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 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 Isolation, cloning and molecular characterization of defensin gene(s) in black pepper (Piper nigrum L.)(Department of Molecular Biology and Biotechnology, College of Agriculture ,Vellayani, 2023-03-22) Abirami, G; Anuradha, TPlant defensins are cationic antimicrobial peptides that function in the innate immune response in plants. Many plant defensins having antimicrobial activity are molecularly characterized in different plants. However, defensin genes present in black pepper are not completely characterized. The study entitled “Isolation, cloning and molecular characterization of defensin gene(s) in black pepper (Piper nigrum L.)” was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture Vellayani, Thiruvananthapuram during 2021-2022. The objective of this study was isolation, cloning and molecular characterization of antimicrobial peptide defensin gene(s) from black pepper variety Panniyur 1. In this study, the full-length gene sequence of a novel defensin gene termed PnDef was isolated for the first time from black pepper var. Panniyur 1 using gene specific primers. The full length of PnDef gene is found to be 356bp. The coding sequences of PnDef was deduced using Genscan software, which was found to contain 231bp open reading frame encoding a protein of 76 amino acid residues. The blast analysis of PnDef amino acid sequence showed 72.60% similarity with the defensin protein of Pyrus ussuriensis × Pyrus communis. The phylogenetic analysis showed a close relationship of PnDef to defensin protein from Rosaceae family. The clustal analysis of PnDef revealed eight conserved cysteine residues in similar positions. The putative PnDef mature protein showed striking similarity with other plant defensins representing a small molecular weight of 5.213 kDa, theoretical isoelectric point (pI) of 8.95 with an aliphatic index and GRAVY of 60.26 and 0.154 respectively. It has a potential 29 residue signal peptide and the remaining 47 residues belong to the mature peptide region. The secondary structure analysis of PnDef revealed that it has 46% alpha helix and 25% of β-sheet structures. The three-dimensional structure modeling of PnDef showed a striking resemblance (81%) with the antifungal defensin of Medicago trancatula implying it may have similar antimicrobial activity. PnDef was found to have three conserved disulfide bonds formed between six cysteine residues. In silico analysis of the PnDef indicated antibacterial, antiviral and antifungal properties of the peptide.Using the sequence data identified in this study one can further go for expression profiling of black pepper defensin in different plant parts under biotic and abiotic stress conditions, recombinant protein can be isolated by inserting into the expression vector and using the recombinant protein one can identify the inhibition activity of the protein against plant pathogens under in vitro studies. Molecular docking can also be done prior to the in vitro studies using the 3D structure of black pepper defensin to identify potential pathogen targets.Item Management of a major fungal disease in black pepper (Piper nigrum L.)(Department of Plant Pathology, College of Agriculture,Padanakkad, 2022) Pranavya, A P; Sajeesh P KBlack pepper (Piper nigrum L.) is one of the major spice crops and plantation crops that grow as the sole crop or intercrop in Kerala. In nurseries of black pepper major biotic stress are caused by bacteria, fungus, virus and nematode, out of this fungal infection are the leading one. The seedling blight, anthracnose and collar rot were found to be more severe in the nursery. With this background, the present study was conducted to identify the major fungal pathogen associated with black pepper nursery and formulate disease management strategies against that pathogen. A survey was carried out in black pepper growing nurseries to record the different diseases. Seedling blight, anthracnose and collar rot were observed. The fungal isolates associated with seedling blight in nurseries at Padannakkad and Kanakappalli were designated as Pc1 and Pc2 respectively. Anthracnose affected seedlings were collected from Padannkkad, Balal, Panniyur and Periya black pepper nurseries and the fungal isolates associated were designated as Cg1, Cg2, Cg3 and Cg4 respectively. The collar rot samples were collected from Padannakkad, Periya and Panniyur and isolates were designated as Sr1, Sr2 and Sr3. In the case of seedling blight infection, highest disease incidence (DI) (74%) was recorded in Pc1. The highest percent disease index (PDI) in case seedling blight (32%) was recorded in Pc1 as compared to Pc2 isolate. In the case of anthracnose, higher DI (30%) and PDI (25%) were recorded in Cg1 in comparison with Cg2, Cg3 and Cg4. In case of collar rot, higher DI (20%) was recorded in case of Sr1 isolate. All the isolates were tested for its pathogenicity; in case of seedling blight, the isolate Pc1 recorded higher radial growth (7.90 cm) and recorded lesser time (2days) for the symptom development as compared to Pc2 isolate. In case of anthracnose, isolate Cg1 recorded higher radial growth (8.0 cm) and took lesser time (5days) for the symptom development in comparison with Cg2, Cg3 and Cg4. In case of collar rot isolate Sr1 recorded higher radial growth (8.96 cm) and recorded lesser time (2days) for the symptom development as compared to Sr2 and Sr3. A trial was laid out to identify the most susceptible variety of black pepper as well as the most virulent strain of the pathogen among different isolates on pepper varieties viz., Panniyur (P) 1-6, P8, P9, Vijay and Karimunda. Panniyur 2 was identified as most susceptible variety and Pc1 was the virulent isolate. Based on cultural and morphological characters and molecular characterization, Pc1 was identified as Phytophthora capsici. In vitro evaluation of biocontrol agents, chemical fungicides and organic preparations were carried out to study the efficacy against P. capsici. Under in vitro conditions biocontrol agent T. harzianum (2%) exhibited maximum inhibition (55.56%) followed by P. fluorescens (53.33%). Among the chemical fungicides copper oxychloride (0.3%) and metalaxyl (8%) + mancozeb (64%) at 0.3 percent recorded 100 per cent inhibition. Among the organic preparations, ready to use neem oil garlic soap (3%) and garlic extract at 0.3 percent recorded maximum inhibition 31.11% percent and 28.88% percent respectively in the mycelial growth of the pathogen. The best-performing treatments from the in vitro evaluation were selected for pot culture experiment on variety Panniyur 2. After 7 days of inoculation, the least PDI (0.00 %) as well as the least lesion development (0.00%) was recorded in case of metalaxyl (8%) + mancozeb (64%) at 0.6 per cent. The higher PDI (86.66%), as well as maximum lesion development (46.66%), was recorded in ready to use neem oil garlic soap (3%). The study reveals that, the seedling blight caused by Phytophthora capsici can be effectively managed with the foliar application of metalaxyl (8%) + mancozeb (64%) at 0.6 per cent.Item Characterization and quality analysis of black pepper (Piper nigrum L.) genotypes of Kerala(Department of Plantation Crops and Spices, College of Agriculture, Vellayani, 2021) Reshma, P; Sreekala, G SThe present study entitled “Characterization and quality analysis of black pepper (Piper nigrum L.) genotypes of Kerala” was taken up with the objectives of survey, characterization and quality analysis of black pepper genotypes of Kerala based on morphological and biochemical parameters. The survey conducted in black pepper plantations and homesteads in fourteen locations of six Agro-Ecological Units (AEUs) identified twenty one genotypes. Five genotypes designated as G1 (Wayanadan), G2 (Chumala), G3 (Vellayaranmunda), G4 (Nadan), G5 (Nadeshan) from AEU 21 (Wayanad Eastern Plateau), six genotypes designated as G6 (Karimunda), G7 (Neelamundi), G8 (Cheppukulamundi), G9 (Vattamundi), G10 (Thulamundi), G11 (Manjamunda) from AEU 12 (Southern and Central Foothills), three genotypes designated as G12 (Chengannurkodi), G13 (Vellanamban), G14 (Jeerakamunda) from AEU 14 (Southern High Hills), three genotypes designated as G15 (Kuthiravally), G16 (Kottanadan), G17 (Arimulak) from AEU 3 (Onattukara Sandy Plains), two genotypes designated as G18 (Padappan), G19 (Karivilanchy) from AEU 8 (Southern Laterites) and two genotypes designated as G20 (Narayakodi) and G21 (Panniyur 1) from AEU 4 (Kuttanad) were selected for the study. Twenty one genotypes were morphologically characterized for both qualitative and quantitative characters. Thirty four qualitative characters were observed and among those only twenty two characters showed variability. Characters such as runner shoot production, pubescence on stem, type of veining, leaf texture, spike colour, spike orientation, spike shape, spike fragrance, spike proliferation, colour change while fruit ripening, fruit taste and seed texture were uniform in all the selected genotypes. The UPGMA dendrogram divided all the genotypes into 15 clusters at 73 per cent similarity. Sensory evaluation of the selected black pepper genotypes showed a significant variation for colour, odour, taste and flavour. G16 (Kottanadan) was significantly superior in taste while G15 (Kuthiravally) was superior in odour and flavour. Thirty four quantitative characters were recorded for plant, leaf, inflorescence, fruit and seed characters. Summarizing these quantitative characters using descriptive statistics revealed wide range of variability in number of well developed fruits per spike, number of spikes per 30 cm2 , number of spikes per vine, bulk density and specific leaf area. Principal component analysis was undertaken to examine the variation and to estimate the relative contribution of various trait for total variability. The principal component analysis for twenty three yield related characters revealed seven principal components at 85.53 per cent variability. The eleven quantitative characters such as juvenile leaf length, leaf length, leaf width, number of spikes/30 cm2 , number of spikes per lateral branch, number of spikes per vine, number of nodes per lateral branch, number of well developed berries per spike, berry diameter, support height and vine column diameter contributed more to the yield. The score plot identified fifteen clusters in which genotypes with superior yield namely, G1 (Wayanadan), G4 (Nadeshan), G5 (Nadan), G15 (Kuthiravally) and G21 (Panniyur 1) formed unique clusters. Minimal data set generated for black pepper included four characters namely, number of nodes per lateral, number of well developed berries/spike, number of spikes/30 cm2 and berry diameter. The principal component analysis for five physiological parameters revealed one principal component at 73.34 per cent variability. Genotypes with high leaf thickness, high relative water content, high epicuticular wax, low specific leaf area and low stomatal density were associated with drought tolerance in black pepper. Sixteen clusters were identified based on score plot. Biplot analysis identified genotypes G16 (Kottanadan), G13 (Vellanamban), G6 (Karimunda), G7 (Neelamundi), G18 (Padappan) and G20 (Narayakodi) as most desirable genotypes for drought tolerance. The principal component analysis for six quality attributes revealed two principal components at 66.62 per cent variability. The genotypes were grouped into seventeen clusters based on score plot while the biplot identified genotypes G16 (Kottandan), G15 (Kuthiravally), G21 (Panniyur 1), G19 (Karivilancy), G17 (Arimulak), G13 (Vellanamban) and G6 (Karimunda) as the high quality ones. The genotypes studied for qualitative, quantitative, physiological and quality traits showed a moderate variability and can be used in the selection of suitable parents for breeding purpose and gene mapping studies.