1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Evaluation of plant growth promoting rhizobacteria for pokkali rice(Department of Agricultural Microbiology, College of Agriculture,Vellanikkara, 2023-02-17) Bony Cyriac.; Surendra Gopal, KPokkali rice farming is a time-honoured, environmentally beneficial, traditional and organic method of growing rice. Neither chemical fertilizers nor plant protection agents are used on the crop in this conventional rice farming technique. The present study was undertaken to evaluate a native plant growth promoting rhizobacteria (PGPR) consortia with an objective to screen PGPR for functional efficiency under in vitro and evaluate for plant growth promotion in Pokkali rice. Isolation, characterization and screening of plant growth promoting rhizobacteria such as nitrogen fixers, phosphate solubilizers, potassium solubilizers, fluorescent pseudomonads and Bacillus sp. were carried out from three Pokkali rice growing regions of Ernakulam district (Vytilla, Varapuzha and Kadamakudy). Maximum population of 4 x 103 , 4.66 x 103 and 16.67 x 103 cfu per g of soil were recorded for nitrogen fixers, phosphate solubilizers and Bacillus sp. respectively and fluorescent pseudomonads and potassium solubilizers were absent. A total of 25 predominant isolates were obtained of which 17 isolates belonged to Bacillus sp. and remaining 4 isolates each were nitrogen fixers and phosphate solubilizers. Cultural, morphological and biochemical characters of all the 25 isolates of rhizobacteria showed varied colony morphology and Gram reaction. These isolates were evaluated under in vitro for plant growth promotion activities (production of indole acetic acid, nitrogen fixation, phosphate and potassium solubilization), antagonistic mechanisms (NH3, HCN and siderophore production) and its activities against Xanthomonas oryzae. Quantity of IAA produced by rhizobacterial isolates ranged from 0.46 to 31.27 μg ml-1. Quantity of nitrogen fixed and phosphate solubilized by rhizobacterial isolates varied from 5.72 to 25.21 mg of N g-1 sucrose utilized and 1.98 to 53.19 μg ml-1, respectively. There was no significant difference between potassium solubilized by isolates. Among the rhizobacterial isolates all Bacillus sp. isolates and two isolates each of nitrogen fixers and phosphate solubilizers were found positive for ammonia production. In case of screening of isolates for siderophore production, isolate VYP1 produced moderate orange halo zone on CAS agar media. Isolates VYN1, VYN2, VRB1 and VRB3 recorded poor siderophore activity. Remaining all isolates in the study screened negative for siderophore production. All isolates in the study were found negative for HCN production. Antagonistic activities of isolates to inhibit the rice pathogen Xanthomonas oryzae was evaluated and twenty one isolates screened exhibited antagonistic response against pathogen in which Bacillus isolates KDB4 and KDB5 exhibited excellent antagonism against pathogen. Based on the plant growth promotion activities and antagonistic activities, four most efficient isolates each of nitrogen fixers, phosphate solubilizers and Bacillus sp. were selected for salinity tolerance studies. Among the isolates, nitrogen fixers and Bacillus sp. exhibited maximum salinity tolerance of 12% whereas, phosphate solubilizers exhibited salinity tolerance up to 4% salt concentration. After salinity tolerance studies, three most tolerant nitrogen fixers (VYN2, KDN1 and KDN2), phosphate solubilizers (VYP1, VRP1 and KDP1), and Bacillus sp. (VYB3, VYB6 and KDB1) were selected for compatibility studies. Three most promising PGPR based consortia, PGPR consortia1 (KDN2 + KDP1 + VYB6), PGPR consortia-2 (KDN1 + KDP1 + VYB6) and PGPR consortia-3 (KDN2 + VYP1 + KDB1) were formulated. The three most promising consortia were evaluated under pot culture experiment, along with KAU commercial formulation (PGPR Mix-1) and Organic Package of Practices Recommendations of KAU (2017), at RRS, Vytilla, with Pokkali rice (variety Vytilla-6) as the test crop. All the three native PGPR consortia (T1, T2 and T3) exhibited significant increase in plant height and plants treated with T1 (PGPR consortia 1- KDN2 + KDP1 + VYB6) and T2 (PGPR consortia 2 - KDN1 + KDP1 + VYB6) recorded significantly higher results. Higher number of grains per panicle, grain yield (g per pot) and theoretical grain yield (kg ha-1) were observed in PGPR consortia 1 consisting of isolates KDN2 (N-fixer) + KDP1 (P- solubilizer) + VYB6 (Bacillus sp.). Significantly higher test weight of seeds were observed in T1, T2 and T3 inoculated with native PGPR consortia compared to other treatments. However, the treatments did not have any significant effect on number of tillers, number of panicles per plant and the percentage of chaffy grains produced. The present study indicated that saline tolerant PGPR consortia-1 consisting of Enterobacter cloacae strain KDN2 (N-fixer) + Bacillus subtilis strain KDP1 (Psolubilizer) + Bacillus altitudinis strain VYB1 (Bacillus sp.) was the most promising biofertilizer in enhancing the production of Pokkali rice of Kerala. However, further evaluation of the most promising consortia needs to be done under field conditions to confirm the results.Item Seed pre-treatment with endospore -forming rhizobacteria in Kiriyathu (Andrographis paniculata (Burm.f.) wall. Nees) for enhancing growth and quality(Department of Plantation Spices Medicinal and Aromatic Crops, College of Agriculture ,Vellayani, 2024-01-30) Anu ,Ambadi; Sonia, N SThe study entitled “Seed pre-treatment with endospore-forming rhizobacteria in Kiriyathu (Andrographis paniculata (Burm. f.) Wall. ex. Nees) for enhancing growth and quality” was carried out at the Department of Plantation, Spices, Medicinal and Aromatic Crops, College of Agriculture, Vellayani, Thiruvananthapuram, Kerala during 2021-2023 with the objectives to evaluate the effect of seed treatment with endospore-forming rhizobacteria on seed storage; seed germination; growth promotion and secondary metabolites production in Kiriyathu. The seed of A. paniculata variety Anand Kalmegh 1 (AK-1) was pre-treated with bacterial cultures viz., B. pumilus VLY 17 (T1), B. amyloliquefaciens VLY 24 (T2), B. velezensis PCSE 10 (T3) and bacterial consortium [B. pumilus VLY17+ B. amyloliquefaciens VLY 24+ B. velezensis PCSE 10] (T4) at 1014 cfu mL-1. Seed without bacterial pre-treatment was served as the control (T5). The treated seeds were evaluated during seed storage for bacterial survival. Moreover, the pre-treated seeds were evaluated for seed germination characteristics and planted in polybags for assessing growth and quality. All bacteria species as well as its combination found to survive in the pre-treated seed up to six months after storage. The bacterial count was found to be highest for B. pumilus VLY 17 during 6 MAS (6.66 log cfu g-1). Germination characteristics of bacteria pre-treated seed were found to be superior than the control seed. The Kiriyathu seed pre-treated with B. pumilus VLY 17 exhibited the highest germination percentage (85.00 %), survival percentage (85.00 %), germination index (2.60), shoot length (6.54 cm), root length (5.04 cm), seed vigour index (926.60), allometric index (0.77) and lower mean germination time (3.30 days). Seed pre-treatment with bacteria showed significant improvement in plant growth characteristics viz., morphological, phenological and yield parameters during harvest at 120 DAS compared to the control. B. pumilus VLY 17 pre-treated seed recorded the highest plant height (83.03 cm), number of branches (33.42), number of nodes (472.44), number of flowers (104.00), shoot weight (fresh-120.27 g, dry-38.81 g), whole plant weight (fresh -138.21 g, dry -43.02 g), root length (29.29 cm), seed yield (3.43 g) and thousand seed weight (2.22 g). Also, B. pumilus VLY 17 pre-treatment took lesser days to flower initiation (95.88 days), days to fruit set (16.06 days) and days to fruit maturity (28.25 days) as well as the highest number of fruits (309.44) and fruit weight (fresh 13.54 g). Highest root dry weight (4.01 g) and fruit dry weight (3.95 g) were in combination treatment and B. velezensis PCSE 10 respectively. Seed pre-treatment showed no significant difference for stem girth, root fresh weight and root volume of Kiriyathu plants. Phytochemicals viz., total alkaloids, total flavonoid content, total phenol content, total saponins and cardiac glycosides were found to be superior in Kiriyathu herbage (ethanolic extract) of bacteria pre-treated seed than the control. B. velezensis PCSE 10 pre-treatment recorded highest total alkaloid content, 78.47 µg AE mg-1 whereas highest flavonoid (37.62 µg QE mg-1) and phenol content (349.92 µg GAE mg-1) were observed by B. pumilus VLY 17 treatment. Total saponins content (140.45 µg DE mg-1) was found highest in bacterial combination while the highest cardiac glycosides (169.33 µg DE mg-1) were present in B. amyloliquefaciens VLY 24 treatment. Pharmacological properties of the Kiriyathu herbage ethanolic extract viz., in vitro antioxidant activity and cytotoxicity against liver cancer cell line (HepG2) were highest in B. pumilus VLY 17 seed pre-treatment, 94.00 % DPPH free radicle inhibition and IC50 value, 50.09 µg mL-1 respectively. Kiriyathu seed could be pre-treated with B. pumilus VLY 17 as a liquid spray at 1014 cfu mL-1 concentration for ensuring enhanced seed germination, plant growth, phytochemical quality and in vitro pharmacological properties. The bacteria survival in the pre-treated seed is ensured up to six months after storage under ambient condition.Item Characterization and evaluation of plant growth promoting rhizobacteria from rice soils of Wayanad(Department of Agricultural Microbiology, College of Agriculture, Vellanikkara, 2021) Wickramasinghe, W R K D W K V; Girija, DPlant growth promoting rhizobacteria (PGPR) are a group of bacteria that colonize the plant rhizosphere and enhance the growth and yield of plants. The present investigation entitled “Characterization and evaluation of plant growth promoting rhizobacteria from rice soils of Wayanad” was undertaken at the Department of Agricultural Microbiology” during the year 2018-2020, with the objective of isolation, characterization and evaluation of plant growth promoting rhizobacteria from rice soils of Wayanad and formulation of a consortium to improve the growth and yield of rice. Isolation of rhizobacteria with potential plant growth promoting (PGP) activities was attempted from rice rhizosphere soils collected from ten locations in Wayanad district of Kerala. Selective media were used for the isolation of PGPRs including nitrogen fixers, solubilizers of phosphate, K and Zn and fluorescent pseudomonads. A total of 149 isolates obtained on different media were subjected to preliminary screening for growth on selective media, which yielded 32 N-fixers, 16 phosphate solubilizers, four K solubilizers, six Zn solubilizers and two fluorescent pseudomonads. These isolates were evaluated in vitro for PGP activities (production of IAA, NH3, HCN and siderophore) and antagonistic activities against R. solani and X. oryzae. Twenty promising isolates were selected based on their functional efficiency for further characterization using cultural, morphological, biochemical and molecular methods. Four isolates were found to be Gram-positive rods and sixteen isolates were Gram-negative short rods. Eighteen isolates were identified based 16S rRNA gene sequencing and the sequences of all the eighteen isolates deposited in the GenBank of the NCBI. Phylogenetic analysis using MEGA 7 software showed two major clusters and several sub-clusters. A few of the native isolates stood out distinctly from the available accessions in the database, showing that they are genetically diverse. Based on the efficiency of N fixation, P, K and Zn solubilization and other PGP activities, isolates were ranked. Based on ranking, three N-fixers (Bacillus sp. AkNF3, Pseudomonas sp. PkNF4 and Pseudomonas putida KgNF1), three phosphate solubilizers (Bacillus megaterium PkPS1, Acinetobacter schindleri AkPS4 and Achromobacter sp. AvPS1), two K-solubilizers (Microbacterium sp. MvKS1 and Acinetobacter calcoaceticus MvKS3) and two zinc solubilizers (Achromobacter marplatensis ThZnS2 and Cytobacillus kochii PkZnS3) were selected for consortial formulation. Compatibility of ten promising isolates was tested by cross streaking and dual culture methods. Three PGPR based consortia (Consortium 1, 2 and 3) were formulated, each consisting of 5 native isolates (two N-fixers, one each of phosphate, K and Zn solubilizers). These consortia were evaluated in pot culture experiment, along with KAU commercial formulation (PGPR mix-1), at RARS, Ambalavayal, with rice (variety Valichoori) as the test crop. PGPR application was combined with two levels (50% and 75%) of recommended dosage of inorganic fertilizers (RDF). Population of total bacteria, N fixers, P, K and Zn solubilizers was higher in combined application of biofertilizer with inorganic fertilizers than uninoculated treatments and this was indicative of better colonization of native PGPRs in the rice rhizosphere. Growth and yield parameters indicated that application of PGPR consortium with 75% RDF was statistically on par with PoP (KAU) and 100% RDF. Results suggested that 25% inorganic N, P and K can be replaced by using native PGPR consortium without affecting plant growth, yield, plant nutrient content and soil nutrient content. Considering the above parameters, two best consortia (Consortium 2 and Consortium 3) were selected for further field evaluation. Field evaluation was carried out to assess the efficiency of two selected native PGPR consortia at RARS, Ambalavayal. Five treatments included were, consortium 2 + 75% RDF, consortium 3 + 75% RDF, reference biofertilizer PGPR mix-1 + 75% RDF, 100% RDF and farmer’s practice (farm yard manure 5t ha-1 ). Results suggested that root colonization of total bacteria, N fixers, P, K and Zn solubilizers was higher in all treatments of combined application of biofertilizers with 75% inorganic fertilizer than 100% RDF alone. Growth and yield parameters suggested that combined application of Consortium 2 with 75 % RDF was statistically on par with 100% RDF. Therefore, it can be concluded native PGPR strains in consortium 2 (Bacillus sp. strain AkNF3, Pseudomonas putida strain KgNF1, Bacillus megaterium strain PkPS1, Acinetobacter calcoaceticus strain MvKS3 and Cytobacillus kochii PkZnS3) successfully colonized the rice rhizosphere, increased nutrient availability to the plants and produced higher yield. The results also emphasized on the importance of exploiting native, location specific microorganisms as biofertilizer consortium, rather than a common consortium for the entire State. Native PGPR based consortia 2 reduced the 25% of inorganic fertilizer (N, P and K) without affecting the growth and yield of rice. This would be more cost effective and ecofriendly when compared with the use of chemical fertilizers alone. Further multi-locational field trials are required to validate the results before commercialization of this consortium, as a biofertilizer.Item Management of bacterial wilt disease of tomato by the root endophytic fungus piriformospora indica, rhizobacteria and bacterial endophytes(Department of Agricultural Microbiology, College of Agriculture, Vellayani,Thiruvananthapuram, 2018) Athira, S; Anith, K NThe study entitled “Management of bacterial wilt disease of tomato by the root endophytic fungus Piriformospora indica, rhizobacteria and bacterial endophytes” was conducted during 2016-2018 at Department of Agricultural Microbiology, College of Agriculture, Vellayani with the objective of assessing the potential of root endophytic fungus Piriformospora indica, plant growth-promoting rhizobacteria and bacterial endophytes in suppressing bacterial wilt incidence in tomato. The bacterial wilt pathogen, Ralstonia solanacearum was isolated from infected tomato plants on Semi selective medium from South Africa (SMSA). Koch’s postulates were proved by artificial inoculation of tomato seedlings. A functional relationship between population and optical density of the broth culture was worked out and was used for determining the inoculum density for challenge inoculation in the biocontrol experiment. In vitro antagonistic interaction between the bioagents and the pathogen was worked out both by direct and indirect assays. In all direct assays which involved cross streak plating, agar plug diffusion technique, disc diffusion and spot on lawn method, it was found that Bacillus amyloliquefaciens VLY24, Bacillus velezensis PCSE10 and Streptomyces leeuwenhoekii KBT004 exhibited antagonism against Ralstonia solanacearum. Indirect antagonism was checked by agar well diffusion and disc diffusion methods using culture filtrate. Out of the eight bacterial bioagents tested, only two i.e., Bacillus velezensis PCSE10 and Bacillus amyloliquefaciensVLY24 had inhibitory effect on the pathogen. Dual culture plate assay on PDA has shown that three bacterial bioagents, Rhizobium radiobacter PCRE10, Bacillus megaterium NAT001 and Streptomyces leeuwenhoekii KBT004 were compatible with Piriformospora indica. However, when the compatible bacteria were co-cultured in a single fermentor system along with Piriformospora indica it was observed that Bacillus megaterium NAT001 failed to grow along with the fungal endophyte. Suppression of bacterial wilt incidence by the individual and combined application of bacterial bioagents and fungal endophytes were tested with the wilt susceptible tomato variety Naveen (Indo-American hybrid seeds Pvt. Ltd, Bengaluru) and the moderately tolerant KAU variety Vellayani Vijay. Bioagents were applied during the nursery production of seedlings and the 21 days old seedlings were transplanted to pots filled with unsterile garden soil. Challenge inoculation with the pathogen was done five days after transplanting by drenching the pots with 10 ml each of the bacterial suspension (107cfu/ml). The disease incidence was scored at weekly intervals for 21 days. When the bioagents were tested individually for the suppression of bacterial wilt incidence in the hybrid variety Naveen, maximum disease suppression was observed in plants treated with Rhizobium radiobacter PCRE10 (15 percent) after 21 days. The disease suppressive ability of Bacillus velezensis PCSE10 (25 percent) was also significantly superior to all other treatments. Selection of bacterial bioagents for combined application with Piriformospora indica was done based on compatibility with the fungal endophyte and inhibitory action against Ralstonia solanacearum. Combined application of the fungal endophyte and Bacillus amyloliquefaciens VLY24 suppressed the wilt incidence to the highest extent (40 percent). When bacterial wilt suppression in Vellayani Vijay was tested by the individual application of bacterial bioagents, maximum disease suppression was recorded in plants treated with Streptomyces leeuwenhoekii KBT004 (30 percent), when observed 21 days after challenge inoculation. The combination of Piriformospora indica with Rhizobium radiobacter PCRE10 (50 percent) and Bacillus velezensis PCSE10 (50 percent) showed significantly lower disease incidence compared to the rest of the treatments. In the plant growth promotion experiment done with the variety Vellayani Vijay in the nursery stage, maximum plant height was observed in plants treated with Rhizobium radiobacter PCRE10 (14.49cm). However, those plants treated with Bacillus pumilus VLY17 had the highest number of leaves per plant (4.58). Combined inoculation of Piriformospora indica and Bacillus amyloliquefaciens VLY24 resulted in improved shoot fresh weight (2.08 g per plant) which was on a par with the plants treated with Bacillus velezensis PCSE10 (1.95 g plant-1). However, shoot weight on dry weight basis was the maximum for plants treated with Bacillus velezensis PCSE10 (122.55 mg plant-1) which was at par with those treated with combination of Piriformospora indica and Bacillus amyloliquefaciens VLY24 (104.78 mg plant-1). The present study revealed that biological management of bacterial wilt in tomato could be a feasible strategy under controlled conditions. The same has to be validated under field conditions before making any recommendations.Item Exploitation of indigenous bacterial antagonists against root-knot nematode, meloidogyne incognita (kofoid and white ) chitwood(Department of Nematology, College off Agriculture, Vellayani, 2018) Vishnu, J S; Nisha, M SItem Standardization of liquid formulation of PGPR MIX-1 and its evaluation for plant growth promotion in amaranthus(amaranthus tricolor L.)(Department of Agricultural Microbiology,College of Agriculture, Vellayani,Thiruvananthapuram, 2018) Gokul K Gopi; Meenakumari, K SItem Standardisation of nursery management practices in pachotti (Symplocos cochinchinensis (Lour.) S. Moore)(Department of Plantation Crops and Spices, College of Agriculture, Vellayani, 2018) Ajil, M S; Deepa S NairThe study entitled “Standardisation of nursery management practices in pachotti (Symplocos cochinchinennsis (Lour.) S. Moore)” was carried out in the Department of Plantation Crops and spices, College of Agriculture, Vellayani durng 2017-18. The objective of the study was to evaluate the propagation efficiency of different propagules viz., seeds, stem cuttings and root cuttings and to standardise the potting media for the nursery plants of pachotti. The propagules viz., seeds, stem cuttings and root cuttings for the study were sourced form Jawaharlal Nehru tropical Botanical Gardens and Research Institute, Palode, Thiruvananthapuram and from Wayanad district. The seeds were subjected to in vivo and in vitro germination studies. In in vivo study, among the pretreatments tried, viz., physical treatments, chemical priming and bio priming, only physical treatment of scarification (with sand paper) responded with a very low germination of 2 per cent. The germination commenced after two months of the treatment. Other in vivo pretreatments as well as in vitro treatments did not give any germination. In vegetative propagation, stem cuttings were exposed dto hormone/chemicals (auxins, phloroglucinol and salicylic acid (SA) pretreatments for two hours before planting. When pretreated with SA @ 10 and 20 mg L -1, at three months after planting, the hardwood cuttings responded with 30 pere cent survival, whith a shoot length of 2.99 mcm and 3.62cm, respectively. The semihardwood cuttings pretreated with SA@ 20 mg L-1 responded with 23.33 per cent survival with a higher shoot length of 3.72 cm. Both the hardwood and semi hardwood cuttings pretreated with SA 20 mgL-1 had on par values with respect to shoot length. Root cuttings were pretreated with different concentrations of various types of auxins. Root cuttings pretreated with IAA @ 250 mg L -1, after three months of planting responded with 33.33 per cent survival with a shoot length of 5.73 cm. Though root cuttings had slightly higher survival percent and shoot length, hardwood cuttings were selected for the valuation of potting media due to better availability and ease in procurement. The three month old hardwood cuttings pretreated with SA @ mgL-1 were then transplanted to ten different potting media comprising of two basal media viz., soil:coipith compost :cowdung (1:1:1) (B1) and soil : soirpith compost : vermicompost (1:1:1) (B2), and each in combination with biofertilisers @ 5g plant -1 viz., PGPR (Plant Growth Promotng Rhizobacteria) Mix I, Azospirillum, PSB (Phosphorus Solubilising Bacteria ) and AMF (Arbuscular Mycorrhizal Fungi). At fourth month after transplanting, B2 in combination with biofertilisers were found to be significantly superior to B2, B1 and B1 in combination with biofertilisers with respect to morphological parameters. B2 +PGPR Mix I recorded highest shoot length (11.50 cm) and number of leaves (10.50) which was on par with B2+Azospirillum, B2+PSB and B2+AMF; the highest number of branches (1.92) was observed in B2+ Azospiriillum which was on par with the treatments , B2+ PGPR Mix I, B2+PSB and B2+AMF. The fresh and dry weight of shoots were the highest (21.35 g and 4.78 g respectively) in B2 +PGPR Mix I which was on par with B2+ AMF. B2+ AMF recorded highest values (4.77 cm, 0.30 mm, 3.28 g and 0.0092 g, respectively) with respect to root growth parameters viz., root length, root girth , fresh and dry weight of roots. The physiological parameters, leaf area index (1.36) and leaf area duration (34.63 days) were the highest in B2+ PGPR Mix I which was on par with B2 in combination with other biofertilisers. The phytochemical analysis indicated that carbohydrate content (80.9 mg g-1) of plant tissue was the highest in B2+PGPR Mix 1, which was on par with B2+PSB, B2+Azospirrillum and B2+AMF. Chlorophyll content was found to the highest (1.20 mg g-1) in B2+ Azospirillum which was on par with B2+PGPR Mix I. The same treatment recorded the highest soluble protein content (20.31mg g-1) and it was on par with B2 in combination other biofertilisers. The nutrient analysis of plant tissue showed that nitrogen (2.22 percent ) and potassium (2.15 per cent ) content was significantly higher in B2+ Azospirillum. B2+PSB Recorded higher phosphorus content (0.26 per cent) among the treatments. The study indicated that nursery plants in the potting media B2 in combination with biofertilizers gave better performance with respect to morphological parmeters, physiological parameters, phytochemicals and plant nutrients. Among the various potting media tried, B2+AMF recorded significantly higher plant growth potential (0.522) followed by B2 + PGPR Mix I (0.428). In the study, hardwood cuttings were identified as the preferred planting material for the nursery establishment of pachotti. The cuttings could be treated with salicylic acid @ 20 mg L-1 for initial establishment of nursery plants. The preferred potting media for transplanting the established cuttings for raising the nursery plants of pachotti is Soil : Coirpith compost : Vermicompost (1:1:1) +AMF (5g/plant).