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    ThesisItemOpen Access
    Plant growth promoting actinobacteria from rhizosphere soils of Black pepper in Wayanad
    (Department of Agricultural Microbiology, College of Agriculture, Vellanikkara, 2021) Rineesha Backer, A; KAU; Girija, D
    Black pepper (Piper nigrum L.) known as the ‘King of spices’ is one of the important export-oriented spice crops that provides major source of income and employment for rural households in Kerala. Wayanad is one of the main pepper growing tracts in Kerala. However, the production of black pepper in Wayanad has been declining over the past many years, mainly due to the poor soil health status, improper land management and changes in climatic factors. An increasing demand for low-input agriculture has resulted in a greater interest in soil microorganisms which are able to enhance plant health and soil quality. However, actinobacteria have not yet been exploited for this purpose. Hence the present study focused on isolation, screening and characterization of actinobacteria from rhizosphere soils of black pepper in Wayanad and evaluation of their plant growth promoting activity. Rhizosphere soil samples were collected from five different locations of flood affected and non-flood affected black pepper growing areas of Wayanad district. Enumeration of actinobacteria on three different media revealed that the population of actinobacteria ranged from 1.00 x104 cfu to 12.00 x104 cfu g -1 soil in non-flooded soil. Actinobacteria could not be detected in flooded soils, even at a dilution of 10-1 , except in Meppadi. Among the different media, starch casein agar recorded higher population of actinobacteria. A total of 35 isolates were purified and maintained for characterization and screening for plant growth promoting activities. Cultural, morphological and biochemical characters of all 35 isolates of actinobacteria were studied. All the isolates were Gram positive, and they varied in the colony morphology, colour of aerial mycelium, spore chain morphology and pigmentation. Two isolates Vlt-K and Plp-B produced yellow water-soluble pigment and Vlt-I produce purple water insoluble pigment. Screening of all the 35 isolates under in vitro conditions for plant growth promoting (PGP) activities revealed that the isolates were highly variable. Two isolates (Ptd-A and Amb-C) were superior to other isolates in IAA production. Significantly higher N-fixation was noticed in four isolates (Ptd-A, Ptd-E, Ptd-B and Ptr-A). Six isolates solubilized insoluble phosphate to available P, and also reduced the pH of the medium. Reduction in pH by phosphate solubilizers has been reported earlier and this is attributed to the production of organic and inorganic acids. None of the isolates solubilized K or Zn. Three isolates with multiple PGP activities were selected, based on ranking for PGP activities in vitro and identified by 16S rRNA gene sequencing (Ptd-A and Ptr-A as Streptomyces sp. and Ptd-E as Actinobacteria bacterium). These actinobacterial isolates evaluated in-planta for PGP activities with black pepper cuttings (variety Panniyur-1). Treatments also included PGPR mix-1 (KAU commercial formulation) and Organic Package of Practices Recommendations (2017). All the three native isolates exhibited significant increase in shoot length, number of leaves and internode length throughout the growth period from planting to five months. Significantly higher root growth was observed in treatment T2: Ptd-A (Streptomyces sp.), with significantly higher root volume, fresh and dry root weight. Significantly higher plant fresh weight was observed in T2: Ptd-A (Streptomyces sp.) and significantly higher plant dry weight was observed in T2: Ptd-A (Streptomyces sp.) and T3: Ptr-A (Streptomyces sp). The present investigation revealed that native actinobacteria have the potential to improve the growth of black pepper. These isolates may further be evaluated under field conditions before commercialization. Screening for other beneficial traits like disease suppression and drought tolerance may also be carried out. The compatibility of the actinobacteria with other PGP microorganisms may also be evaluated. Actinobacterial spores survive in soil for longer periods and this may be an added advantage. Actinobacterial diversity and the biochemical molecules produced by actinobacteria could be exploited to build up sustainable and eco-friendly agriculture.
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    ThesisItemOpen Access
    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; KAU; Girija, D
    Plant 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.
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    ThesisItemOpen Access
    Comparative assessment of climate resilient capacity of two indigenous goat breeds based on changes in both phenotypic and genotypic traits
    (Academy of Climate Change Education and Research, Vellanikkara, 2020) Reshma Nair, M R; KAU; Sejian, V
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    ThesisItemOpen Access
    Exopolysaccharide producing bacteria from soil based nesting structures of insects
    (Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2022-03-14) Sruthi, Suresh; KAU; Chitra, N
    The study entitled ―Exoploysaccharide producing bacteria from soil based nesting structures of insects‖ was conducted during the year 2019-2021 in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram with the objective to isolate and characterize exopolysaccharide producing bacteria from mud wasp and termitaria and the ability of the best two isolates in soil aggregate stability study. Thirty three bacterial isolates were obtained from different termitaria and mud dauber wasp nest. Among them colonies of fifteen isolates had slimy mucoidal appearance and were identified as exopolysaccharide producers. The exopolysaccharide production of the isolates ranged from 25.78 µg mL-1 (Klebsiella pneumoniae KWP23) to 2.58 µg mL-1 . The total carbohydrate content in exopolysaccharide was maximum in bacterial isolate Paenibacillus polymyxa KTM17 (14.48 µg mL-1 ). The best five isolates were selected for molecular characterization by 16s rRNA sequencing and identified as Priestia aryabhattai TWP12, Priestia aryabhattai TWP13, Klebsiella pneumoniae KWP23, Priestia megaterium KTM4, Paenibacillus polymyxa KTM17. They were screened for phosphate, potassium and silicate solubilization. Klebsiella pneumoniae KWP23 showed solubilization zone for phosphate (4 mm) and potassium (1 mm). None of the isolates chosen were silicate solubilizers. Two bacterial isolates with maximum EPS production viz. Paenibacillus polymyxa KTM17 and Klebsiella pneumoniae KWP23 were selected for the further studies. Effect of carbon source and carbon: nitrogen ratio on exopolysaccharide production was studied. Glucose was found to be the best carbon source for EPS production in Klebsiella pneumoniae KWP23 at C: N ratio of 10:1. In Paenibacillus polymyxa KTM17, highest EPS production was recorded when sucrose was used as carbon source at a C: N ratio of 50:1. The effect of temperature on EPS production of the isolates was evaluated. Increase in incubation temperature resulted in a corresponding increase in EPS production in case of Klebsiella pneumoniae KWP23. The highest EPS 3 production by Klebsiella pneumoniae KWP23 was (23.00 µg mL-1 ) at 50oC and Paenibacillus polymyxa KTM17 at 30oC (31.50 µg mL-1 ). The effect of salinity on EPS production by Klebsiella pneumoniae KWP23 and Paenibacillus polymyxa KTM17 isolates was also assessed. For Klebsiella pneumoniae KWP23, the EPS production was maximum without NaCl (18.0 µg mL-1 ) and for Paenibacillus polymyxa KTM17, the EPS production was maximum at 6% (1 M) NaCl (26.25 µg mL-1 ). Klebsiella pneumoniae KWP23 and Paenibacillus polymyxa KTM17 were further subjected to dual culture plate assay to study the biocontrol potential of the isolates. Paenibacillus polymyxa KTM17 showed a clear inhibition zone on dual culture assay with Rhizoctonia solani, but Klebsiella pneumoniae KWP23 did not inhibit the growth of the fungus. Both Klebsiella pneumoniae KWP23 and Paenibacillus polymyxa KTM17 inhibited the growth of Helminthosporium sp. The potential of the isolates to form soil aggregates and its aggregate stability was studied. The mean weight diameter of dry aggregates obtained was highest for the soil amended with Paenibacillus polymyxa KTM17 (1.068 mm). The study on wet aggregate stability on water immersed revealed that the best soil aggregate stability was given by the isolate Paenibacillus polymyxa KTM17. The stability of the aggregate lasted for more than 24 hours when immersed in water whereas that in the aggregate kept as control disintegrated immediately on pouring water. The effect of agitation and water immersion on the aggregate stability was also analyzed. On oscillation of the aggregates in water the aggregates in control soil showed maximum turbidity (0.254 nm). The dry weight of the disintegrated soil was minimum in the soil aggregate amended with Paenibacillus polymyxa KTM17 (0.014 g). Based on the results of the present study, it can be concluded that among the selected isolates Klebsiella pneumoniae KWP23 had maximum production of EPS. Both Klebsiella pneumoniae KWP23 and Paenibacillus polymyxa KTM17 has biocontrol potential against plant pathogenic fungi. Paenibacillus polymyxa KTM17 is a potential isolate which can effect soil aggregate stability in dry and wet condition.
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    ThesisItemOpen Access
    Isolation and characterization of beneficial rhizosphere microorganisms from ragi grown in Attappady hill tract of Kerala
    (Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2021) Gayathri, M; KAU; Chitra, N
    The study entitled “Isolation and characterization of beneficial rhizosphere microorganisms from ragi grown in Attappady hill tract of Kerala” was conducted during the year 2019-2021 in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram with the objective to isolate nitrogen fixing and phosphate solubilizing bacteria from soil and root samples collected from Attapady and select efficient isolates through in vitro screening process and assess the plant growth promotion activity by roll towel assay. Thirty six nitrogen fixing bacteria and eight phosphate solubilizing bacteria were isolated from root and soil samples from ragi grown in Attappady region. The isolates were designated as ATY1-ATY36 for NFB isolates and PSB1-PSB8 for PSB isolates. Twenty isolates were selected after eliminating isolates with similarities in colony morphological characters, in order to avoid repetition among the isolates obtained from the same sample. Both NFB and PSB isolates were characterized by morphology and biochemical tests. All selected isolates were subjected to plant growth promotion activity studies. The Indole Acetic Acid (IAA) production showed a wide range of variation from 101.22 µg mL-1 to 3.26 µg mL-1 . Gibberellic Acid (GA) production of all the twenty isolates were done and the results ranged between 10.07 µg mL-1 and 2.18 µg mL-1 . Maximum IAA and GA production was recorded in isolate ATY10. Extracellular ammonia production of the bacterial isolates ranged between 176.53±5.07 μmol mL−1 and 75.59 μmol mL−1. The nitrogen content of NFB isolates ranged between 30.75 µg mL-1 and 9 µg mL-1 . The isolate ATY33 had maximum nitrogen content. The selected PSB isolates were subjected to plate assay and broth assay using Pikovskaya’s medium. The isolate PSB1 recorded maximum zone of solubilization (18.23 mm), phosphate solubilizing index (2.82) and quantification of phosphate solubilization (53.41 mg L-1 ). The bacterial isolates were screened based on weighted average ranking. In NFB, ATY10, ATY34 and ATY35 were selected for further studies and in PSB, the isolates PSB1, PSB3 and PSB4 were selected. The selected isolates were 164 identified as Pantoea agglomerans ATY10, Rhizobium sp. ATY34, Ensifer adhaerens ATY35, Burkholderia territorii PSB1, Burkholderia cepacia PSB3 and Aeromonas hydrophila PSB4 by 16s rRNA sequencing. Effect of these isolates were assessed for plant growth promotion in vitro in ragi seeds using roll towel assay. Maximum germination percentage was recorded by Aeromonas hydrophila PSB4 (81.24 per cent) and the minimum number of days taken for germinationwas recorded by Pantoea agglomerans ATY10 treated seeds. Also, the treatment Pantoea agglomerans ATY10 recorded maximum shoot length (3.31 cm), root length (7.76 cm), seedling length (11.08 cm), seedling vigour index (876.62), root dry weight (0.443 mg) and root shoot ratio (0.50). The treatment Burkholderia territorii PSB1 recorded maximum root fresh weight (3.762 mg) and shoot dry weight (0.877 mg). The six isolates were subjected to assess the multiple traits such as phosphorus, potassium and silicate solubilizing capacity, siderophore production and antifungal activity. Burkholderia territorii PSB1 showed maximum zone of inhibition against Rhizoctonia solani (3.16 mm) and Sclerotium rolfsii (0.58 mm). The maximum antagonistic activity against Fusarium sp. and Helminthosporium sp. was recorded by Burkholderia cepacia PSB3 with 4.13 mm and 5.66 mm of zone of inhibition respectively. Burkholderia territorii PSB1 and Burkholderia cepacia PSB3 solubilized potassium alumino silicate in agar also, with clearance zone of 11.00 mm and 12.66 mm (in diameter), respectively. None of the isolates solubilized magnesium trisilicate in Bunt and Rovira medium. The siderophore production was observed in Aeromonas hydrophila PSB4 isolate in Chrome Azurol S (CAS) agar medium plates. Based on the results of the present study, it can be concluded that Pantoea agglomerans ATY10 is the superior isolate among NFB isolates for plant growth promotion. Also, the isolate Burkholderia territorii PSB1 is best among PSB isolates for plant growth promotion and biocontrol activity.
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    ThesisItemOpen Access
    Development of encapsulated formulation of PGPR mix-I and IRS evaluation
    (Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2020) Ayisha, Y L; KAU; Meenakumari, K S
    The study entitled “Development of encapsulated formulation of PGPR mix-I and its evaluation” was conducted during 2018-2020, in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram, with the objective to develop calcium alginate based encapsulated formulation of PGPR mix-I and its evaluation for slow release and biodegradation. The component cultures of PGPR mix-I were procured from the Department of Agricultural Microbiology, College of Agriculture, Vellayani for standardization of protocol for preparation of calcium alginate based encapsulated bead formulation of PGPR mix-I. Encapsulated bead formulation of PGPR mix-I was prepared by standard procedures. An experiment was carried out to standardize the protocol for preparation of calcium alginate based encapsulated formulation of PGPR mix–I in completely randomized design with different treatments such as 10% Standard starch, 15% Standard starch, 10% Wheat flour, 15% Wheat flour, 10% Talc, 15% Talc and control treatment as 2% Sodium alginate alone in three replications. Consistent viable count was recorded in encapsulated formulation amended with 10% Standard starch. It exhibited maximum viable count of each of the component cultures of PGPR mix-I as a result of three month population study. A significant decline of total viable population in control treatment was observed in each month compared to encapsulated formulation amended with 10% Standard starch. Based on the population study, encapsulated formulation of PGPR mix-I 10% Standard starch amended was adjudged as the best combination of filler material and hence the shelf life studies of the same had to be continued at monthly intervals at room temperature and refrigerated conditions for six months by serial dilution and plate count method. Significant viable count was recorded in encapsulated beads stored at room temperature condition throughout the shelf life study. The moisture content of beads were also monitored during standardization and shelf life study. During standardization study, moisture content of PGPR mix-I encapsulated beads of each treatment was monitored for a period of three months at monthly intervals at room temperature and it showed a significant variation among treatments in each month. A reduction in moisture content of beads was observed from first month to the end of sixth month in all treatments. Beads amended with 10% Standard starch showed a moisture content of 13.37%, 12.07%, 11.72% and 11.45% after 24 hours of drying, first, second and third month respectively. During shelf life study, 10% Standard starch combination at refrigerated condition showed moisture content in the range of 12.83% to 11.45% while at room temperature the same has recorded values in the range of 12.07% to 10.70%. Evaluation of rate of release of immobilized bacteria from encapsulated beads was determined as per the procedure described by Bashan (1986) and the number of released bacteria was determined by the plate count method in respective selective medium. The higher cfu of component cultures of PGPR mix-I was observed after gentle shaking at 32⁰C for 24hours (T1) in75ml of sterile saline solution. Evaluation of biodegradation of encapsulated beads was studied at weekly intervals in sterile and non-sterile soil with PGPR mix-I inoculated and non-inoculated beads with three replications each (Bashan, 1986). Both the sets were observed weekly for their rate of biodegradation. As per biodegradation scale values like 0, ˃0-0.5, ˃0.5-1, ˃1-2, ˃2-2.5 or 3 was assigned according to the degree of visible degradation which indicates no visible degradation,onset of degradation, slight visible degradation on bead edges, one-half to three-fourth of the beads degraded, 90% of beads become mushy, full degradation (beads are disintegrated into small pieces or not found in the nylon bag) repectively (Bashan, 1986). The PGPR mix-I inoculated beads with bacteria in non-sterile soil showed highest scale of biodegradation throughout the biodegradation study (mean value 1.34) and beads without bacteria in sterile soil showed the lowest scale (mean value 0.52). Kruskal-Wallis rank sum test was done and there was a significant difference between treatments and so multiple comparison was done using Dunn test. During all the four weeks of biodegradation study, treatment T1 (beads with PGPR mix-I in non sterile soil) recorded the highest biodegradation and T4 (beads without PGPR mix-I in sterile soil) recorded the least biodegradation. Treatments T2 (beads with PGPR mix-I in sterile soil) and T3 (beads without PGPR mix-I in non sterile) were on par with both the treatments T1 and T4 in all the four weeks. Treatment wise evaluation of biodegradation of beads was done with Kruskal-Wallis rank sum test and gives a chi-squared value of 46.205 with df = 15 and p-value = 4.932e-05. There was a significant difference between treatments and so multiple comparison was done using Dunn test. Treatment T4 (beads with bacteria in non sterile soil during fourth week) showed significantly different from treatment T13 (beads without bacteria in sterile soil during first week). In the present investigation, calcium alginate based encapsulated beads of PGPR mix-I amended with 10% Standard starch exhibited maximum viable count of component cultures of PGPR mix-I throughout the three months period of standardization study. In terms of evaluation of shelf life and moisture retention during storage, beads stored under room temperature condition was found to be better. The rate of release of component cultures of PGPR mix-I from the encapsulated formulation was more during the first 24-48 hours. Biodegradation studies of encapsulated beads of PGPR mix-I revealed that the beads inoculated with PGPR mix-I in non sterile soil showed highest biodegradation throughout the period of investigation.
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    ThesisItemOpen Access
    Field evaluation of abiotic stress tolerant strains of Trichoderma harzianum and Pseudomonas fluorescens for Phytophthora disease management in black paper (Piper nigrum L.)
    (Department of Agricultural Microbiology, College of Horticulture, Vellanikkara, 2020) Rima, K R.; KAU; Surendra Gopal, K
    Black pepper (Piper nigrum L.), known as “the king of spices” is an important spice commodity of commerce and trade in India since pre-historic period. It is highly sensitive to abiotic stresses like climate changes. The abiotic stresses in plants can be overcome by the use of beneficial microorganisms. However, the microorganisms themselves are vulnerable to abiotic and biotic stresses. Therefore, abiotic stress tolerant strains of beneficial microorganisms have to be developed for black pepper to overcome changes due to micro-climatic variables and soil parameters. In an earlier study in the Department of Agricultural Microbiology, College of Horticulture, native abiotic stress tolerant isolates of Trichoderma harzianum (CKT isolate) and Pseudomonas fluorescens (PAP isolate) were identified for growth promotion and disease management in black pepper (Piper nigrum L.) under pot culture studies. As the performance of these strains varies under field conditions, a study on “Field evaluation of abiotic stress tolerant strains of Trichoderma harzianum and Pseudomonas fluorescens for Phytophthora disease management in black pepper (Piper nigrum L.)” was undertaken with an objective to study the effect of micro- climatic and soil parameters on abiotic stress tolerant strains under field conditions. Before field evaluation, pot culture experiment was conducted to evaluate the efficiency of the abiotic stress tolerant Trichoderma harzianum (CKT) and Pseudomonas fluorescens (PAP) for growth promotion and Phytophthora disease management in black pepper during April – October, 2017. T. harzianum (CKT) was the most promising isolate for growth promotion and disease management based on biometric (number of leaves, plant height, number of lateral branches, number of nodes, intermodal length), biological and disease management characters under pot culture studies. It was found that population of Trichoderma harzianum (CKT), Pseudomonas fluorescens (PAP), Trichoderma viride (KAU reference culture) and Pseudomonas fluorescens (KAU reference culture) was negatively correlated with soil temperature and positively correlated with soil moisture. There was no significant correlation between the population of microbes and soil pH and soil respiration. Field evaluation revealed that Trichoderma harzianum (CKT) + KAU POP was the most promising treatment for growth promotion under field conditions based on biometric and biological characters. The population of Trichoderma harzianum (CKT), Pseudomonas fluorescens (PAP), Trichoderma viride (KAU reference culture) and Pseudomonas fluorescens (KAU reference culture) were negatively correlated with soil temperature and positively correlated with soil moisture content. However, soil pH and soil respiration did not have any correlation with the population of Trichoderma sp. and Pseudomonas fluorescens. Micro-climatic and soil parameters did not show any effect on the production of ammonia, HCN and siderophore but had significant effect on production of IAA in Trichoderma sp. and Pseudomonas fluorescens. Soil temperature (26.8 to 30.6 °C), soil moisture content (14 to 17.6 %), soil pH (5.7 to 6.1) and soil respiration (8.1 to 9.8 mg CO2 g-1 day-1) were found to be the optimum micro-climatic and soil parameters for IAA production. Trichoderma harzianum (CKT) was the most promising isolate for growth promotion under field conditions. However, multilocational field trials at different agro- ecological zones of Kerala need to be conducted before commercialization of Trichoderma harzianum (CKT).
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    ThesisItemOpen Access
    Isolation and in vitro screening of silicate solubilizing bacteria from paddy rhizosphere
    (Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2020) Akhila P, Subash.; KAU; Meenakumari, K S
    The study entitled “Isolation and in vitro screening of silicate solubilizing bacteria from paddy rhizosphere”, was conducted during 2018-2020, in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram, with the objective of isolation and in vitro screening of bacteria which are capable of solubilizing insoluble form of silicate. Bacteria capable of solubilizing silicates were isolated from the rhizosphere soils collected from different upland and low land paddy fields by serial dilution and plate count method using Bunt and Rovira medium supplemented with 0.25 per cent magnesium trisilicate. Based on the clear halo zone formed around the bacterial colonies on solid media, they were identified as Silicate Solubilizing Bacteria (SSB). Twenty seven isolates of bacteria capable of solubilizing insoluble form of silicate (magnesium trisilicate) were obtained from different locations and were allotted code numbers from SSB 1 to SSB 27. These isolates were subjected to plate and broth assay in Bunt and Rovira medium supplemented with 0.25 per cent magnesium trisilicate. After three days of incubation of test plates at room temperature, all the twenty seven isolates solubilized magnesium trisilicate and produced clearing zone around the bacterial colonies on solid media. The size of clearance zone ranged from 3 mm to 13 mm in plates. The maximum clearance zone of 13 mm was recorded with the isolate SSB 14 which was significantly superior to all other isolates. In broth culture, SSB 20 showed the highest silicate solubilization of 94.65 mg L-1. Based on plate as well as broth assay of all the twenty seven isolates obtained, five isolates viz., SSB 3, SSB 14, SSB 18, SSB 20 and SSB 22 which showed the maximum clearance zone in plate and silicate solubilization in broth were selected as superior isolates. All the isolates obtained were subjected to plate and broth assay for phosphate solubilization in Pikovskaya’s medium and potassium solubilization in Aleksandrov medium. Among them, fourteen isolates showed phosphate solubilization in plates and the clearance zone ranged from 0.87 mm to 5.50 mm. The maximum clearance zone of 5.50 mm was recorded with the isolate SSB 22 which was on par with SSB 23 with 5 mm clearance zone in plate and highest solubilization of 41.92 mg L-1 in broth was shown by SSB 22 which was significantly superior to all other isolates. Twelve isolates showed potassium solubilization in plates and the clearance zone ranged from 2.25 mm to 5.50 mm. Maximum clearance zone of 5.50 mm was recorded with the isolate SSB 8 which was significantly superior to all other isolates. The highest potassium solubilization of 37.50 mg L-1 in broth was observed with isolates SSB 1, SSB 2, SSB 7, SSB 8, SSB 13, SSB 18, SSB 21 and SSB 22 which were found to be statistically on par. Acid production by the five superior SSB isolates, SSB 3, SSB 14, SSB 18, SSB 20 and SSB 22 was detected as a yellow halo around the bacterial colonies in bromophenol blue amended Bunt and Rovira medium. All the five superior isolates tested showed positive results for acid production. The antagonistic activity of the five superior SSB isolates were assessed against major pathogens of paddy viz., Rhizoctonia solani, Magnaporthe grisea, Helminthosporium oryzae and Xanthomonas oryzae pv. oryzae following dual culture method. Out of the five isolates tested, three isolates (SSB 18, SSB 20 and SSB 22) inhibited Rhizoctonia solani. Three isolates (SSB 3, SSB 18 and SSB 22) showed antagonism against Magnaporthe grisea and four isolates (SSB 3, SSB 18, SSB 20 and SSB 22) inhibited Helminthosporium oryzae. The bacterial pathogen, Xanthomonas oryzae pv. oryzae was inhibited by three isolates (SSB 18, SSB 20 and SSB 22). Among all the five isolates tested against different phytopathogens, SSB 18 was found superior with the maximum zone of inhibition of 9.65 mm, 14.45 mm, 10.80 mm and 11.50 mm against Rhizoctonia solani, Magnaporthe grisea, Helminthosporium oryzae and Xanthomonas oryzae pv. oryzae respectively. The five superior isolates were characterized based on morphological and biochemical characters. The results revealed that all the isolates were rod shaped, Gram positive endospore formers. Based on the results of present study, it can be concluded that SSB 3, SSB 14, SSB 18, SSB 20 and SSB 22 are the superior silicate solubilizing bacterial isolates. Among them, SSB 18 showed the highest antagonistic activity against major pathogens of paddy.
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    ThesisItemOpen Access
    Development of encapsulated formulation of PGPR mix-I and its evaluation
    (Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2020) Ayisha, Y L; KAU; Meenakumari, K S
    The study entitled “Development of encapsulated formulation of PGPR mix-I and its evaluation” was conducted during 2018-2020, in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram, with the objective to develop calcium alginate based encapsulated formulation of PGPR mix-I and its evaluation for slow release and biodegradation. The component cultures of PGPR mix-I were procured from the Department of Agricultural Microbiology, College of Agriculture, Vellayani for standardization of protocol for preparation of calcium alginate based encapsulated bead formulation of PGPR mix-I. Encapsulated bead formulation of PGPR mix-I was prepared by standard procedures. An experiment was carried out to standardize the protocol for preparation of calcium alginate based encapsulated formulation of PGPR mix–I in completely randomized design with different treatments such as 10% Standard starch, 15% Standard starch, 10% Wheat flour, 15% Wheat flour, 10% Talc, 15% Talc and control treatment as 2% Sodium alginate alone in three replications. Consistent viable count was recorded in encapsulated formulation amended with 10% Standard starch. It exhibited maximum viable count of each of the component cultures of PGPR mix-I as a result of three month population study. A significant decline of total viable population in control treatment was observed in each month compared to encapsulated formulation amended with 10% Standard starch. Based on the population study, encapsulated formulation of PGPR mix-I 10% Standard starch amended was adjudged as the best combination of filler material and hence the shelf life studies of the same had to be continued at monthly intervals at room temperature and refrigerated conditions for six months by serial dilution and plate count method. Significant viable count was recorded in encapsulated beads stored at room temperature condition throughout the shelf life study. The moisture content of beads were also monitored during standardization and shelf life study. During standardization study, moisture content of PGPR mix-I encapsulated beads of each treatment was monitored for a period of three months at monthly intervals at room temperature and it showed a significant variation among treatments in each month. A reduction in moisture content of beads was observed from first month to the end of sixth month in all treatments. Beads amended with 10% Standard starch showed a moisture content of 13.37%, 12.07%, 11.72% and 11.45% after 24 hours of drying, first, second and third month respectively. During shelf life study, 10% Standard starch combination at refrigerated condition showed moisture content in the range of 12.83% to 11.45% while at room temperature the same has recorded values in the range of 12.07% to 10.70%. Evaluation of rate of release of immobilized bacteria from encapsulated beads was determined as per the procedure described by Bashan (1986) and the number of released bacteria was determined by the plate count method in respective selective medium. The higher cfu of component cultures of PGPR mix-I was observed after gentle shaking at 32⁰C for 24hours (T1) in75ml of sterile saline solution. Evaluation of biodegradation of encapsulated beads was studied at weekly intervals in sterile and non-sterile soil with PGPR mix-I inoculated and non-inoculated beads with three replications each (Bashan, 1986). Both the sets were observed weekly for their rate of biodegradation. As per biodegradation scale values like 0, ˃0-0.5, ˃0.5-1, ˃1-2, ˃2-2.5 or 3 was assigned according to the degree of visible degradation which indicates no visible degradation,onset of degradation, slight visible degradation on bead edges, one-half to three-fourth of the beads degraded, 90% of beads become mushy, full degradation (beads are disintegrated into small pieces or not found in the nylon bag) repectively (Bashan, 1986). The PGPR mix-I inoculated beads with bacteria in non-sterile soil showed highest scale of biodegradation throughout the biodegradation study (mean value 1.34) and beads without bacteria in sterile soil showed the lowest scale (mean value 0.52). Kruskal-Wallis rank sum test was done and there was a significant difference between treatments and so multiple comparison was done using Dunn test. During all the four weeks of biodegradation study, treatment T1 (beads with PGPR mix-I in non sterile soil) recorded the highest biodegradation and T4 (beads without PGPR mix-I in sterile soil) recorded the least biodegradation. Treatments T2 (beads with PGPR mix-I in sterile soil) and T3 (beads without PGPR mix-I in non sterile) were on par with both the treatments T1 and T4 in all the four weeks. Treatment wise evaluation of biodegradation of beads was done with Kruskal-Wallis rank sum test and gives a chi-squared value of 46.205 with df = 15 and p-value = 4.932e-05. There was a significant difference between treatments and so multiple comparison was done using Dunn test. Treatment T4 (beads with bacteria in non sterile soil during fourth week) showed significantly different from treatment T13 (beads without bacteria in sterile soil during first week). In the present investigation, calcium alginate based encapsulated beads of PGPR mix-I amended with 10% Standard starch exhibited maximum viable count of component cultures of PGPR mix-I throughout the three months period of standardization study. In terms of evaluation of shelf life and moisture retention during storage, beads stored under room temperature condition was found to be better. The rate of release of component cultures of PGPR mix-I from the encapsulated formulation was more during the first 24-48 hours. Biodegradation studies of encapsulated beads of PGPR mix-I revealed that the beads inoculated with PGPR mix-I in non sterile soil showed highest biodegradation throughout the period of investigation.