PhD Thesis

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End date: 2025Subject: search.filters.subject.Banana

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    Process optimization and utilization of resistant starch from banana.
    (Department of Community Science ,College of Agriculture,Vellanikkara, 2025-09-11) Sruthy,P M; Sharon, C L
    Banana (Musa spp.), often hailed as the common man’s fruit, holds a special place in the scenario of agriculture and nutrition, besides having immense economic significance in India and across the globe. Banana, known for its year round availability, affordability, and rich nutrient profile, has transitioned from a commonly consumed fruit to a functional ingredient of growing interest in modern health and food industry. Resistant starch (RS) found in unripe green banana is a promising bioactive compound with potential health benefits. India is leading the global banana production, hence there lies untapped potential in harnessing banana starch, especially its resistant form, for the development of value added food products. However, native banana starch faces limitations in its functional properties, which can be effectively addressed through modification techniques such as Heat Moisture Treatment (HMT). This approach not only enhances the nutritional and functional quality of banana starch but also paves the way for its incorporation into a wide range of health oriented food formulations. Hence, the current study “Process optimisation and utilisation of resistant starch from banana”, was carried out with the objective of optimising the conditions for resistant starch formation in banana and its quality evaluation. The study also aimed to develop value added products incorporating various proportions of banana resistant starch. The popular GI (Geographical indication) granted Kerala banana variety Chengalikodannendran banana and the worldwide popular Cavendish variety Grand Nainebanana were selected for this study. Starch extracted from their flours yielded 26.89% (Chengalikodan) and 27.40% (Grand Naine), with purity of 88.62% and 89.73%, respectively. Resistant starch content was 20.02% in Chengalikodan and 20.62% in Grand Naine. Starch samples of Chengalikodan and Grand Naine banana were modified by autoclaving at 110℃ and 121℃ with different moisture content (10, 20, and 30 %) at three different time periods (15, 30 and 60 minutes). Resistant starch (RS) content in Chengalikodan banana starch was significantly affected by autoclaving conditions. Maximum RS (25.84%) was observed at 121°C for 30 minutes with 10% moisture. In Grand Naine starch, autoclaving enhanced RS from a control value of 20.62% to a peak of 30.52% (at 121°C for 30 minutes with 10% moisture). Repeated autoclaving and cooling cycles further increased RS in Chengalikodan, which reached 54.56% after four cycles, while Grand Naine peaked to 59.63%. Repeated autoclaving and cooling cycles were applied to the isolated banana starch. As a result of this treatment, RS3 was formed. The presence of RS3 confirms that the modification process was effective. The samples with maximum RS content from Chengalikodan and Grand Naine were selected for further analysis and product development. Two treated banana starch (RS3) samples (Chengalikodan and Grand Naine), along with corresponding flour and isolated starch, were initially analysed for colour index and granular morphology. Morphological analysis showed that flour and isolated starch granules varied in shape, size, and surface texture based on processing. Chengalikodan and Grand Naine flours had irregular, oval, and elongated particles with smooth to rough surfaces, while isolated starch granules were smooth and more uniform. Autoclaving altered native starch structure, forming compact, aggregated granules due to retrogradation, with resistant starch showing larger, fused clusters and rougher surfaces. Chengalikodan flour had higher lightness and whiteness, which decreased isolated starch and treated starch. In contrast, Grand Naine showed consistently lower lightness across all forms, reflecting the impact of processing and variety on visual traits. The physicochemical properties of flour, isolated starch, and autoclaved starch samples from Chengalikodan and Grand Naine banana were evaluated over three months. Autoclaved starch samples of both Chengalikodan and Grand Naine showed higher pH (5.36 and 5.34), moisture (7.08 and 7.02%), water holding capacity (4.91 and 5.00%), and gelatinisation temperature (77.02 and 75.67℃), but lower bulk density (0.48 and 0.51g/ml), swelling power (8.86 and 8.88%), solubility (3.65 and 3.58%), syneresis (27.41 and 28.22%), dispersibility(75.14 and 76.31%), and peak viscosity (487cp and 571cp), which are important properties for the formulation of functional, shelf stable and healthy food products. This study assessed the nutritional, functional, and glycemic characteristics of flour, isolated starch, and treated starch (RS) samples of Chengalikodan and Grand Naine banana. Treated starch had the lowest total soluble solids (2.56 and 2.41ºBrix), with lower total and reducing sugars. Treated starch showed the highest carbohydrate content (87.72 g and 87.33 g/100 g), highest slowly digestible starch (18.88% and 18.59%) and lowest rapidly digestible starch (24.56% and 22.53%) for Chengalikodan and Grand Naine, respectively. It also contained high amylose of 35.42% (Chengalikodan) and 38.92% (Grand Naine) and lower amylopectin. Treated starch showed the lowest in vitro starch digestibility of 44.56% and 40.65% and the lowest glycemic index of 37.56 and 36.65, slightly increasing after three months, for Chengalikodan and Grand Naine, respectively. Instant custard mixes were developed using 10-35% banana resistant starch, 5-35% corn flour, and other ingredients. Both Chengalikodan (T4) and Grand Naine (T10) banana based custard mix with 20% RS had the highest overall acceptability of 8.55 and 8.22 respectively. Control custard mix (T0) had the highest brightness (L* = 83.93) and whiteness (WI = 79.66), while T4 and T10 showed reduced lightness (81.90 and 77.74) and whiteness (77.32 and 74.12). Banana resistant starch mixes had lower initial swelling power, solubility, water absorption capacity, and dispersibility, which declined further during storage. Bulk density and moisture were higher and increased over time. Carbohydrate, protein, and fat contents were initially lower and gradually decreased. Starch content was lower in T4 and T10 (56.21% and 54.95%) than in the control (62.91%). Rapidly and slowly digestible starch were also lower in T4 (57.62% and 10.23%) and T10 (58.23% and 10.25%), while resistant starch was higher (32.15% in T4 and 31.52% in T10) compared to the control. In vitro starch digestibility was lower in T4 and T10 (41.28% and 42.40%) than in the control (62.70%). Vermicelli was formulated using 25-45% resistant starch from Chengalikodan and Grand Naine banana, combined with banana flour and whole wheat flour (30-70%). Vermicelli with 100% refined wheat flour served as control. Sensory evaluation showed that T3 (35% Chengalikodan resistant starch + 15% banana flour + 50% whole wheat flour) scored highest overall acceptability (8.28). Correspondingly, uppuma made from T3 vermicelli also received superior sensory scores, with overall acceptability of 8.42. Products with Grand Naine resistant starch (35% Grand Naine resistant starch + 15% banana flour + 50% whole wheat flour) had slightly lower scores (8.24 for vermicelli and 8.15 for uppuma than Chengalikodan based products, though both were well accepted. The study compared physicochemical, nutritional, and starch digestibility properties of vermicelli made with RS from Chengalikodan and Grand Naine banana to a refined wheat flour control. Banana RS vermicelli had lower lightness and whiteness, with higher moisture content (6.89% in Chengalikodan, 6.95% in Grand Naine) than the control (6.33%). It also had higher carbohydrate and energy content, while protein and fat decreased over storage. Rapidly digestible starch was lower in banana RS vermicelli (41.45% and 43.09%) than the control (84.42%), while slowly digestible starch and RS were higher. Glycemic index was also lower (46.23 in Chengalikodan, 45.55 in Grand Naine) compared to the control (76.23). Instant uppuma mixes were made using Chengalikodan and Grand Naine banana resistant starch vermicelli (35% RS + 15% banana flour + 50% whole wheat flour). Among treatments with varying water volumes, T3 (100g vermicelli with 300 ml water) received the highest sensory scores for both banana varieties. The mixes showed better stability over three months of storage. The production cost of banana flour was ₹ 33/100g (Chengalikodan) and ₹ 19/100g (Grand Naine), while isolated banana starch cost ₹ 34/100g and ₹ 20/100g, respectively. Treated starch production cost was ₹ 23/100g (Grand Naine) and ₹ 37/100g (Chengalikodan). Custard mixes made with banana RS cost ₹ 40/100g (Chengalikodan) and ₹ 36/100g (Grand Naine), while vermicelli and instant uppumamixes with Chengalikodan RS cost ₹ 42/100g and ₹ 54/100g, compared to ₹ 33/100g and ₹ 45/100g for Grand Naine. Banana starch is a promising functional ingredient that can contribute to the development of health enhancing food products. Heat moisture treatment enhances its functional and physicochemical properties, opening new opportunities for health focused applications. This research highlights banana starch as a sustainable, cost effective ingredient with transformative benefits, poised to meet the growing demand for nutritious, innovative food solutions and shape the future of the food industry.
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    Development of multi nutrient formulation and pellets for organic farming and their evaluation in Banana (Musa AAB cv. Nendran)
    (Department of Soil Science and Agricultural Chemistry, College of Agriculture, Vellayani, 2024-11-05) Lucy Taki; Biju Joseph
    A study entitled “Development of multi nutrient formulations and pellets for organic farming and their evaluation in banana (Musa AAB cv. Nendran)” was carried out at the Department of Soil Science and Agricultural Chemistry, College of Agriculture, Vellayani during 2019-2023, with the objective to develop multi nutrient formulations and pellets using nutrient sources permitted under National Programme for Organic Production (NPOP) and to evaluate them in relation to nutrient release characteristics and productivity of nendran banana in agro-ecological unit 8 of Kerala. The study comprised of four parts viz., preparation and characterization of organic multi nutrient formulations, development of organic multi nutrient pellets and their quality evaluation, incubation study to investigate the nutrient release characteristics of organic multi nutrient formulations and pellets and a field study to evaluate the organic multi nutrient formulations and pellets in nendran banana. Organic multi nutrient formulations were prepared using organic nutrient sources like blood meal (BM), soybean meal (SM), rock phosphate (RP), steamed bone meal (SBM), potassium sulfate (SOP), langbeinite (L), epsom salt (ES) and borax (B) permitted in NPOP. Formulations were prepared by mixing nutrient sources considering the nutrient requirement of nendran banana (N:P2O5:K2O @ 300:115:450 g plant-1) and the fertility status of the experimental soil. The multi nutrient formulations prepared were F1 (BM+RP+SOP+ES+B), F2 (BM+RP+L+ES+B), F3 (BM+SBM+SOP+ES+B), F4 (BM+SBM+L+ES+B), F5 (SM+RP+SOP+ES+B), F6 (SM+RP+L+ES+B), F7 (SM+SBM+SOP+ES+B) and F8 (SM+SBM+L+ES+B). The multi nutrient formulations were characterized for their physical, chemical and biochemical properties. The results of characterization study were subjected to principal component analysis which revealed that the formulation F1 containing blood meal, rock phosphate, potassium sulfate, epsom salt and borax was superior to other formulations with index mean value of 13.07. Formulation F1 had 3.73 g cm-3 bulk 348 density, 2.67% moisture content, 6.5 pH, 3.23 dSm-1 EC, 29.43% OC, 7.21% N, 2.71% P, 10.78% K, 5.98% Ca, 0.35% Mg, 4.45% S, 1174.11 mg kg-1 Fe, 4.53 mg kg-1 Mn, 13.55 mg kg-1 Zn, 8.65 mg kg-1 Cu and 93.67 mg kg-1 B. It also contained 45.04% crude protein, 2.95% humic acid and 3.55% fulvic acid. Based on the result of the PCA, formulations F1 (BM + RP + SOP + ES + B), F2 (BM + RP + L + ES + B), F3 ((BM + SBM + SOP + ES + B) and F6 (SM + RP + L + ES + B) with index value of 13.07, 9.82, 11.93 and 7.35 were selected for further pelletization studies. Four selected organic multi nutrient formulations were mixed with 2 binding agents viz. bentonite (Bn) and starch (St) at 2 levels (2% and 4%). The formulations were mixed with binding agents as per the treatments and moistened with deionized water and compressed into pellet form. The 16 multi nutrient pellets prepared were P1 (BM+RP+SOP+ES+B+2%St), P2 (BM+RP+SOP+ES+B+4%St), P3 (BM+RP+SOP+ES+B+2%Bn), P4 (BM+RP+SOP+ES+B+4%Bn), P5 (BM+RP+L+ES+B+2%St), P6 (BM+RP+L+ES+B+4%St), P7 (BM+RP+L+ES+B+2%Bn), P8 (BM+RP+L+ES+ B+4%Bn), P9 (BM+SBM+SOP+ES+B+2%St), P10 (BM+SBM+SOP+ES+B+4%St), P11 (BM+SBM+SOP+ES+B+2%Bn), P12 (BM+SBM+SOP+ES+B+4%Bn), P13 (SM+RP+L+ES+B+2%St), P14 (SM+RP+L+ES+B+4%St), P15 (SM+RP+L+ES+B+2%Bn) and P16 (SM+RP+L+ES+B+4%Bn). The results of laboratory analysis of multi nutrient pellets were also subjected to PCA. Four different groups were made out of the 16 multi nutrient pellets with group 1 consisting of P1 to P4; group 2 consisting of P5 to P8; group 3 consisting of P9 to P12 and group 4 consisting of P13 to P16. PCA was performed separately on different groups to select one best multi nutrient pellet from each group. After one-way ANOVA of index values, the mean values of pellets P3 (11.23), P5 (8.21), P11 (11.36) and P15 (6.17) were greater found in their respective groups. However, the pellets P4 (BM+RP+SOP+ES+B+4%Bn), P6 (BM+RP+L+ES+B+4%St), P12 (BM+SBM+SOP+ES+B+4%Bn) and P16 (SM+RP+L+ES+B+4%Bn) with next higher 349 mean values of 11.03, 8.02, 11.16 and 6.05 were selected for further studies because of their superior physical qualities and stability. In the third part of the study a laboratory incubation experiment was carried out for 300 days to evaluate the nutrient release pattern of the 4 selected formulations (F1, F2, F3 and F6) and pellets (P4, P6, P12 and P16) upon addition to soil. Soil samples were drawn at 60 days interval (60th, 120th, 180th, 240th and 300th day of incubation) and nutrient release characteristics were studied. The result revealed that the soil organic carbon, water soluble carbon, labile carbon and particulate carbon increased till 60th day in all treatments then decreased till the end of incubation period. Significantly higher water soluble carbon (49.07 mg kg-1), labile carbon (1084.33 mg kg-1) and particulate carbon (3950.00 mg kg-1) was observed throughout the incubation period in T4 containing soyabean meal, rock phosphate, langbeinite, epsom salt and borax which was on par with T8 containing same formulation with 4% bentonite. The available N, NH4 +-N and NO3 --N content increased till 120th day irrespective of treatments and thereafter it decreased. Treatment T1 having blood meal + rock phosphate + potassium sulphate + epsom salt + borax recorded highest available N (246.46 kg ha-1), NH4 +-N (84.00 mg kg-1) and NO3 --N (46.67 mg kg-1) content throughout the incubation period followed by its corresponding pellet T5 having blood meal + rock phosphate + potassium sulphate + epsom salt + borax + 4% bentonite. The available P, labile P and non labile P increased upto 240th day for all the treatments. T1 showed significantly higher level of available P (111.41 kg ha-1), labile P (22.27 mg kg-1) and non labile P (42.89 mg kg-1), throughout the incubation period which was on par with T5. It was observed that available K, water soluble K, exchangeable K and non exchangeable K increased till 240th days of incubation and then declined in all treatments. Formulation T3 having blood meal + steamed bone meal + potassium sulphate + epsom salt + borax had significantly higher available K (972.93 kg ha-1), water soluble K (81.67 mg kg-1) and exchangeable K (352.68 mg kg-1) throughout the incubation period. However, the highest non exchangeable K varied during different 350 incubation period. Secondary nutrients Ca, Mg and S showed maximum release in all treatments till 240th day and thereafter declined with T2 formulation containing BM+RP+L+ES+B showing significantly higher content throughout the incubation period. The highest available micronutrient (Fe, Mn, Zn, Cu) was in treatment T5 which was on par with T1. A field experiment was conducted during 2021-2022 to evaluate the effect of organic multi nutrient formulations and pellets on soil fertility and crop productivity using nendran banana as test crop. The results of soil analysis revealed that application of multi nutrient formulations and pellets did not have much influence on physical properties of soil in both basin and 1m away from the plant. However, the chemical and biological attributes were significantly improved in both the soil. Application of T2 containing formulation BM+RP+SOP+ES+B and T6 containing pellet BM+RP+SOP+ES+B+4%Bn improved the availability of N, P, K, Fe, Mn and Zn content in both the soils. Dehydrogenase activity and microbial population was enhanced by application of KAU organic POP (T1), formulation SM+RP+L+ES+B (T5) and pellet SM+RP+L+ES+B+4%Bn (T9) in basin soil. Crop growth parameters like plant height, girth of pseudostem and no of functional leaves per plant were significantly higher in T2 which was on par with T6. Yield parameters like length, girth and weight of index finger, bunch weight, no. of hands per bunch, no. of fingers per bunch and no. of fingers in D-hand was also significantly higher in T2 and T6 compared to other treatments. T2 recorded the highest yield of 34.17 t/ha which was on par with T6 (31.25 t/ha). The crop receiving T9 (SM+RP+L+ES+B+4%Bn) had the longest crop duration and the shortest was observed in T2. The fruit quality parameters like TSS, TSS/acidity ratio, total sugar, reducing sugar, non reducing sugar, total sugar/ acidity ratio, carbohydrate, pulp to peel ratio was significantly higher in treatments T2 and T6. The foliar N, P, K, Ca, Fe content at harvest was highest in T2 containing formulation BM+RP+L+ES+B, foliar Mg, S content was highest in T3 containing formulation BM+RP+L+ES+B and foliar 351 Mn, Zn, Cu, B content was highest in T4 containing formulation BM+SBM+SOP+ES+B. Total uptake of N, Zn and B was highest in T2 while the total uptake of P, K, Ca, Mg, Fe, Mn, Cu was highest in T6. Fruit quality parameters like TSS, total sugar, reducing sugar, non reducing sugar and pulp/peel ratio was significantly higher in T2 which was on par with T6. Shelf-life of nendran fruit increased significantly in T4 receiving the formulation BM+SBM+SOP+ES+B and T8 receiving the same formulation with 4% bentonite. Nutrient use efficiency was higher in T2 and T6. The highest B:C ratio of 2.21 was recorded in T2. From the results of the study, it can be concluded that multi nutrient formulation prepared using blood meal, rock phosphate, potassium sulphate, epsom salt and borax (T2) and multi nutrient pellet prepared using blood meal, rock phosphate, potassium sulphate, epsom salt, borax and 4% bentonite (T6) was superior to other formulations and pellets. The nutrient release from above formulation and pellet were maximum at incubation period of 120th day for N and 240th day for P, K, secondary and micronutrients. The treatment T2 applied @ 4.2 kg plant-1 was found to be superior to other treatments with respect to available nutrient status of soil, nutrient uptake, growth parameter, yield and quality of nendran banana, which was found to be on par in the case of the most of the parameters with the treatment T6 applied @ 4.5 kg plant-1. T2 was also the most economical treatment. Hence, the application of organic multi nutrient formulation prepared using blood meal, rock phosphate, potassium sulphate, epsom salt and borax at the rate of 4.2 kg plant-1 as 3 equal split doses at 2MAP, 4MAP and 6MAP can be recommended for nutrient management in organically grown nendran banana.
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    Management of banana bract mosaic virus using beneficial fungal root endophyte, Piriformospora indica
    (Department of Plant Pathology, College of Agriculture ,Vellayani, 2024-03-20) Sinijadas, K.; Joy, M
    The research work entitled “Management of Banana bract mosaic virus using beneficial fungal root endophyte, Piriformospora indica” was carried out at the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during the academic year 2018-2022. The objective was to evaluate the beneficial fungal root endophyte, P. indica for the management of Banana bract mosaic virus (BBrMV); and to elucidate the role of antioxidants and plastids in this tripartite interaction. The characteristic symptoms of BBrMV viz., reddish spindle shaped streaks on pseudostem, black necrotic streaks on peduncle, chlorotic spindle lesions on leaves and underdeveloped fruits were observed during the survey conducted in five agro-climatic zones (ACZ) of Kerala. Percent disease incidence (PDI) and vulnerability index (VI) of the disease recorded from different zones showed the highest in banana var. Nendran (PDI - 61.66 & VI – 44.03) from southern zone and lowest in var. Poovan (PDI – 7.29 & VI – 6.29) in central zone. Serological and molecular detection confirmed the presence of BBrMV in banana var. Nendran. The sequence similarity analysis of the coat protein (CP) gene of BBrMV southern zone isolate (Vellayani) showed 99.12 percent genetic closeness to its Tamil Nadu isolates compared to the Kerala isolates (98.38 percent). Screening of the most virulent strain of BBrMV from five different ACZ of Kerala was done based on early symptom development in TC banana plants var. Nendran. The BBrMV strain from southern zone could produce the symptoms on var. Nendran within 30 days (lowest) followed by central zone (55 days) on artificial inoculation with viruliferous aphids, Pentalonia nigronervosa. The cross-infection study of virulent strain of BBrMV (southern zone) of Nendran on Nendran developed chlorotic spindle lesions on leaves at 30 days after inoculation followed by Nendran on Robusta (75 days). P. indica-colonization in banana var. Nendran was carried out using standardized medium. The chlamydospores of the fungus were observed in roots at 20 days after colonization (DAC). In both in vitro and in vivo experiments, P. indicacolonized plants showed reduced disease severity irrespective of the virusinoculation stage with a vulnerability index of 6.7 percent in BBrMV (+Pi / +V); and 20.0 percent in the virus-infected plants post-colonized with P. indica (+V / +Pi) compared to 53.3 percent in the virus alone infected plants. Further, PCR analysis with BBrMV coat protein specific primer yielded amplicon of low intensity in P. indica-colonized plants inoculated with the virus compared to the control plants indicating the ability of the fungus to inhibit the virus. Further, P. indica precolonized plants inoculated with BBrMV had improved growth and yield parameters compared to non-colonized plants. Field trial was laid out with two treatments (P. indica-colonized and noncolonized banana plants var. Nendran) at Instructional Farm, College of Agriculture, Vellayani. P. indica-colonized plants recorded a drastic reduction in the severity of BBrMV by 33 to 58 percent compared to non-colonised control plants. Enhanced plant height (30 percent), collar girth (45 percent), number of leaves (25 percent), leaf length (30 percent), leaf width (27 percent), fresh weight of shoot (34 percent), number of secondary roots (62 percent), number of tertiary roots (76 percent) and root weight (86 percent) were observed in P. indica-colonized plants at 90 days after treatment. P. indica-colonization also improved the bunch weight (32.9 percent) and fruit quality. Biochemical detection of superoxides using nitroblue tetrazolium (NBT) and H2O2 with diamino benzidine (DAB) stains at 5, 10, 15, 30 and 45 days revealed a reduction in reactive oxygen species (ROS) accumulation in both P. indica-colonized plants challenged with BBrMV (+Pi / +V) and virus infected plants post-colonized with P. indica (+V / +Pi) compared to BBrMV alone. The decrease in ROS production and disease severity in the endophyte-colonized plants inoculated with the virus were attributed to the increased activities of antioxidant enzymes viz., peroxidase, superoxide dismutase, catalase, glutamate synthase and ascorbic acid oxidase. The molecular analysis of genes involved in the symptom development indicated the beneficial effect of P. indica on BBrMV infection in banana. P. indica reduced the symptoms by up-regulating chlorophyll biosynthesis gene (chlorophyll synthase-CHLG) and down-regulating chlorophyll degradation genes (chlorphyllase CLH1 & CLH2; and pheophytin pheophorbide hydrolase - PPH), carotenoidbiosynthesis genes (phytoene synthase-PSY1 and PSY2), carotenoid degradation gene (Phytoene desaturase - PDS) and the virus specific genes responsible for symptom development (Hc-Pro and P3). Thus, the present study reveals that P. indica enhances tolerance against BBrMV in addition to improved growth promotion, yield and fruit quality in banana plant.
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    Dissipation and distribution of fipronil, carbosulfan and their metabolites in banana var. Nendran (AAB) and soil
    (Department of Soil Science and Agricultural Chemistry, College of Agriculture, Vellayani, 2021) Visveswaran, S; Thomas George
    The study entitled “Dissipation and distribution of fipronil and carbosulfan and their metabolites in banana (Musa spp) and soil” was carried out at the Department of Soil Science and Agricultural Chemistry, College of Agriculture, Vellayani during the period from August 2016 to April 2017. The objective was to assess the dissipation, metabolism and persistence of fipronil and carbosulfan in banana, cv. Nendran (AAB), grown under red loam soils (AEU 8-southern laterites) of College of Agriculture, Vellayani, and its impact on soil organisms. Banana variety Nendran, grown as per package of practices recommendations, KAU in randomized block design at the Instructional Farm, Vellayani, with five treatments viz., T1- absolute control (No application of carbosulfan and or fipronil), T2- recommended practice of 30 mg ai of fipronil per plant per application, applied 3 times on 0, 60 and 150 days of planting, T3 – double dose of T2, T4- recommended practice of 400 mg ai of carbosulfan per plant per application, applied 3 times on 0, 60 and 150 days of planting and T5- double the dose of T4. Samples viz, soil, leaves, fingers bunches and flower bud, central core of pseudo-stem and corm were collected and analyzed for residue at definite time intervals. The residue estimation of the target pesticide molecules and their toxic metabolites were performed by adopting standard extraction and clean up procedure viz., conventional acetone extraction followed by hexane partitioning as well as by QuEChERS method. Method validation was conducted by spiking with analytical standards from which recovery, (accuracy 70-120% of spiked values), standard deviation of recoveries, RSD value (below 20%), linearity (calibration curve), for the adopted method were worked out and compared for compliance. Acetonitrile extracted samples were analyzed using “Waters Acuity UPLC 137 system” with suitable column and then subjecting the effluent to triple quadrupole API 3200 MS/MS system equipped with electrospray ionization interface (ESI) operating in required mode as per the molecule. Extraction using QuEChERS method gave satisfactory values for validation parameters and hence adopted for the studies. The presence of carbosulfan in the 1st to 4th leaves till day 20th and subsequent dissipation pattern prediction for BDL in 22.5 day indicated that, at recommended dose of application, it is not safe to use the leaves within 23 days of application for serving or food packing (as commonly practiced in many households of Kerala). Sample matrices revealed the presence of metabolites each for fipronil and carbosulfan with variation from below detectable limit (BDL) to a highest content of 3.804 μg/g carbosulfan at 2 hours of application in the soil. Blossom bud, flower bract alone, bunch on 15th day of emergence, bunch on 30th day of emergence, peel, bunch on harvest, pseudo stem and corm did not register any detectable level of fipronil or carbosulfan and their metabolites and even with an additional application of treatment on the day of bunching also did not register any detectable level of fipronil or carbosulfan and their metabolites. Residue of fipronil and their toxic metabolites in the first, second and third leaves of banana on penultimate day of completion of pre-bunching application was found to be below the detectable levels throughout the period of sampling and this may be attributed to low absorption, fast metabolism and mobility. However, on 40th day the fipronil was detected in the 4th leaf to the extent of 0.034 μg g-1 and was not detectable on 50th day. Metabolites of Carbosulfan residue existed in the first, second and third leaves between 5th and 20th day and it dissipated to below detectable limit on 40th day of application. The content of residue under treatment T5 was distinctly higher than T4 during these periods and dissipated to BDL on 25th day. 138 In soil, the fipronil though dissipated to BDL before 50th day, persisted from 2 hours of application till 40th day. However, carbosulfan and its metabolites were early to dissipate to BDL on 7th day of completion of application. Sample in experiment with 5 times the recommended dose of application as injection into pseudostem at the time of bunch emergence also did not record any residue above detectable level in the flower bud, flower bract alone, bunch on 15th day of emergence, bunch on 30th day of emergence, peel, bunch on harvest and pseudostem. Soil urease activity on 10th day was significantly influenced by the treatment where T1, T3 and T5 are significantly lower than T2 and T4. However, dehydrogenase activity and acid phosphatase activity were not significantly influenced by the treatment. Bacterial population was higher in T1. Treatments were found to significantly influence the weight of blossom bud on dehorning, pseudostem, bunches and corm. Other biometric parameters were not influenced by the treatment. The above results show complete dissipation of fipronil and carbosulfan to safe limits in soil and banana leaf, when applied as per package of practices recommendation for banana cultivation (fipronil 30 mg a.i. and carbosulfan 400 mg a.i. per plant applied thrice viz., on 0, 60 and 150 days of planting), within 50 and 23 days of application. Also, the application of fipronil and carbosulfan as per the above dose in no way results in the accumulation of residue on any of the edible plant parts of banana and hence it is safe for human consumption.
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    Development of recombinant coat protein for immunodiagnosis of banana bunchy top and bract mosaic diseases
    (Department of Plant Pathology, College of Agriculture, Vellanikkara, 2021) Darsana Dilip, K C; Vimi Louis
    The present investigation was undertaken to develop recombinant coat protein (rCP) of Banana bunchy top virus (BBTV) and Banana bract mosaic virus (BBrMV) for immunodetection of the viruses. The experiments were conducted at the Virology Lab, Banana Research Station, Kannara; Department of Plant Pathology, College of Agriculture, Vellanikkara, Kerala Agricultural University and Indian Institute of Science, Bengaluru during the period of 2016-2020. A roving survey in 10 districts of Kerala, divided into population subsets viz., North, Central and Southern zones were conducted for sample collection. After a preliminary DAC-ELISA, 17 and 12 representative samples respectively were selected and carried forward for further evaluations. The CP gene of BBTV was amplified from the total DNA isolated using reported primers by Polymerase Chain Reaction (PCR) and that of BBrMV by Reverse Transcriptase-PCR (RT-PCR). The CP gene sequences of these isolates were determined and submitted in the NCBI-GenBank Database. The 17 BBTV isolates were designated as MT174314-MT174330 and the 12 BBrMV isolates as MT818176- MT818187. It was inevitable to evaluate the molecular diversity of the viruses prior to devising nucleic- acid based and serological detection methods. The phylogeographic analysis depicted a clear demarcation of BBTV Kerala isolates based on geography whereas no such clustering was observed in the case of BBrMV isolates. Being an RNA virus, the molecular diversity of BBrMV (ranging between 1-12 %) was higher than BBTV. However, the 5’ and 3’ terminal of BBrMV CP gene was hypervariable and found unsuitable to be targeted for nucleic-acid based detection. Hence, forward primer was designed from the NIb region of ssRNA genome of BBrMV and reverse primer from 3’ UTR region upstream and downstream to the CP gene respectively. For nucleic-acid based detection of BBTV, highly conserved non-coding region of DNA-S upstream and downstream to the CP ORF was targeted. The primers were validated by detecting virus from the field samples collected from various parts of the state. The rCPs were chosen as a potential antigen for raising antibodies in order to develop serodiagnostic assays for the early detection of the viruses. The BBTV CP gene was clonedin to three expression vectors viz., pRSET-C, pGEX-4T-2 and pET32a(+) and transformed to expression hosts like BL21 (DE3) pLysS, Rosetta (DE3) pLysS and C41 strains of E. coli after amplification in DH5α. The 20 kDa recombinant BBTV CP (rBBTV CP) cloned in to pRSET-C, and overexpressed in various E. coli hosts had a hexa histidine (6X His) tag at the N terminal. Similarly, a 37 kDa fusion protein (pET/rBBTV CP) was overexpressed from pET/BBTVCP clone had a thioredoxin (Trx) tag (17 kDa) along with the 6X His tag. Whereas, a 45 kDa fusion protein (pGEX/rBBTV CP) with GST tag was overexpressed from pGEX/BBTVCP clone. These affinity tags in the fusion rCP enabled purification from other E. coli proteins. Although pRSET/rBBTV CP was soluble, the 20 kDa protein was highly unstable and partially degraded during purification at 4 °C. Curiously, pGEX/rBBTV CP dissociated from its GST affinity tag and the rCP without the tag degraded. On evaluating the protease cleavage sites in the fusion protein, trypsin cleavage sites were present between the C terminal of GST and N terminal of BBTV CP which might be the reason for cleavage of the ~20 kDa protein from its affinity tag. Thus, it was impossible to purify the protein from the pool of E. coli proteins. Restriction free (RF) cloning of BBTV CP to pGEX-4T-2 was attempted not only to replace these trypsin cleavage sites but also the thrombin cleavage site present in the vector with Tobacco etch virus (TEV) NIa protease site. Thrombin is a specific enzyme used to cleave off the tag from the fusion protein after purification. However, its specificity is not universal. Furthermore, the commercially available enzyme is costly. TEV protease on other hand was produced in the laboratory and was highly specific. However, the cleavage using TEV protease was unsuccessful apparently because of a steric hindrance contributed by the two extremely ordered regions flanking the TEV cleavage site present in the disordered region of the fusion protein. pET/rBBTV CP was highly soluble like ΔpGEX/rBBTVCP. Likewise, BBrMV CP gene was cloned into pRSET-C and pGEX-4T-2 to obtain pRSET/rBBrMV CP and pGEX/rBBrMV CP of size 34 kDa and 60 kDa respectively. The 34 kDa pRSET/rBBrMV CP was insoluble. Overexpression and purification of the protein was standardized in various conditions to increase solubility. On the contrary, pGEX/rBBrMV CP was highly soluble and was purified by GSH Sepharose affinity column chromatography. 360 μg/ml of untagged protein was obtained from 1 l culture. However, like any other potyviral CP, the exposed N and C terminal of BBrMV CP was also prone to proteolytic cleavage. It partially degraded when incubated with thrombin atroom temperature for GST tag cleavage. All these bands were detected by potyviral CP specific antibody in Western blot. Further on storage complete degradation of the protein was observed. Further standardisation of the protocol is necessary to either stabilise monomeric CP or develop BBrMV VLPs in vitro for immunising animal in order to raise the antiserum. The immunogenicity of the antigens (rBBTV CP and rBBrMV CP) was confirmed by Western blot using BBTV CP specific and potyvirus CP specific antibody procured from NRC, Banana and IISc, Bangalore respectively. The rCPs were also characterized by fluorescence spectroscopy, sucrose gradient ultra centrifugation and electron microscopy. The fluorescent spectra of tagged and tag less rBBrMV CP deviated from 330 nm which is typical for a partially disordered protein. However, the spectra of pET/rBBTV CP and ΔpGEX/rBBTV CP were different. The former depicted the spectra of a mostly globular protein. There were two λmax for the fluorescence spectra of ΔpGEX/rBBTV CP. The epitope prediction of BBTV CP with Trx tag gave interesting insights. A single linear epitope of 80 residues were detected in pET/rBBTV CP comprising of C terminal of the affinity tag and the N terminal of BBTV CP. This was expected to increase the immunogenicity of the antigen and administered for production of antiserum. The titre value of polyclonal antiserum produced against the 37 kDa pET/rBBTV CP was evaluated by DAC-ELISA and was found to be 1:128000. Titre value for serological assays of field samples was standardized as 1:10000 to be more inclusive for detecting virus even at early stages of infection. A total of 247 tissue culture samples and 10 field samples were screened for the presence of the virus using the antiserum and was compared with the procured antiserum. Seemingly, the latter non-specifically reacted with plant proteins which gave a higher absorbance value in negative control and correspondingly high absorbance in the infected samples. The polyclonal antiserum raised against rBBTV CP was used to standardize serological detection assays like IC-PCR, DIBA and TAS-ELISA apart from DAC-ELISA. DIBA and TAS-ELISA were the most sensitive assays which could detect up to 1:80 dilution of the antigen. In conclusion, due to the higher nucleotide variability of the CP gene, serological detection is preferred over nucleic acid based assays. However, the quality of antigen used for raising the antibody plays a major role in serodiagnostics. Hence, high quality rCPs of both BBTV and BBrMV were developed in the laboratory in various vector/host systems. ThepET/rBBTV CP overexpressed in C41 strain of E.coli (1.1 mg/ ml obtained from 1 L culture) was used for immunisation of the animal. A highly sensitive antiserum specific to BBTV with a titre ten fold higher than that of the commercially available antiserum was obtained. Using this antiserum raised against rBBTV CP, various serodiagnostic assays were standardised in the laboratory. Among these, TAS-ELISA was the most sensitive, detecting antigen even at higher dilution.
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    Characterization and integrated management of fusarium oxysporum f.sp. cubense (E.F. Smith) synder and hansen causing fusarium wilt disease of banana
    (Department of Plant Pathology, College of Horticulture, Vellanikkara, 2020) Lishma, N P; Anita Cherian, K
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    Nutrient and moisture optimization in banana (Musa AAA. Grand Nain)
    (Department of Agronomy, College of Agriculture, Vellayani, 2016) Bindhu, J S; Girija Devi, L
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    Studies on certain chemical constituents of banana leaves in relation to incidence of leaf spot diseases: A note on the fungi occurring on banana
    (Division of Plant Pathology ,Agricultural College and Research Institute ,Vellayani, Trivandrum, 1966) Chandrasekharan Nair, M K; Sam Raj, J
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    Development of juice- based beverage and ripe- fruit powder from banana(musa SPP.)
    (Department of Processing Technology, College of Horticulture, Vellanikkara, 2005) Evelin Mary, A; Jacob John, P
    The study on "Development of juice-based beverage and ripe-fruit powder from banana (Musa spp.)" was carried with the objective of developing a drying and packaging regime for ripe banana powder and to develop a beverage based on clarified banana juice. Technology for spray and drum dried banana powder was standardized through drying and packaging experiments. Good quality spray dried banana powder could be obtained with an inlet air temperature of 150°C, keeping the outlet temperature at 100°C with two per cent maltodextrin and three per cent soluble starch on wet basis as additive. Successful drum drying could be achieved with drum temperature of 152°C with drum speed of three rpm and soluble starch 2.5 per cent on wet basis as additive. Drum drying was found better in terms of higher recovery, lesser additive, better quality and lesser cost of production. Moisture sorption studies revealed that banana powder was highly hygroscopic requiring special packaging, techniques. Gas Chromatography-Mass Spectrometry analysis revealed that the caramelized fruity fig like aroma of banana powder was due to Furaneol and Ethyl einnamate. Banana powder could be stored for one year under ambient conditions without deterioration when packed in aluminium foil laminated pouches with nitrogen. Banana powder can be used as an ingredient in baby food, health food, instant beverage mix, breakfast cereals, flavourings, bakery and dairy products. The cost of production of 100 grams of spray and drum dried banana powder worked out to be Rs.26.61 and Rs.18.66 respectively. The technology for banana juice based beverage was also standardized. Clear banana juice could be extracted using commercial pectinase enzyme @ 5 mll kg pulp and incubating for four hours at room temperature. Robusta juice was superior in terms of colour, flavour and overall acceptability compared to Poovan, karpooravalli and Palyankodan juices. Blended banana-mango RTS beverage in the ratio of 50:50 was preferred by the taste panel over other fruit blends. The disadvantages in banana juice viz., lack of colour, consistency and acidic taste could be overcome by mixing equal amounts of mango juice. Blending banana juice with mango pulp helped in incorporating the excellent flavour of banana and reduced the cost of production of mango R TS beverage. The cost of production of banana-mango RTS beverage when worked out came to be Rs.3.84 per 200 ml bottle while that of pure mango RTS beverage was RsA.23. The blended RTS beverage could be stored for six months under ambient condition without deterioration. With liberalization in industrial policy and globalization, more opportunities are created for export of value added and nutritious products from banana. India being the largest producer of banana in the world can tap this potential by exporting products' like ripe banana powder and juice based beverages.
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    Evaluation of selected banana(musa spp.) varieties grown in Kerala for post harvest attributes
    (Department of Processing Technology, College of Horticulture, Vellanikkara, 2005) Reni, M; Sheela, K B
    The present investigation on ‘Evaluation of selected banana (Musa spp) varieties grown in Kerala for postharvest attributes was conducted in the Department of Processing Technology, College of Horticulture, Vellanikkara, Thrissur, Kerala. Twenty five accessions of banana belonging to six genomic groups were catalogued as per IPGRI description based on 21 fruit characters. Of the twenty five accessions, maximum value for finger weight, finger length, volume of finger, pulp weight and peel weight was recorded by Zanzibar and lowest by Matti. Other varieties with higher finger weight were Nedunendran, Chengalikodan, Manjeri Nendran, BRS I, Monthan, Batheesa and Kanchikela. Varieties Zanzibar and Chinali recorded highest value for TSS (32.5°brix). Varieties Chengalikodan, Koompillakannan, Nendunendran, Manjeri Nendran, Matti and Njalipoovan also recorded higher values for TSS (TSS >30°brix). Culinary varieties recorded lower TSS. Nendran clones recorded higher values for total and reducing sugars, vitamin C, ß carotene and starch. Physiological loss in weight was highest in Grand Naine and lowest in Batheesa at the end of storage period. Longer shelf life was recorded by the varieties Bodles Altafort, Chengalikodan, Red Banana, Grand Naine, Manjeri Nendran and Poomkalli. On organoleptic evaluation higher scores for sweetness and taste was recorded by Kadali and Koompillakannan. The physical, chemical and physiological changes during ripening were recorded in four stages viz., mature green unripe stage, colour changing, full ripe and over ripe stage in eight varieties. Pulp percentage and pulp/peel ratio showed an increasing trend whereas peel percentage and fruit firmness showed a decreasing trend during the postharvest period. TSS, total, reducing and non reducing sugars and moisture content increased progressively in the various varieties from stage I to stage IV, whereas starch, pectin and tannin content decreased during ripening. Acidity in the various varieties increased up to colour changing stage whereas on full ripening decreased. The activity of starch hydrolysing enzymes -amylase, ß-amylase and invertase increased during the postharvest period in the various varieties. The rate of respiration showed a climacteric peak at full ripe stage and after that it slightly decreased. The effect of different postharvest treatments on enhancing the shelf life varied with different varieties. Precooling with ice flakes and tap water was effective in extending shelf life in Palayankodan fruits. In Njalipoovan and Chengalikodan longer shelf life was obtained for fruits vacuum infiltrated with 1 per cent CaCl2. Maximum shelf life in the case of Robusta was for fruits coated with edible wax at cut ends and precooling with cold water. Precooling treatments in general improved the TSS and total sugars in Palayankodan, Njalipoovan, Robusta and Chengalikodan. Higher scores for sensory qualities were obtained for precooled fruits in Palayankodan, Robusta and Chengalikodan and for fruits vacuum infiltrated with 1 per cent CaCl2 in Njalipoovan. Effect of different packaging treatments varied significantly in the various varieties. In Palayankodan, Njalipoovan and Chengalikodan minimum physiological loss in weight and maximum shelf life was observed for vacuum packed fruits. Maximum yellow life was observed in fruits packed in corrugated fibre board boxes and CFB box + polyethylene lining + ethylene absorbent. Higher values for TSS, total sugars and higher scores for taste, flavour and overall acceptability was for fruits packed in CFB boxes.