female plants and highest fruit set (86.27 %). However, application of 100 % RD of N and K through fertigation and foliar sprays of 0.5 % ZnSO4 and 0.3 % borax at 4 th, 8th, 12th and 16th MAP (T5) initiated earliness in flowering (142.67 days) and harvest (275.00 days). Treatments T4 and T5 were on par in fruit length, fruit girth, fruit volume, pulp percentage, flesh thickness and yield contributing characters like fruit weight, number of fruits per plant and yield per plant (38.30 kg plant⁻1 and 37.60 kg plant⁻1 respectively). Fruit quality parameters viz., TSS, carotenoids, ascorbic acid, total sugar, reducing sugar and non reducing sugar were found highest in T5. Fruits from T5 also registered longest shelf life (5.78 days) and highest mean sensory score for all parameters. Index leaf analysis at 6 MAP revealed highest nitrogen and potassium content of leaf in T10 (150 % RD of N and K through fertigation and foliar sprays of 1.0 % 19:19:19). Calcium, magnesium and sulphur content of leaves were highest in T5, whereas boron and zinc content were highest in T8 (125 % RD of N and K through fertigation and foliar sprays of 0.5 % ZnSO4 and 0.3 % borax). Highest nitrogen and potassium of fruits were observed in T12 (150 % RD of N and K through fertigation with water spray). Highest nitrogen content in soil was noticed in T11 (150 % RD of N and K through fertigation and foliar sprays of 0.5 % ZnSO4 and 0.3 % borax) and T10 witnessed highest potassium content. T4 and T5 were at par regarding B : C ratio (2.58 and 2.54 respectively). In part II of the investigation, postharvest management for extending shelf life of papaya was carried out with nine treatments replicated thrice. Different postharvest treatments given were precooling - hydro cooling (S1), surface sanitization with 150 ppm sodium hypochlorite (S2), external coating with 1% chitosan (S3), precooling followed by external coating with 1% chitosan (S4), packaging with ethylene scrubber 8% KMnO₄ (S5), precooling followed by packaging with ethylene scrubber 8% KMnO₄ (S6), precooling followed by external coating with 1% chitosan and packaging with ethylene scrubber 8% KMnO4 (S₇), hot water treatment followed by waxing with 6% carnauba wax and packaging with ethylene scrubber 8% KMnO₄ (S8) and control (S9). Papaya variety Surya was raised at Instructional Farm, Vellayani and fruits at fully mature green stage were harvested, subjected to different postharvest treatments and packaging was done in CFB boxes and stored under ambient conditions till the end of shelf life. Papaya fruits subjected to precooling followed by external coating with 1% chitosan and packaging with ethylene scrubber KMnO₄ (S7) recorded the longest shelf life of 9.67 days, which was at par with S8 (hot water treatment followed by waxing with 6% carnauba wax and packaging with ethylene scrubber KMnO₄). Physiological loss in weight, ion leakage, percentage disease index, bacterial and fungal count were significantly lowest in S7 and S8 after three days of storage. These treatments also recorded maximum total carotenoids, total soluble solids, total sugar, reducing sugar and minimum acidity after nine days of storage which indicated extended shelf life. Highest mean rank score for sensory attributes were also recorded in treatments S7 and S8 after nine days of storage. In conclusion, application of 100 % recommended dose of N and K (406.52 g urea plant⁻¹ year⁻¹ and 568.33 g plant⁻¹ year⁻¹ respectively) through weekly fertigation from one to 20 months after planting and foliar sprays of 0.5 % ZnSO4 + 0.3 % Borax at 4 th, 8th, 12th and 16th MAP, along with basal application of 850g rock phosphate and 15 kg FYM resulted in increased growth, yield and quality characters of papaya variety Surya with highest B: C ratio. Fruits at fully mature green stage when subjected to precooling followed by external coating with 1% chitosan and packaging with ethylene scrubber 8% KMnO₄ in CFB boxes exhibited a shelf life of 9.67 days in storage under ambient condition in papaya variety Surya.Item Bio-ecology and management of papaya mealybug on mulberry(Department of Agricultural Entomology, College of Agriculture, Vellayani, 2014) Rajan, V P; Krishnakumar, R