1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Elucidation of role of small RNA mediated gene regulation in secondary metabolite pathway of black pepper (Piper nigrum L.)(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2024-07-19) Shruthy, N S; Asha, SThe study entitled “Elucidation of role of small RNA mediated gene regulation in the secondary metabolite pathway of black pepper (Piper nigrum L.)” was conducted in the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2023-2024. Piperine, an amide alkaloid that contributes the unique spicy flavor is the major economic secondary metabolite of black pepper which is renowned for its pharmacological activities and therapeutic efficacies. The biosynthetic pathway of various secondary metabolites was highly influenced by a regulatory factor, microRNA- ‘the master regulators of gene expression’. They are small, non-coding RNAs of typically 18-24 nucleotides and can be a useful tool in metabolic engineering. Therefore, a better understanding of the mode of action of miRNA is very essential to exploit them in improving the plant for enhanced secondary metabolite production. Hence, the present study focuses on ‘identification and characterization of small RNAs involved in piperine biosynthesis from black pepper (Piper nigrum L.) In this study, the microRNAs targeting the key genes involved in the piperine biosynthesis pathway were analyzed by combined in silico-experimental method. From the 19 million reads of small RNA transcriptome, 303 conserved families of MiRNA were reported in black pepper. Among these, potential candidates of small RNAs were predicted to target the key genes involved in piperine biosynthesis such as Piperine synthase, piperamide synthase, and Piperoyl CoA Ligase genes. From these candidates, we characterized conserved and novel microRNAs. Precursor miRNAs for each candidate were also predicted based on their distinctive features such as length, ability to form hairpin stem-loop structure, and minimal folding energy. Among the potential candidate miRNAs, ‘Pni_miR5654’ and novel miRNAs such as ‘Pni_miR19’ and Pni_miR33’ were predicted for experimental validation. The tissue-specific expression of these miRNAs and their cognate targets in different parts of the black pepper variety Panniyur-1 were evaluated. The result indicates that the immature spike has the highest expression of microRNAs compared to leaf and mature berries whereas the highest expression of cognate target genes was observed in mature berries compared to leaf and immature spikes, that is, the miRNA: target pairs were inversely correlated. Further to analyse the expression of miRNA: target pairs in bio-elicitated callus cultures, initially the callus initiated from berries were treated with the culture filtrate of Pirimorsphora indica and R/NA was isolated at 0, 24, and 48 hrs after treatment. The study sowed and upregulation in the expression of both the miRNAs and their cognate pairs in 24 hour treatment. In this present study, the microRNA candidates Pni_miR5654, Pni_miR19’ and ‘Pni_miR33’ were identifies to have gene regulatory role in piperine biosynthesis by targeting the key genes in the pathway. Although miRNA medicated regulation was detected in the spike and berry stages, the inverse correlation of miRNA: target pairs was not found in in vitro cultures of black pepper, indicating the cellular reprogramming of miRNAs during callogenesis. The data generated from this study could be helpful in modifying the target genes, constructing artificial miRNAs, and performing inhibition of miRNA in order to enhance the production of piperine in black pepper.Item Interaction between root-knot nematode and fungal pathogens in black pepper, Piper nigrum L.(Department of Nematology, College of Agriculture, Vellayani, 2024-04-09) Amruth Hari; Nisha, M SThe study entitled " Interaction between root-knot nematode and fungal pathogens in black pepper, Piper nigrum L." was conducted at the Department of Nematology, College of Agriculture, Vellayani, Thiruvananthapuram during 2021- 2023. The objectives were documentation of nematodes and fungal pathogens in the rhizosphere of black pepper, the interaction between root-knot nematode and fungal pathogens, histopathological and biochemical changes due to nematode-fungal disease complex in black pepper. A survey was conducted in the major black pepper growing areas of Kannur (AEU-15), Idukki (AEU-16) and Wayanad (AEU-21) districts to document the nematode fauna and fungal pathogens associated with black pepper. The plant parasitic nematodes identified were Meloidogyne incognita (Kofoid and White) Chitwood, Rotylenchulus reniformis Linford and Oliveira, Radopholus similis (Cobb), Helicotylenchus dihystera (Cobb) Sher and Macroposthonia sp. The highest population of M. incognita and R. reniformis was recorded in Padiyoor and Malapattam areas in Kannur district with prominence value (PV) of 14.99 and 12.29 respectively. In Idukki and Wayanad districts highest population of M. incognita was recorded in Marayoor and Panamaram with PV of 14.76 and 15.48 respectively. Fungal pathogens viz. Fusarium solani, F. oxysporum, Phytophthora sp. Rhizoctonia sp. and Pythium sp. were also isolated from soil and plant samples. Occurrence of F. solani and high population of M. incognita was recorded in Padiyoor and Irikkur areas in Kannur, Marayoor and Mattukatta areas of Idukki and Panamaram and Pulpally regions of Wayanad. Severe galling and yellowing were observed in plants infested with nematode and fungi. A pot culture study was conducted to investigate the interaction between M. incognita and F. solani, since F solani was observed to be the major fungal pathogen associated with nematode infested plants in surveyed locations. The interactive effect of nematode and fungus in plant growth parameters, nematode multiplication, and disease incidence in black pepper were observed. The experiment was laid out in a completely randomized design and the treatments were M. incognita alone, F. solani alone, simultaneous inoculation of M. incognita and F. solani, M. incognita + F. solani two weeks after nematode inoculation, F. solani + M. incognita two weeks after fungus inoculation, and uninoculated control. M. incognita was inoculated @one J2 g soil-1. F. solani was mass multiplied in sand maize medium and mixed with soil (0.5% w/w). After 45 days of inoculation, 55.00 to 70.21 per cent reduction in in plant growth parameters over uninoculated control was observed in plants inoculated with M. incognita fourteen days prior to fungus inoculation. Number of galls, females, egg 119 masses and nematode population were found significantly higher in plants inoculated with nematode alone followed by M. incognita + F. solani fourteen days after nematode inoculation. Reproduction factor of nematode was higher (4.12) in the treatment where, M. incognita was inoculated alone and in all other treatments it ranged from 2.72 to 3.39. Plants inoculated with M. incognita fourteen days prior to F.solani exhibited highest disease incidence (100 per cent) and early symptom development (24 days). The results point out that the nematode infestation can predispose plants to fungal infection and thereby can increase the disease severity in field conditions. After 45 days of inoculation, histopathological and biochemical changes due to M. incognita and F. solani was analysed. Largest (909.2 µm2) and highest number (5) number of giant cells in vascular bundles was observed in plants inoculated with M. incognita alone followed by M. incognita inoculated two weeks prior to fungus inoculation. Fungal mycelium plugging in xylem vessels were observed in all the treatments in which the M. incognita and F. solani were inoculated sequentially and simultaneously. The plugging in xylem vessels were comparatively less in the treatment where F. solani was given alone, showing that presence of nematode increases the activity of fungus. Regarding biochemical changes, the highest amount of phenol was observed in leaves (0.49 mg g tissue -1) and roots (1.75 mg g tissue-1) of plants inoculated with M. incognita + F. solani fourteen days after nematode inoculation. Defence enzymes viz., peroxidase (PO) (8.93 fresh weight), phenyl alanine ammonia lyase (PAL) (16.31 µg of cinnamic acid fresh weight) and polyphenol oxidase (PPO) (6.98 fresh weight) in leaves also increased in plants inoculated with M. incognita + F. solani fourteen days after nematode inoculation. Similar trend was observed in root samples also. Higher activity of phenol and defense related enzymes can be attributed to the defense induced in plants due to the increased infection by nematode and fungus. The protein content was found higher in leaves (3.22 µg of BSA g-1 fresh weight) and roots (3.46 µg of BSA g-1 fresh weight) of the uninoculated plants compared to plants inoculated with nematode and fungus. The decrease in protein content on inoculated plants can be due to the increased use of amino acids by galls or giant cells thereby reducing their availability for protein synthesis. Investigations of the present study highlighted that plant parasitic nematodes viz. M. incognita, R. reniformis, R. similis, H. dihystera and Macroposthonia sp and fungal pathogens viz. F.solani, F. oxysporum Phytophthora sp., Pythium sp. and Rhizoctonia sp. were found associated with black pepper grown in Kannur, Idukki and Wyanad districts of Kerala. Prior inoculation of M. incognita enhanced nematode multiplication and 120 disease incidence as evidenced by increased number of giant cells, distortion of vascular bundles and plugging of fungal mycelia in xylem vessels.Item Tablet formulation of plant growth promoting microbes (PGPM) for the management of foot rot of black pepper(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2025) Hiba Abdurahiman, U.Plant pathogens and pests pose significant challenges to agricultural productivity. While synthetic agrochemicals provide rapid and reliable solutions, their excessive use has led to environmental and health concerns, driving the search for more sustainable alternatives. Biological control, an eco-friendly and cost-effective strategy, employs beneficial organisms such as plant growth promoting microbes (PGPM) to combat plant pathogens through diverse mechanisms. However, conventional formulations of biocontrol agents, including talc and liquidbased products, have limitations such as short shelf life, bulkiness, transportation difficulties, and contamination risks. Thus, the current study entitled ‘Tablet formulation of plant growth promoting microbes (PGPM) for the management of foot rot of black pepper’ is undertaken during the period from 2022-24 at Department of Plant Pathology, College of Agriculture, Vellanikkara to develop tablet formulation of plant growth promoting microbes (PGPM) and evaluate its biocontrol efficacy under in vivo conditions. The PGPM used in this study included Bacillus cereus (BPB 16) as the bacterial isolate and Trichoderma asperellum (BPT 8) and Trichoderma harzianum (CT 30) as the fungal isolates. These were characterized through cultural, morphological, and molecular methods. Media optimization was carried out by culturing the isolates in basal media - Nutrient Broth (NB) for bacteria and Potato Dextrose Broth (PDB) for fungi, supplemented with additives such as sugar sources [mannitol, trehalose], wetting agents [polyvinyl pyrrolidone (PVP), poly ethylene glycol (PEG)], adhesives [glycerol, carboxy methyl cellulose (CMC)], and surfactant (tween-80). Nine treatment combinations were tested, with population density recorded from 15 days after inoculation (DAI) to six months after inoculation (6 MAI). The treatments T4 (NB + mannitol, PEG, CMC, Tween-80) for Bacillus cereus, T3 (PDB + mannitol, PEG, glycerol, Tween-80) for Trichoderma asperellum, and T6 (PDB + trehalose, PEG, glycerol, Tween-80) for Trichoderma harzianum demonstrated the most stable population densities over six months varying from 24.67 x 1012to 18 x 108cfu ml-1, 11 x 1012 to 9.67 x 108cfu ml-1 and 40.50 x 1012 to 11.33 x 108cfu ml-1respectively. The best additive combinations for each microbe were selected for preparation of tablet formulation of PGPM. The carrier material used for preparation of tablet was talc and these tablet formulations were optimized by adjusting the carrier material's moisture content to 5 per cent and 8 per cent. Stability tests revealed that 5 per cent moisture tablets stored under refrigerated conditions had the highest population density (15.50 × 10⁸ cfu ml⁻¹), followed by those stored at ambient conditions (12.33 × 10⁸ cfu ml⁻¹) after three months of storage (3 MAS). While standardizing the recommended dosage of the formulation, it was noticed that one tablet dissolved in 100 litres of water produced population densities of 7 × 10⁶ cfu ml⁻¹ and 4 × 10⁶ cfu ml⁻¹ for 5 per cent and 8 per cent moisture tablets, respectively. The biocontrol efficacy and plant growth promotion of the PGPM tablets were assessed in a pot culture experiment against Phytophthora sp. causing foot rot disease in black pepper variety, Panniyur-1. Tablet formulations of PGPM, conventional liquid and talc formulations of Pseudomonas and Trichoderma, talc formulation of PGPM, copper oxychloride as chemical check and an absolute control were the treatments laid out in the experiment. The polybags were challenge inoculated with the pathogen after 15 days of first treatment application and on symptom appearance, treatments were applied thrice as soil and foliar application at 10 days interval. Plant growth parameters such as height, number of leaves, number of nodes of black pepper cuttings as well as disease incidence, and severity were recorded. On analysis of the biometric parameters, it was noticed that plants treated with PGPM tablet formulations exhibited the highest plant height, number of nodes and leaves and was comparable with the talc based formulation of PGPM. Likewise, observations on disease incidence and severity revealed that the tablet formulation of PGPM was superior among all treatments followed by the liquid formulation of Trichoderma and these results were comparable to the chemical check, copper oxy chloride (0.25 %). Observations on enumeration of viable count of fungi, bacteria and actinomycetes in the soil were recorded before and after treatment application by serial dilution and plating technique and it was noticed that the tablet formulation of PGPM consistently showed the highest soil microbial count. It was further noticed that the bacterial population was comparatively higher in all treatments compared to the population of fungi and actinomycetes. The study has successfully developed and standardized a protocol for producing tablet formulation of plant growth promoting microbes (PGPM) that exhibit both stability and efficacy. These tablets demonstrated the ability to retain high microbial population densities under refrigerated and ambient storage conditions for a minimum period of three months. Furthermore, the formulation has been optimized to be user-friendly, with a recommended application rate of one gram dissolved in 100 liters of water, ensuring an effective microbial concentration for practical use. To ensure wider applicability and adoption, multi-locational field trials should be conducted in different crops against various diseases to validate the findings and once validated, the PGPM tablet formulation holds the potential for commercialization as a sustainable biocontrol solution, offering farmers an ecofriendly alternative to chemical pesticides for managing plant diseases and promoting crop health.Item Bio- elicitation of secondary metabolites in callus cultures of black pepper (Piper nigrum L.)(Department of Molecular Biology and Biotechnology, College of Agriculture , Vellayani, 2024-02-21) Edupulapati Nandini; Asha, SThe present study on “Bio-elicitation of secondary metabolites in callus cultures of black pepper (Piper nigrum L)” was carried out at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, during the period from 2021-2023. The objective of the study was development of callus culture of black pepper (Piper nigrum L.) for secondary metabolite production and its bio-enhancement using derivatives of Piriformospora indica. Callus cultures were successfully initiated from the leaves and mature ripe berries of the Panniyur 1 variety. The use of 0.1% mercuric chloride for 3 minutes proved effective in achieving healthy, contamination-free callus initiation from leaf explants, while a duration of 5 minutes was found to be effective for healthy callus initiation from berry explants. Callus induction was carried out using Murashige and Skoog's (MS) medium, incorporating various combinations and concentrations of plant growth regulators (PGR). The percentage of callus induction varied depending on the type of explant and media composition used. In this study, it was observed that leaf tissue exhibited a higher responsiveness to callus induction compared to berry. Out of the four treatments, the callus induction was observed on MS media supplemented with 5.0 mg/L Napthelene Acetic Acid (NAA) and MS media supplemented with 1.0 mg/L 6benzyladenine (BA). Among these, the best response and friable callus was noted from MS media with 5.0 mg/L NAA, with 75% callus induction percentage from leaf explant within 4weeks. Better Callus proliferation with mean diameter of 1.094cm was also found in this media. Callus cultures from leaves were subjected to 24 hrs, 48 hrs and 72hrs of elicitation with Piriformospora indica cell wall extract (PiCWE) (1:1) and culture filtrate (PiCF) (1:1) and the effect of elicitation on secondary metabolite production was studied by detecting the presence of piperine by High Performance Liquid Chromatography. The maximum improvement in the accumulation of piperine was observed in callus cultures at early stages (at 24hrs) of elicitation with P. indica cell wall extract and P. indica culture filtrate. Meanwhile, P. indica CWE and CF treatment at 48hrs and 72hrs treatment was found lower performance compared with 24hrs of bioelicitation. The expression profiling of piperine synthase and Pip CoA ligase genes showed upregulation of the piperine synthase (2.49fold), and pip CoA ligase genes (63.87fold) in the 24hrs treatment with PiCF in leaf derived callus, Berry derived callus showed upregulation of piperine synthase gene with PiCWE treatment at 24 hrs with a fold change of (21.06) while pip coa ligase showed fold change of 5.13. The expression of genes were found to be decreased in later stages. The results of the study indicated that P. indica culture filtrate and cell wall extract acts as effective elicitors in boosting the production of secondary metabolites in the leaf and berry derived callus cultures of black pepper (Piper nigrum L.) respectively. But the expression of key genes and subsequent production of piperine decreased in the leaf callus, indicating the possibility of site-specific regulation of metabolites. Future studies will be directed towards the elucidation of tissue specific regulation of metabolites in black pepper.Item Plant growth promotion and foot rot disease management in black pepper by bacillus spp.(Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2022-02-11) Anju, A B; Anith, K NThe study named “Plant growth promotion and foot rot disease management in black pepper by Bacillus spp.” was conducted in Department of Agricultural Microbiology, College of Agriculture, Vellayani and Coconut Research Station, Balaramapuram, during the academic year 2019-2022. The objective of the study was to assess the effects of endospore-forming Bacillus spp. on growth and foot rot disease suppression in black pepper in the nursery. Experiments comprised both in vitro and in vivo studies. In in vitro interaction, dual culture plate assay was done to assess the antagonistic effect of bacterial isolates against P. capsici. All the three bioagents used were proven to be antagonistic to P. capsici. B. pumilus VLY17 showed the maximum zone of inhibition whereas B. amyloliquefaciens VLY27 showed the least zone of inhibition. No inhibition on mycelial growth of P. capsici was observed on treatment with culture filtrate in agar well diffusion assay. In detached leaf assay screening, significant difference was observed in lesion size developed on leaves treated with bacterial suspension compared to that on control leaves treated with sterile water. On 2DAI, leaves dipped in B. pumilus VLY17 showed the least lesion size whereas on 3 DAI, leaves applied with consortium of the bacterial isolates showed the least lesion size. Bio agents were characterized to check the production of plant growth promoting and disease suppressing metabolites. All the three isolates showed significant amount of IAA production. B. velezensis PCSE10 produced higher IAA (15.10 ppm) when estimated without adding tryptophan whereas B. amyloliquefaciens VLY27 produced maximum IAA (23.34 ppm) when medium was supplemented with L-Tryptophan. None of the strains could produce HCN under in vitro conditions. B. amyloliquefaciens VLY27 and B. velezensis PCSE10 used produced Ammonia. None of the bacterial isolates used showed siderophore production and phosphate solubilization. Bacterization with B. pumilus VLY17 showed the maximum establishment of cuttings, maximum number of leaves per plant, maximum shoot length and longest root length, maximum root volume and maximum fresh weight and dry weight of newly emerged shoot and leaves as well as roots throughout the experimental duration. Even though fresh weight of roots increased on bacterization, no significant difference was observed in statistical analysis compared to control. Plants treated with B. amyloliquefaciens VLY27 showed the maximum number of roots per plant. Suppression of Phytophthora foliar infection on bacterization was evaluated in the nursery. On analysis significant difference was observed in lesion size on treatment with bacterial suspension compared to pathogen inoculated control. The least lesion size was observed on treatment with B. pumilus VLY17. The treatments also showed significant effect on disease index. Abundance of root colonization by endospore forming bacterial isolates was checked to ascertain if the plant growth promotion as well as the disease suppression was brought about by the endospore-forming bacterial isolates. Colonization of endospore formers was found to be maximum on roots of plants treated with consortium of bacterial isolates. From the present study, it is concluded that endospore-forming bacteria used in the study have plant growth promotional activities and biocontrol potential against the pathogen P. capsici, which causes foot rot in black pepper.Item Comparative analysis of price forecasting models for black pepper(Department of Agricultural Statistics, College of Agriculture,Vellanikkara, 2024-02-17) Akshaya Ajith.; Sajitha Vijayan, M; Srinivasan, KBlack pepper, the ‘king of spices’ is one of the most popular and widely consumed spices which shares a place on most dinner tables with salt. India is the third largest producer in the world (International Pepper Community, 2023), also a significant consumer and exporter of black pepper, with Kerala and Karnataka producing the majority of the nation's output. Kerala ranked second in terms of black pepper acreage (76,160 ha), and production (33290 metric tonnes) but seventh in terms of productivity (0.44 metric tonnes/ha) (GOI, 2023). Historically, the market value of pepper contributed to the development of the city of Kochi as a centre of international commerce. Kochi has the first exclusive pepper exchange in India which was established by the Indian Pepper and Spice Traders Association, IPSTA and the exchange was well regulated by the traditional players here, without any default on supply or delivery of the commodity and volatility. As per latest records of Spices Board, the price of black pepper surpassed Rs.500/Kg which was the maximum price in the past years since 2022 in Kochi market and this indicated that black pepper prices are highly volatile. Being a perennial crop, the large variation in prices of black pepper within a year is a major problem faced by farmers as well as consumers. Hence, analysis of time series data of prices of black pepper is of prime importance. In this context, the present study was undertaken to evaluate different time series models for prices of black pepper and to suggest suitable forecast models for Kochi market. Time series data on monthly and weekly average prices of garbled and ungarbled black pepper at Kochi market from January 2000 to December 2020, collected from Spices Board, Kochi formed the database for the study. Analysis of price pattern revealed that wide fluctuation exists in the prices of black pepper in Kochi market. In order to have a general idea about trend of prices of black pepper, models like linear, exponential and quadratic, were fitted. From among several models tried, exponential model was found to be best fit for the monthly and weekly prices of both garbled and ungarbled black pepper. To examine the time series data for the price of black pepper, a multiplicative model was employed for decomposition. Seasonal indices for the 12 months from January to December was calculated for both garbled and ungarbled black pepper prices as the seasonal variation were present in monthly and weekly data. Different traditional time series models such as exponential smoothing models (single, double, Holt-Winters’ multiplicative models (HWMS)), Auto Regressive Integrated Moving Average (ARIMA), Seasonal ARIMA (SARIMA), Auto Regressive Conditional Heteroskedasticity (ARCH) and Generalized Auto Regressive Conditional Heteroskedasticity (GARCH) were applied to the price data using R software. The Augmented Dickey-Fuller test and Heteroscedasticity Lagrange’s Multiplier test were used to test the stationarity and volatility of the time-series respectively. In addition to the traditional method of price forecasting, machine learning techniques like Artificial Neural Network (ANN), and Recurrent Neural Network (RNN) were applied to the data. An artificial neural network (ANN) is a mathematical model that aims to replicate the functionality and architecture of biological neural networks. Time- delay Neural Network (TDNN) which is a major type of ANN was employed for this temporal data as it uses time delays at the input layer of the network to build a short-term memory model for forecasting the prices of black pepper. An artificial neural network with a recurrent topology is called a recurrent neural network. Long Short-Term Memory (LSTM) neural network, a specialized type of RNN, as it possesses the capability to learn patterns with long dependencies and is adept at detecting complex patterns. The price data was split into training and testing data in the ratio of 80:20. The best forecasting model was determined based on the lower values of the Root Mean Square (RMSE) and Mean Absolute Percentage Error (MAPE). The predictability performance of the selected model was also evaluated using MAPE. The HWMS model was observed as the finest among the different exponential smoothing models for this time series data on prices of garbled and ungarbled black pepper. SARIMA(2,1,2)(3,0,2)12 , SARIMA(2,1,2)(2,0,2) 12, SARIMA(2,1,2) (1,0,0)52 and SARIMA(1,1,1) (1,0,1)52 were identified best among several ARIMA models tried for monthly and weekly garbled and ungarbled black pepper respectively. The GARCH (1,1) was considered best among the different ARCH family models for this price series data. TDNN (6:2s:1l), TDNN(6:3s:1l), TDNN (13:8s:1l) and TDNN (12:7s:1l) models were found to be the pinnacle artificial neural network model with lower MAPE values 4.29, 4.63, 1.99 and 2.22 in the case of monthly and weekly prices of garbled and ungarbled black pepper respectively. The results revealed that the TDNN model showed superiority in price forecasting of black pepper in all the cases when compared with all other models. Thus, the TDNN model was used to forecast the prices from January 2021 to December 2022. The MAPE value between the actual and forecasted prices for 2021 and 2022 was 4.19 and 4.86 respectively for monthly price of garbled black pepper, while for monthly price of ungarbled black pepper it was 4.09 and 5.05 respectively. The MAPE value between the actual and forecasted prices for 2021 and 2022 was 3.11 and 3.16 respectively for weekly price of garbled black pepper, while for weekly price of ungarbled black pepper it was 3.36 and 3.58 respectively. In conclusion, the analysis suggested that the TDNN model proves to be a reliable forecasting tool for predicting prices of black pepper in the Kochi market. The robustness of the TDNN model offers a plethora of opportunities for understanding the future price pattern of black pepper which enables, various stakeholders such as producers and traders to adapt with the price fluctuations and for policymakers to ensure market stability. The TDNN model's reliability in forecasting black pepper prices not only enhances market transparency fostering overall market efficiency in India.Item Phaeophycean seaweed extracts(PSWE): exploration of its antifungal and bio-elicitor properties against major fungal diseases of black pepper(Department of Plant Pathology, College of Agriculture, Padannakkad, 2023-09-15) Abinaya, B; Sajeesh, P KThe present study entitled “Phaeophycean seaweed extracts (PSWE): Exploration of its antifungal and bio-elicitor properties against major fungal diseases of black pepper” was conducted in the Department of Plant Pathology, College of Agriculture, Padannakkad during 2021-2023. Plant samples of black pepper showing typical symptoms of foot rot and anthracnose were collected, and identified through cultural, morphological, and molecular characterizations, as Phytophthora capsici in case of foot rot and Colletotrichum sp. in case of anthracnose. Marine brown algal samples were collected, and the extracts were prepared through four different methods, including acid, alkali, solvent and hot water extraction. The antifungal activity of the phaeophycean seaweed extract (PSWE) at different concentrations was tested using the poisoned food technique (PFT). Hot water extract at 5000 ppm (PSWE (HW 5000)) was recorded as the highest inhibition (100%) of mycelial growth against P. capsici. In case of Colletotrichum sp., hot water extract at 5000 ppm exhibited 20 per cent inhibition. Further, the stability of the active principle in PSWE against high temperature and exposure to sunlight was tested using PFT and agar well diffusion assay (AWDA) against P. capsici. It was found that the active principle was degraded by the high temperature at (121℃ for 20 min.) and sunlight (min. temp. 28℃ and max. temp. 37℃). In PFT, zero per cent inhibition was recorded in high temperature, and 24.76 per cent inhibition was observed in sunlight-exposed PSWE. 17.11 per cent of mycelial inhibition was recorded when treated with high temperature, and 36.6 per cent of inhibition was recorded when exposed to sunlight. The protein and non-protein fractions of the extract were separated using the chloroform: methanol method and tested for antifungal activity. The protein fraction of the extract contributed to 40 per cent of mycelial inhibition through PFT. The molecular weight of the protein fraction was found to be more than three kDa, and the protein fraction with less than three kDa size recorded 42.5 per cent of mycelial inhibition. The size of protein fractions was analysed through SDS-PAGE as 75 kDa to 150 kDa. PSWE (HW 5000) was selected for a pot culture experiment with 12 treatments using the variety Panniyur 1 against foot rot pathogen P. capsici. The treatments were applied, and seven days after inoculation, the least per cent lesion development (PLD) of 33.33 per cent, as well as the least lesion size (3.42 cm), was recorded in PSWE (HW 5000) treatment with pre-inoculation of the pathogen through soil drenching and foliar spray. The highest PLD, 73.33 per cent, and lesion size (5.77 cm) was recorded in PSWE HW (5000) with post-inoculation of the pathogen via soil drenching. Biochemical basis of defense induction in the host plant by PSWE (HW5000) was analysed through the expression of defense-related enzymes. Application of PSWE (HW 5000) through soil drenching and foliar application with pre-inoculation of the pathogen (T5) and same treatment without pathogen inoculation (T9) expressed higher activity of defense-related enzymes such as polyphenol oxidase, peroxidase, superoxide dismutase and catalase. The presence of reactive oxygen species (ROS) was assessed using nitroblue tetrazolium (NBT) and diaminobenzidine (DAB) staining techniques. The build-up of superoxide ions and H2O2 were observed in the leaves of pathogeninoculated plants by their corresponding colour development. In both staining techniques, T5 and T6 exhibited reduced stain intensity compared to plants in control. It was found that PSWE (HW 5000) has noticeable growth promotion activity in black pepper. The growth promotion was highest in the application of PSWE (HW 5000) via soil drenching and foliar application (T9), followed by soil drenching (T7) of PSWE (HW 5000) and soil drenching and foliar application with pathogen inoculated plants (T5). The number of leaves (36.44), shoot and root length (165 and 21.83 cm), shoot and root biomass in fresh weight basis (47.66 and 14.33 g) and in dry weight basis (15.85 and 5.25 g) was higher in T9. The present study revealed that the PSWE (HW 5000) has effective inhibition activity against P. capsici and Colletotrichum sp., and it is effective in managing foot rot disease in black pepper under controlled conditions through the induction of host defense enzymes. Moreover, it enhances plant growth in black pepper. The signaling pathways associated with defense actions and growth promotion mechanisms have to be investigated.Item Efficacy of silver nanoparticles in eliminating systemic contamination in the in vitro culture of black pepper (Piper nigrum L.)(Department of Plant Biotechnology, College of Agriculture , Vellayani, 2023-04-05) Savio Franklin; Swapna AlexThe study entitled “Efficacy of silver nanoparticles in eliminating systemic contamination in the in vitro culture of black pepper (Piper nigrum L.) was carried out at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2019 to 2021. The objective of the study was to evaluate the efficacy of silver nanoparticles for eliminating the systemic contamination and improving the efficacy of micropropagation protocol in black pepper Piper nigrum L. Single nodal cuttings of Piper nigrum L. var. Panniyur 1 collected from Instructional Farm, College of Agriculture, Vellayani were used as explants. Among the five surface sterilization treatments tried with different concentrations of mercuric chloride, ethyl alcohol and pretreatments with bavistin, surface sterilization with 70 % ethyl alcohol for 1 min followed by 0.2 % mercuric chloride for 10 min showed maximum percentage of uncontaminated cultures for a period of two weeks (26.6 %), Phenolic exudation was observed in all the cultures within a period of two weeks. No contamination was observed when the uncut surface sterilized nodes were stabbed on Nutrient Agar (NA) and basal Murashige and Skoog (MS) media, thereby confirming that surface sterilization treatment was effective and contamination observed in the cut nodes after a period of two weeks was endophytic in nature. All the cultures were incubated on the illuminated racks at 25 ◦C with 16 h photoperiod. Tissue indexing by pricking the tissue with a sterile needle at three different positions (portions below medium, above the medium, and also at the tip of the shoot) followed by stabbing the needle on NA and MS media did not show any contamination. Plant tissue homogenate indexing by grinding one-gram tissue homogenate in one millilitre of sterile distilled water followed by serial dilution and plating showed the presence of 5.84 x 106 cfu in NA medium on the second day of inoculation and 4.88 x 106 cfu colonies in MS medium on the fourth day of inoculation. On medium indexing by placing the two vertical halves of the node in horizontal position on NA and MS media, endophytic contamination was observed on seventh day and fourteenth day of inoculation on NA and MS media respectively. Serial dilution of the endophytic contamination was done and the colonies picked from 10-6 dilution were used for bacterial in vitro inhibition assay. In vitro inhibition assay was carried out using sterile discs loaded with different concentrations (10 mgL-1 and 20 mgL-1) of 20 nm and 100 nm silver nanoparticles alone and in combination with streptopencillin, 150 mgL-1 of streptopenicillin alone, and sterile distilled water as control. No inhibition zone was observed on treatments with silver nanoparticles alone whereas an inhibition zone of 28 ± 2 mm was observed for streptopenicillin (150 mgL-1), 21 ± 1 mm for streptopenicillin with 20 nm silver nanoparticles and 18 ± 1mm for combination of streptopenicillin with 100 nm silver nanoparticles. To conclude, surface sterilization with 70 % alcohol for 1 min followed by 0.2 % mercuric chloride for 10 min was effective in controlling surface contaminants in black pepper var. Panniyur 1. Endophytic contaminants could not be effectively removed by 20 nm and 100 nm of silver nanoparticles at concentrations of 10 and 20 mgL-1 and hence is not recommended as an alternative to incorporation of 150 mgL-1 of streptopencillin in controlling the systemic contamination of black pepper in tissue culture.Item Pink pigmented facultative methylotrophs (PPFMs) from black pepper (Piper nigrum L.) for drought stress mitigation and plant growth promotion(Department of Agricultural Microbiology, College of Agriculture,Vellayani, 2024-04-04) Yarlagadda Manju Bhargavi.; Anu Rajan, SThe study entitled “Pink Pigmented Facultative Methylotrophs (PPFMs) from black pepper (Piper nigrum L.) for drought stress mitigation and plant growth promotion" was carried out during the period from 2021-2023 in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram, with the objective to isolate, screen and characterize Pink Pigmented Facultative Methylotrophs (PPFMs) for plant growth promotion and abiotic stress mitigation with special emphasis on drought management in black pepper (Piper nigrum L.) under Kerala conditions. The study comprised an in vitro screening experiment and a pot culture experiment with black pepper variety Karimunda. A total of 60 PPFM cultures were isolated from phyllosphere and rhizosphere by leaf imprint method and serial dilution and plating method on Ammonium Mineral Salt (AMS) agar medium supplemented with 0.5% methanol and 10 µg cycloheximide. These isolates were tentatively identified as PPFMs based on the characteristic pink pigmented colonies. Characterization of the isolates by colony morphology, gram reaction and biochemical tests were carried out. 30 PPFM isolates were selected for in vitro screening of PPFM isolates for plant growth promotion. IAA production by the different isolates of methylotrophs showed wide variations ranging from 10.02 to 51.25 µg mL-1 culture filtrate (without tryptophan) and from 10.06 to 60.00 µg mL-1 culture filtrate (with tryptophan). Maximum IAA production of 51.25 µg mL-1 and 60.00 µg mL-1 culture filtrate (without and with tryptophan, respectively) was recorded by PNPPFM 59. GA production by different isolates were ranging from 29.19 to 1.81 µg mL-1. Maximum GA production of 29.19 µg mL-1 was recorded by PNPPFM 60. Extracellular ammonia production by the different isolates of methylotrophs ranged from 1.43 to 7.07 µmol mL-1. Maximum extracellular ammonia production of 7.07 µmol mL-1 was recorded by PNPPFM 59. ACC deaminase activity production by different isolates were ranging from 0.89 to 13.94 µg mL-1. Maximum ACC deaminase activity production of 13.94 µg mL-1 was recorded by PNPPFM 60. For the assessment of drought tolerance potential, these isolates were grown in different concentrations of PEG 6000 viz. 0%, 5%, 10%, 20%, 30% and 40%. All the cultures were able to tolerate up to 30% concentration of PEG 6000. Index ranking was done to select the better performing isolates for the subsequent experiments. Consequently PNPPFM 60, PNPPFM 59 and PNPPFM 44 were selected for the pot culture experiment. The pot culture experiment was undertaken with black pepper cuttings to study the effect of PPFM isolates on plant growth promotion and drought tolerance. The experiment was laid out in CRD with 7 treatments and three replications. The treatments comprised four PPFM isolates (PNPPFM 60, PNPPFM 59, PNPPFM 44 and Ref. culture PPFM 38) and three control treatments (sterile water, uninoculated media and absolute control). These treatments were given as cutting dipping and foliar spray at 45, 75 and 90 DAP. The study revealed that PPFM isolates had significant effect on growth and establishment of black pepper cuttings. Maximum number of leaves (4.66), shoot length (28.30 cm), root length (17.70 cm), number of roots (14.66 and 138.33, primary and secondary roots respectively), fresh weight of roots (1.01 g), dry weight of roots (0.4 mg), fresh weight of newly emerged shoots and leaves (10.74 g) and dry weight of newly emerged shoots and leaves (2.96 g) were recorded with PNPPFM 60 and least was recorded with either one or more of controls. Cell membrane integrity and relative water content were highest with PNPPFM 60 on 4th as well as on 14th day. Highest chlorophyll stability index on 14th day was recorded with PNPPFM 60. Proline content (78.67 µg g-1 tissues), super oxide dismutase activity (0.45 activity g-1 min-1), catalase activity (18.48 activity g-1 tissues), peroxidase activity (47.18 activity g-1 min-1) and malondialdehyde (0.54 µmol g-1 tissues) were highest with PNPPFM 60. Considering the major drought tolerance parameters such as proline content, SOD, catalase, peroxidase and malondialdehyde, PNPPFM 60 was the best among the PNPPFM isolates tested in the pot culture experiment. The present study revealed that the isolate PNPPFM 60 (cutting dip – 1% PPFM culture broth and foliar spray – 1% culture broth at 45, 75 and 90 DAT) were effective in improving the growth parameters and drought tolerance in black pepper.Item Trader preferences based on primary product attributes of black pepper in Kerala: an empirical study(Department of Agricultural Extension Education, College of Agriculture, Vellayani, 2024-12-06) Yaman Chadar; Smija, P KFacilitating integration of primary traders into modern agricultural value chains, known as High-Value Markets (HVMs), presents a promising avenue for improving the sustainability of value chains of black pepper in Kerala. The study entitled “Trader preferences based on primary product attributes of black pepper in Kerala: an empirical study” was undertaken during 2023-24. Analysing the criteria employed by primary level traders for sourcing black pepper and their willingness to participate in high value markets; estimating attribute utility; socioeconomic profiling and constraint analysis of primary level black pepper traders in entering high value markets were the primary objectives of the study. The study area selection focused on the importance of Idukki and Wayanad districts in Kerala's black pepper production. Eight Agro-Ecological Units (AEUs), specifically 12, 14, 15, 16, 17, 19, 20, and 21 were purposively chosen for their extensive pepper cultivation, and 120 primary traders were randomly selected for the research. The survey employed Best-Worst Scaling (BWS) choice experiment in selected AEUs to comprehend the preferences of respondents regarding quality attributes that could promote sustained participation in HVMs. The choice experiment used fractional factorial design to create multiple profiles and balanced incomplete block design to create the survey questions. The results indicated that traders predominantly favour a flexible incentive-based pricing model and long-term formal relationships with buyers. Profile 1(farm owner, spot price, place of origin certificate and sun drying) stands out with a notably high mean BWS score of 3.37. This indicates that it possesses a highly desirable attribute that strongly appeals to respondents, consistently receiving positive evaluations. In addition to Profile 1, Profiles 9 (FPOs, fixed premium price, organic certificate and sun drying), Profile 4 (Absentee owner, spot price, organic certificate and blanching and machine drying), and Profile 3(farm owner, fixed premium price, fair trade and blanching and machine drying) also demonstrated positive BWS scores indicating their effective participation in HVMs. Profile 2 (farm owner, seasonal highest price, organic certificate and machine drying) is the least preferred with BWS score of -2.92. A utility function was developed for the purpose of fitting a conditional logistic model to the attribute level responses. The highly significant coefficient (𝛽1) of 1.10 associated with attribute level D1 (sun drying) indicates a substantial positive impact on preference followed by B1(spot price), D3 (blanching and machine drying) and B3 (fixed premium price). In contrast, attribute levels A1 (farm owner), A3 (FPOs), C2 (fair trade certificate) and C3 (organic certificate) demonstrate significant negative effects on preference with odds ratio less than 1(one) when compared to reference level, indicating that respondents are less inclined to favour these levels compared to the reference level. A questionnaire-based survey was also conducted to throw light on the factors affecting willingness of primary traders to participate (WTP) in HVMs. A percentage analysis of the same showed that the majority (63.33%) of traders were open to participating, while (36.67%) were hesitant. The logistic regression analysis demonstrated that variables such as digital literacy, the number of transactions involving the commodity (green/black/white pepper), and the extent of value chain linkages (intra-state, inter-state, and international) are influencing the WTP in HVMs. Primary traders in the black pepper and spice markets view high market volatility and risk exposure as the major constraints in entering HVMs followed by delayed payment, low production, inadequate financial support from the government for black pepper and other spice crops, insufficient quality testing centres in key production areas, lack of proper machinery for threshing, cleaning, sorting and drying, lack of organised commodity specific associations for primary traders, and lastly lack of proper market information. The research indicated that primary traders have a limited understanding of the factors influencing their entry into HVMs. Focused training in responsible sourcing, international standards, post-harvest handling, branding, data skills, risk management, and group dynamics will equip stakeholders with essential competencies. A pilot project on digital transformation will further help evaluate effective models for industry advancement. It is also crucial to implement robust local audit systems and origin certification, supported by collaborations with private labs and NGOs. Strengthening trader associations, and distinguishing between bulk and direct consumption markets will elevate domestic quality control. Prioritizing participatory research with farmers and traders to develop affordable quality testing, small-scale threshers, and advanced dryers will enhance post-harvest processing and will do wonders in lifting the HVMs of black pepper in Kerala.