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Subject: Agricultural Microbiology × Author: KAU × End date: 2019 × Start date: 2019 × Type: Thesis ×
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    ThesisItemOpen Access
    Alginate based consortial formulation of native microbial fertilizers
    (Department of Agricultural Microbiology, College of Horticulture, Vellanikkara, 2019) Alfiya Beegum, A; KAU; Surendra Gopal, K
    At present, the available biofertilizers are bulky and have short shelf life due to contamination problem. Hence, a suitable formulation needs to be developed which is less bulky and has increased shelf life. Alginate is one of the most commonly used polymers for microbial encapsulation. It is commercially extracted from seaweeds like Macrocystis pyrifera, Ascophyllum nodosum, Laminaria etc. The present study was undertaken in the Department of Agricultural Microbiology, College of Horticulture, Vellanikkara to develop an alginate based formulation of nitrogen, phosphorus, and potash biofertilizers consortia and evaluate for growth enhancement using tomato as the test crop. Five isolates each of nitrogen fixers, phosphorus and potassium solubilizers from Wayanad district were obtained from the repository maintained in the Department of Agricultural Microbiology, COH, Vellanikkara. The bacterial isolates were screened for nitrogen fixation, phosphorus and potash solubilization along with indole acetic acid production. Amount of nitrogen fixed (22.63 mg of N g -1 sucrose utilized) and indole acetic acid production (6 μg ml-1) were highest for Microbacterium arborescence. Similarly, Burkholderia cepacia recorded the highest amount of phosphorus solubilization (64.83 μg ml-1) and indole acetic acid production (8.67 μg ml-1). Among the potassium solubilizers, Acinetobacter calcoaceticus solubilized the highest amount of potassium (41.63 μg ml-1) under in vitro conditions. Microbacterium arborescence, Microbacterium testaceum and Nguyenibacter vanlangensis were selected as the three most promising nitrogen fixers. Similarly, the phosphorus solubilizers selected were Burkholderia cepacia, Bacillus subtilis (KASB5) and Bacillus subtilis (H4). The potassium solubilizers selected were Acinetobacter calcoaceticus, Burkholderia sp. and Brevibacterium sp. Compatibility studies were conducted among the selected bacterial isolates using cross streak method which showed no inhibition at the intersection of two bacterial isolates. The compatible isolate was further confirmed by dual culture method. The efficient isolates selected for consortia preparation were Microbacterium arborescence + Burkholderia cepacia + Acinetobacter calcoaceticus (N1P1K1), Microbacterium arborescence + Bacillus subtilis (KASB5) + Acinetobacter calcoaceticus (N2P2K2) and Microbacterium testaceum + Burkholderia cepacia + Burkholderia sp. (N3P3K3). In order to prepare a sterile and uniform sized alginate beads, a protocol was standardized for temperature, time and concentration of sodium alginate solution with calcium chloride solution. The optimum concentration of sodium alginate solution and temperature required for alginate beads preparation was 3% and 95 oC for 15 minutes. The diameter of alginate beads ranged between 2 mm – 2.7 mm. The rate of release of nitrogen fixers from alginate based consortia-1 during the initial 24 hours was high (41.67 x 106 cfu -1 g of beads) which reached to a population of 21 x 106 cfu -1 g of beads at 90th day. Population of nitrogen fixers, phosphorus solubilizers and potassium solubilizers decreased towards the 90th day when compared with the initial count of bacteria released from the alginate beads. A pot culture experiment using tomato as a test crop was conducted under sterile and unsterile potting mixture separately to evaluate Alginate based consortia (T1, T2 and T3), combination (T1 + T2 + T3), Talc based consortia (T5, T6 and T7) and POP (T8), Organic POP (T9) and Absolute control (T10). In sterile soil, the population of nitrogen fixers were higher for all the treatments receiving bacterial consortia. The population of P-solubilizers were higher in alginate based consortium-1 (13.0 x 106 cfu g-1) while the population of K-solubilizers were higher in treatments with combined application of alginate based consortia. Under unsterile soil, the population of nitrogen fixers were found to be higher in treatments with alginate and talc based consortia. The population of potassium solubilizers was higher in treatment with combined application of alginate based consortia. Treatments with alginate based consortium showed a higher population of phosphorus solubilizers compared with talc based consortia in unsterile soil. However, the population of N fixers, P and K solubilizers decreased with time in all treatments. Compared to initial soil status, application of alginate based bacterial consortium-1 resulted in a significant increase in available nutrient content among treatments receiving bacterial consortia in sterile soil conditions. Yield and yield attributes were higher in the alginate based bacterial consortia-1(897.0 g/plant) under sterile condition. However, yield was higher in treatment based on POP under unsterile soil conditions (845.0 g/plant). Among alginate based treatments, alginate based consortium-1 recorded a higher yield (707.33g/plant) under unsterile condition also. The present studies indicated that the alginate based consortium could be a potential microbial inoculant formulation which is less bulky, free from contamination, biodegradable and non-toxic. Encapsulation enables slow and controlled release of cells and thus, maintains a uniform bacterial population. However, further studies are required to study its shelf life, its performance under field condition before commercialization and develop a suitable protocol for large scale production.
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    ThesisItemOpen Access
    Soil microclimatic parameters and microbial activities on the population and diverisity of aqrbuscular mycorrhizal fungi
    (Department of Agricultural Microbiology, College of Horticulture, Vellanikkara, 2019) Anusha, K; KAU; Surendra Gopal, K
    Arbuscular mycorrhizal fungi (AMF) are ubiquitous, which promote the plant growth by assisting in nutrient uptake and also mitigate several biotic and abiotic stresses in plants. Soil temperature, soil moisture, soil pH and nutrient availability are the major factors that affect the diversity, distribution and activity of AMF. The arbscular mycorrhizal fungi are obligate symbiont and require a host plant to complete its life cycle. Solenostemon rotundifolius or Chinese potato is one of the important minor tuber crop of Kerala, which is rich in starch, proteins, vitamins and minerals, with 70- 90% mycorrhizal colonization. The present study was undertaken to assess the effect of soil microclimatic parameters and microbial activities on the population and diversity of AMF and also evaluate the influence of AMF on the growth and yield of Solenostemon rotundifolius. The field experiment was conducted in a randomized complete block design (RCBD) with nine treatments and three replications at Agronomy farm, College of Horticulture, Vellanikkara during 2017 to 2019. The treatments consisted of five AMF species viz., Rhizophagus fasciculatus (T1), Funneliformis mosseae (T2), Glomus etunicatum (T3), Acaulospora sp. (T4), and Gigaspora sp. (T5), consortium of AMF (T6), POP recommendations of KAU, 2016 (T7), Organic POP of KAU, 2017 (T8) and absolute control (T9). Per cent AMF root colonization was higher (93.33%) in Rhizophagus fasciculatus (T1), Funneliformis mosseae (T2), Acaulospora sp. (T4) and T6 (T1 +T2 +T3 +T4 +T5). Spore population varied between the months, but highest spore population were recorded at 30 DAP and 120 DAP, whereas lowest was at 60 DAP. However, AMF spore diversity was highest in T6 (T1 +T2 +T3 +T4 +T5) throughout the experiment. AMF spore count and per cent root colonization increased with soil temperature but, not affected by soil moisture and soil pH. However, AMF spore diversity decreased with soil pH. Funneliformis mosseae (T2) and consortium of AMF (T6) recorded highest dehydrogenase activity throughout the experiment. Carbon dioxide evolution was highest at 120 DAP, in Gigaspora sp. (T5), which was on par with Acaulospora sp. (T4) and Funneliformis mosseae (T2). Acid phosphatase activity was highest in Funneliformis mosseae (T2) followed by Acaulospora sp. (T4). Per cent root colonization by AMF was positively correlated with dehydrogenase activity, CO2 evolution and acid phosphatase activity. AMF spore population was positively correlated with dehydrogenase activity and CO2 evolution. AMF spore diversity was positively correlated with CO2 evolution and acid phosphatase activity. Funneliformis mosseae (T2) showed better performance with respect to biometric characters (plant height, root biomass and dry weight) of the plant. AMF consortium (T6) treated plants recorded highest (16.98 t ha-1) tuber yield, which was also on par with T1 (Rhizophagus fasciculatus), T2 (Funneliformis mosseae), T4 (Acaulospora sp.) and T7 (POP recommendations of KAU, 2016). Phosphorus uptake by Chinese potato plants were highest (60.06 kg ha-1) in T2 (Funneliformis mosseae) and lowest in absolute control. Plant biometric characters, tuber yield and P uptake in Chinese potato were enhanced with AMF root colonization. Consortium of AMF (T6), Rhizophagus fasciculatus (T1) and Acaulospora sp. (T4) treated plots were recorded with less nematode population (506.67 to 559 nematodes per 250 g soil). The present study indicated that soil temperature affected AMF root colonization and spore count. Increase in soil microbial activities (dehydrogenase activity, CO2 evolution and acid phosphatase activity) showed increased root colonization, spore count and spore diversity. AMF root colonization enhanced growth, phosphorus uptake and yield of plant. Funneliformis mosseae (T2) was the most promising AMF for improving the growth, yield and phosphorus uptake in Solenostemon rotundifolius. However, extensive field studies are needed under different seasons and agro ecological zones, in order to develop an abiotic stress tolerant AMF for Chinese potato plant.
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    ThesisItemOpen Access
    Growth Promotion in Chilli on Inoculation with Pseudomonas Fluorescens and Piriformosporaa
    (Department of Agricultural Microbiology, College of Agriculture,Vellayani, 2019) Nandana, M S; KAU; Anith, K N
    The study entitled “Growth promotion in chilli (Capsicum annuum L.) on inoculation with Pseudomonas fluorescens and Piriformospora indica” was undertaken during 2017-2019, in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram, with the objective to assess the compatibility of the root endophytic fungus Piriformospora indica and two Pseudomonas fluorescens strains, and to evaluate their effect on growth promotion in chilli variety Vellayani Athulya. The Pseudomonad strains used were Pseudomonas fluorescens PN026 and Pseudomonas fluorescens AMB8. Experiments comprised both in vitro and in vivo studies. For in vitro study a dual culture plate assay was done in potato dextrose agar (PDA) and coconut water agar (CWA) with the fungal and bacterial endophytes to evaluate the direct antagonism. Both Pseudomonas fluorescens PN026 and Pseudomonas fluorescens AMB8 showed antagonism to the root endophyte Piriformospora indica in PDA whereas in CWA, Pseudomonas fluorescens PN026 did not show any antagonistic effect and Pseudomonas fluorescens AMB8 showed a reduced antagonism to Piriformospora indica compaired to that in PDA. Indirect antagonism was evaluated through agar well diffusion method and paper disc diffusion method using culture filtrate of the bacterial strains and the culture filtrate from both the bacterial strains showed antagonism against Piriformospora indica in which the maximum zone of inhibition was observed in culture filtrate of Pseudomonas fluorescens AMB8. A Co-culture experiment involving P.indica and Pseudomonas strains using a single fermentation system was attempted in two different media; potato dextrose broth (PDB) and autoclaved coconut water (ACW). The flasks were incubated under agitation for 48 h and the population of the bacteria was determined at 24 h intervals by dilution plating in Kings B agar medium and it was observed that, when 10 day old cultures of the fungus in ACW and PDB were inoculated with the bacteria, ACW supported the growth of the bacteria similarly to fungus free ACW and KB medium. Co-cultivation in PDB led to a decline in bacterial population and the autoclaved coconut water can be suggested as a better medium for coculturing of P. indica and Pseudomonas fluorescens strains. A pot culture experiment was undertaken to study the effect of the different treatments on growth promotion of chilli. The experiment was laid out in CRD with six treatments and three replications and observation was taken in 15 days. The treatments comprised fungal and bacterial endophytes along with combinations of both fungal and bacterial endophytes and an uninoculated control. Different parameters like plant height, number of leaves, number of branches, number of fruits/plant, fresh fruit yield, fresh shoot weight, dry shoot weight, fresh root weight, dry root weight, days to flowering, days to fruit set and percentage root colonisation by Piriformospora indica were evaluated. Maximum plant height was recorded in the treatment with mixed inoculation of Piriformospora indica and Pseudomonas fluorescens PN026 without any significance in statistical data. A significantly increasing trend was observed in number of leaves with mixed inoculation of Piriformospora indica and Pseudomonas fluorescens PN026 from 45th day after transplanting and it was statistically on par with the treatment consisting of Piriformospora indica alone. Number of branches were found to be higher with mixed inoculation of Pirifomospora indica and Pseudomonas fluorescens PN026. There was no significant difference in the fresh weight and dry weight of shoot, whereas the fresh and dry weight of root (21.13 g and 8.26g respectively) were significantly higher in the plants treated with P. indica along with Pseudomonas fluorescens PN026. Number of fruits per plant (10.25/plant) and fresh fruit yield per plant (37.95g/plant) were recorded significantly higher with mixed inoculation of Piriformospora indica and Pseudomonas fluorescens PN026. The in vivo study disclosed that plants treated with Piriformospora indica along with Pseudomonas fluorescens PN026 were found to perform better than all other treatments. The mixed inoculation of Pseudomonas fluorescens AMB8 and P. indica had no additional advantage in plant growth in chilli. Piriformospora indica was able to successfully colonize in the plant roots applied with the bacterial endophyte. The present study revealed that plant growth promoting rhizobacteria, Pseudomonas fluorescens PN026 can be used along with Piriformospora indica, the root endophyte, for enhancing plant growth in chilli.
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    ThesisItemOpen Access
    Microbial inoculants for enhancing degradation of biosolid waste in aerobic composting
    (Department of Agricultural Microbiology, College of Horticulture, Vellanikkara, 2019) Shilpa, P; KAU; Girija, D
    Solid waste management is a major challenge throughout the world, especially in urban areas, due to the rapid growth of population along with urbanization. Earlier, centralized management of biosolid waste was being practiced. However, due to problems in transportation and segregation, management at source is being promoted. Aerobic composting has been practiced from time immemorial for recycling of biosolid waste, using various processes and containers. Recently, more importance is being given to bio- composting, considering the efficiency of microorganisms in enhancing degradation of organic substrates by their multiple mode of action. Hence, this study was taken up to explore microorganisms which can enhance the process of aerobic composting of biosolid waste. Isolation of potential decomposing microorganisms was attempted from different compost samples including coir pith compost, kitchen waste compost and Oushadhi ayurvedic compost. A total of 14 isolates were obtained from different compost samples. All the isolates were assigned names depending upon the type of microorganism and the source from which they were isolated. Based on the ability to degrade the chemical components in selective medium, four isolates (BaBc-1, BaCp-1, BaOu-1 and AcOu-1) and four reference cultures (Bacillus subtilis, Bacillus niabensis, Gongronella butleri and Trichoderma asperellum) were selected for quantitative assay. Enzyme assay was carried out for selected isolates and the isolate G. butleri exhibited highest cellulase activity. BaBc-1, B. subtilis and BaOu-1 recorded significantly higher β- 1, 3 glucanase activity. Glucosidase activity was found to be significantly high in G. butleri, T. asperellum, BaBc-1 and B. subtilis. Significantly higher laccase, amylase and pectinase activity was recorded in BaOu-1, BaBc-1 and AcOu-1 respectively. Maximum protease activity was recorded in fungal isolates G. butleri and T. asperellum. Potential isolates were further subjected to cultural, morphological, biochemical and molecular characterization. The isolate BaBc-1 showed maximum homology to Bacillus subtilis, BaCp-1 to Bacillus cereus, BaOu-1 to Bacillus sp. and the actinomycete isolate AcOu-1 to Streptomyces roseofulvus. The compatible combinations of selected isolates with high enzyme activity were selected for formulation of microbial consortia and the consortia were evaluated for degrading vegetable waste under in vitro condition. All the inoculated treatments showed faster degradation compared to uninoculated control. Based on visual observations, per cent weight reduction, enzyme activity and microbial population on 21 DAI in flask culture, consortium II (B. subtilis BaBc-1+ T.asperellum+ Bacillus sp. BaOu-1) and consortium IV (B. subtilis+ G. butleri +B. subtilis BaBc-1) were selected for pilot scale experiment. The efficiency of selected consortia was evaluated in KAU smart biobin along with cow dung slurry and uninoculated treatment. In T1 (B. subtilis BaBc-1+ T.asperellum+ Bacillus sp. BaOu-1) compost formation was initiated within 17 days after inoculation. Based on the volume reduction, duration of composting process, yield of compost, microbial population and phytotoxicity of compost, consortium I (B. subtilis BaBc-1+ T.asperellum+ Bacillus sp. BaOu-1) was selected as best performing consortium in KAU smart biobin. Hence, this consortial formulation was selected for large scale experiment in Thumburmuzhi composting units. Cow dung was used as inoculum in positive control and uninoculated treatment served as negative control. The treatment T1 (B. subtilis BaBc-1+ T.asperellum+ Bacillus sp. BaOu-1) recorded maximum temperature (640C) during composting period, faster volume reduction and maximum microbial population in compost. Based on these results, T1 was found to be the best treatment in Thumburmuzhi composting unit. The study revealed that, consortial formulation of B. subtilis BaBc-1, T. asperellum and Bacillus sp. BaOu-1 could be exploited for enhancing degradation of biosolid waste in aerobic composting. This can be used in future for the management of agricultural and municipal solid waste. The plant growth promoting (PGP) activities of these isolates could be an added advantage in improving the growth and yield of plants.