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Subject: Agricultural Microbiology × End date: 2019 ×
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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
    Evaluation of native rhizosphere microflora for plant growth promotion and management of fusarium yellows in ginger
    (Department of Agricultural Microbiology, College of Horticulture, Vellanikkara, 2016) Rekha, K G; KAU; Surendra Gopal, K
    A study was undertaken on “Evaluation of native rhizosphere microflora for plant growth promotion and management of Fusarium yellows in ginger”. The main objectives were to enhance the growth and yield of ginger using native beneficial microorganisms isolated from wayanad district and to manage Fusarium yellows disease in ginger by using antagonistic microorganisms. Rhizosphere soils were collected from ten different locations of healthy and diseased ginger fields of Wayanad district. The predominant beneficial microorganisms namely nitrogen fixing bacteria, phosphate solubilizing bacteria, Bacillus sp., Pseudomonas fluorescens, Streptomyces sp., and Trichoderma sp. were isolated. The highest population of nitrogen fixers (1.4x104 cfu/g), fluorescent pseudomonads (9x104 cfu/g), and Trichoderma sp. (6x103 cfu/g) were obtained from Pulpalli location. Phosphate solubilizing bacteria (9x103 cfu/g) and Streptomyces sp. (6x104cfu/g) were highest in Kappikunnu and maximum population of Bacillus sp. (2.3x104 cfu/g) was recorded in Mayilumpadi. The isolates were screened for plant growth promotion and antagonistic activity under in vitro condition. Maximum nitrogen fixation was recorded in NFMh isolate (0.43 mg/g of sucrose utilized) among nitrogen fixers. Highest IAA (35.02 μg/ ml), HCN and siderophore production were recorded by NFAh (nitrogen fixer) isolate. Among the phosphate solubilizers, highest P- solubilization (450.00%) was by PSBAh isolate where as, IAA (33.07 μg/ ml) and HCN was shown by PSBMh isolate. Among the isolates of Bacillus sp. The maximum P- solubilization index (182.99%) was recorded by BsAh isolate and the least (137.43%) by BsKUh isolate. Highest IAA (14.54 μg/ml) was produced by BsMh isolate (Mayilumpadi) and the least (12.92 μg/ml) by BsAh isolate (Appade). Out of the ten isolates tested for HCN production, only three BsMh, BsKuh and BsPTd isolate resulted moderate level of HCN production. Only one isolates, BsMh positive for siderophore production. All the seven isolates of fluorescent pseudomonads the highest (241.57%) psolubilization was in PfMh isolate and lowest (190.11%) by the isolate PfKh. Highest IAA production was (16.75 μg/ml) by the isolate PfKh from Kappikunnu and the lowest (12.13 μg/ml) by PfPh isolate. Hydrogen cyanide production of all the isolates were tested and that isolates PfPh and PfKUh produced highest HCN under in vitro screening. Siderophore production was produced only by three isolates PfKh, PfMh, and PfPh. Among the eight isolates of Streptomyces sp. the maximum P- solubilization index (176.99%) was recorded by StrAh isolate and the least (153.76%) by StrPh isolate. Highest IAA (11.63 μg/ml) was produced by StrMh isolate (Mayilumpadi) and the least (10.41 μg/ml) by StrKUh isolate (Kurumankotta). Two isolates StrMh and StrPh resulted moderate level of HCN production. Four isolates StrMh, StrPh, StrAh and StrAd isolates resulted siderophore production. StrPh (Pulpalli) isolate resulted excellent siderophore production. All the isolates of Trichoderma sp. did not solubilized phosphorus and IAA under in vitro screening Two isolates TrPh and TrKUh produced moderate HCN under in vitro screening. Siderophore production was produced only by three isolates TrKh, TrMh, and TrAh. Selected isolates of Bacillus sp., Pseudomonas fluorescens, Streptomyces sp., Trichoderma sp., were tested for their antagonistic activity against the major soil borne pathogens of ginger viz., Fusarium oxysporum, Pythium aphanidermatum, Rhizoctonia solani and Ralstonia solanacearum under in vitro condition. The isolate BsAh showed maximum antagonistic activity against F. oxysporum (64.08 per cent) and P. aphanidermatum (15. 09 per cent) where as, BsKh isolate was antagonistic against R. solani (47.09 per cent) and BsMh isolate showed 43.81per cent inhibition against R. solanacearum. Among Pseudomonas fluorescens, PfKh showed 59.43 per cent inhibition against F. oxysporum where as PfAh isolate recorded 33.71 per cent inhibition against P. aphanidermatum, 27.20 per cent aginst R. solani and PfPh isolate showed 31.32 per cent inhibition against R. solanacearum. Among the isolates of Streptomyces sp., StrPh isolate recorded maximum inhibition (67.78 per cent) against F. oxysporum, 43.11 per cent inhibition against R. solani and 34.23 per cent against R. solanacearum. TrAh isolate (Trichoderma sp.,) showed highest (78.93 per cent) inhibition against F. oxysporum, 51.30 per cent inhibition against P. aphanidermatum and 43.27 per cent against R. solani. However, TrMh isolate shown 15.96 per cent inhibition against R. solanacearum. Three most efficient isolates of nitrogen fixers (NFMh, NFPh, NFKh), phosphate solubilizing bacteria (PSBPh, PSBKh, PSBAh), Bacillus sp., (BSMh, BSAh, BSKUh), fluorescent pseudomonads (PFKh, PFPh, PFAh), Streptomyces sp., (StrPh, StrMh, StrKh) and Trichoderma sp., (TrAh, TrKUh, TrPh) obtained under in vitro studies were further screened under pot culture studies. Among the isolates PSBPh (T4) was found to be the most efficient in enhancing the growth and yield of ginger where as the isolate TrKUh (T11) was the most efficient isolate for the management of Fusarium yellows disease. These two isolates were identified as Burkholderia cepacia (PSBPh) and Trichoderma harzianum (TrKUh). The present study clearly showed that Burkholderia cepacia (PSBPh) and Trichoderma harzianum (TrKUh) were effective for plant growth promotion and management of Fusarium yellows in ginger respectively. However, these isolates have to be evaluated for efficiency under field condition
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    ThesisItemOpen Access
    Functional diversity of beneficial microorganisms from the rhizosphere of black pepper in Wayanad
    (Department of agricultural microbiology, College of Horticulture,Vellanikkara, 2015) Athira, P S; KAU; Girija, D
    Black pepper (Piper nigrum L.) is a perennial, woody and flowering climber belonging to family Piperaceae. It is one of the important spice crops which provides major source of income and employment for rural households in Kerala. Wayanad dominated in pepper farming in the state about 20 years ago. Annual production of pepper was 40,000 tonnes in the mid-1980s, which comprised about half of India’s total pepper production. But recently, the production has declined drastically due to the infestation of pests and diseases. Foot rot caused by Phytophthora capsici and yellowing of black pepper are the major diseases devastating most of the plantations in Wayanad. However, some of the plants in the disease affected areas remain healthy which could be due to inherent activity of native rhizosphere microflora. The present study focused on assessing the functional diversity of beneficial microorganisms which could possibly be exploited for the benefit of plant growth. Four healthy gardens, four gardens each affected by foot rot and yellowing were selected for sample collection. Rhizosphere soil samples were collected from five healthy vines in each garden. Population of beneficial microbes in the rhizosphere soils of healthy and disease affected gardens were compared. In general, rhizosphere soil from healthy gardens recorded higher population of bacteria, fungi, phosphate solubilizers and fluorescent pseudomonads. A total of 207 isolates (including 112 bacteria, 32 actinomycetes and 63 fungi) were purified and maintained to study their plant growth promoting and antagonistic activities. Maximum IAA production (292.50 μg ml-1) was recorded by HPLBC-6 followed by HABC-3 (46.43 μg ml-1). The isolate HPLPSB-3 was the most efficient P solubiliser (162.7 μg ml-1) followed by HPLF-5 (161.3 μg ml-1). The isolate YPTN- 3 fixed maximum amount of nitrogen (46.92 mg of N g-1 of sucrose) followed by HVKN-6 (32.62 mg of N g-1 of sucrose). From the invitroexperiment, two most promising isolates each of IAA producers, phosphate solubilizers and nitrogen fixers were selected for preliminary screening for growth promotion on blackpepper cuttings. The isolate HPLPSB-3 (P solubiliser) recorded maximum sprouting, vine length, number of leaves, number of roots and roots fresh weight underinplanta screening for plant growth promotion. However, maximum root length was observed in HPLBC-6 (IAA producer). All the isolates were screened in vitro for their antagonistic activity against foot rot pathogen Phytophthora capsici. Among the bacteria, isolate HPLPSB-6 recorded maximum inhibition (69.27 %) of the pathogen. Among the actinomycetes, HVZACT-1 recorded maximum mycelial inhibiton of 66.66 %. Among the fungal isolates screened, maximum inhibition (75.17 %) was recorded by the isolate FPRF-3. The three most promising PGPM selected from preliminary in planta screening and three antagonists from in vitro screening were further tested for their efficiency in controlling foot rot disease in blackpepper nursery. Minimum disease incidence (6.23%) and severity (4.00 %) were observed in isolate FPRF-3. This was followed by actinomycete HVZACT-1 with disease incidence of 13.20 % and severity of 8.00 %. Maximum disease incidence and severity were observed in control with pathogen alone. In addition to biocontrol activity, FPRF-3 also improved plant growth parameters such as length of vine, number of leaves and roots. The selected growth promoting isolates HPLPSB-3, HPLBC-6 and YPTN-3 were identified as Acinetobacter grimontii, Providencia sp. and Paenibacillus sp. The three selected antagonists HPLPSB-6, HVZACT-1 and FPRF-3 were identified as Paenibacillus polymyxa, Streptomyces termitum and Trichoderma viride respectively. Based on in planta evaluation, Acinetobacter grimontii was considered as the best PGPM and Trichoderma viridethe most promosing antagonist against P. capsici. These isolates could be further exploited for improving the growth and managing foot rot disease, after validation under field conditions. The compatibility of PGPM with antagonists and chemical fungicides may also be evaluated.This is the first report of antagonistic activity of the actinomycete S. termitum against P. capsici causing foot rot disease in blackpepper.
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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
    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; KAU; Anith, K N
    The 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.
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    ThesisItemOpen Access
    Development of inoculant cultures of zinc solubilizing microorganisms
    (Department of Agricultural Microbiology, Vellayani, 2016) Aathira S Kumar; KAU; Anith, K N
    The study entitled “Development of inoculant cultures of zinc solubilising microorganisms” was conducted at College of Agriculture, Vellayani during the period 2014 - 2016 with the objective of isolation, characterization and evaluation of zinc solubilising microorganisms from soils of Kerala and to develop inoculant culture of the best zinc solubilising isolate. Microorganisms capable of solubilising zinc were isolated from Agroecological units 20, 21 (Wayanad) and 8 (Thiruvananthapuram) by serial dilution technique on Bunt and Rovira medium containing 0.1% insoluble zinc oxide. Ten isolates of bacteria capable of solubilising insoluble forms of zinc (zinc oxide) were obtained and allotted code numbers from ZSB-1 to ZSB-10. These were subjected to plate assay and broth assay in media supplemented with 0.1 per cent insoluble forms of zinc as zinc oxide or zinc phosphate. After three days of incubation of test plates, all the ten isolates solubilised zinc and produced clearing zone around the colonies on solid media. The size of the solubilisation zone ranged from 8.67 mm to13.33 mm in zinc oxide and from 1.00 mm to 5.33 mm in zinc phosphate incorporated medium. In broth culture, maximum solubilisation of zinc in both sources was observed on 30th day in the range of 35.91 ppm to 104.08 ppm in zinc oxide supplemented medium and 1.38 ppm to 4.15 ppm in zinc phosphate supplemented medium. The isolate ZSB – 4 showed maximum solubilisation of zinc in plate assay and broth assay. For soil incubation study, the isolate with maximum zinc solubilisation (ZSB – 4) was inoculated in zinc deficient soils and analysed for the soil chemical parameters and population dynamics of the bacterial isolate for a period of three months. The treatments were designed as T1 and T2 with Zn at two levels as ZnO, T3 with Zn solubilising culture alone @ 2 kg ha-1, T4 and T5 with Zn solubilising culture @ 2 kg ha-1 supplemented with Zn at two levels as ZnO and T6, the absolute control had no insoluble zinc supplementation and inoculation with the bacterial isolate. The isolate ZSB – 4 significantly increased the available zinc content in soil from 0.55 ppm to 9.47 ppm in treatment T4 (ZSB – 4 @ 2 kg ha-1 + zinc oxide @ 1 kg ha-1) during the incubation period. The same treatment registered the highest mean value for available phosphorus content, 12.09 kg ha-1 and 12.26 kg ha-1 respectively for the 2nd and 3rd month. There was an increase in oxidisable organic carbon content in the 3rd month for treatments T4 (1.44%), T5 (1.43%) and T3 (1.38%) and it was maximum in T4 which was statistically on par with T3 and T5. On the 3rd month, there was a decrease in the available boron content in soil for the treatments T3 (0.30 ppm) and T5 (0.34 ppm) when compared with previous months and the treatment T4 (0.33) maintained the level of boron. The total zinc content of the soil ranged from 0.05 % to 0.08 % during the entire three months and the mean values had no significant difference among treatments. The maximum colony count of 9.3 x 103 cfu g-1 of soil was recorded in the treatment T4 during the 3rd month which was on par with T5 (9.1 x 103 cfu g-1) and T3 (8.8 x 103 cfu g-1). The best isolate ZSB – 4 was subjected to molecular characterization and it was revealed that the organism is Bacillus cereus. Different carrier materials like talc, lignite, vermiculite, vermicompost and perlite were used in the present study to assess survival of ZSB – 4. Among the different carriers tested, the talc powder supported the maximum population of 3.9 x 108 cfu g-1 during the 3rd month. Based on the results of present study it can be concluded that application of talc based formulation of the zinc solubilising bacteria ZSB – 4 (Bacillus cereus) @ 2 kg ha-1along with zinc oxide @ 1 kg ha-1 was found to increase zinc content in soil after incubation. Based on the survival of the isolates in different carriers for the development of inoculant cultures, talc was found to be most suitable carrier for the formulated product.
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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.
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    ThesisItemOpen Access
    Assessment of soil temperature and soil parameters on the population and functional efficiency of pseudomonas fluorescens in the rhizosphere of Pokkali rice (Oryza Sative L.)
    (Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2018) Reshma Francis; KAU; Surendra Gopal, K
    Pokkali is a umque variety of rice that is cultivated organically in coastal wetlands of Kerala. Pokkali fields are situated close to the sea and therefore prone to flooding and salinity. Pokkali rice has enormous potential for tolerating most of the abiotic stresses. It is the only economic crop which can be grown in waterlogged environment while tolerating salinity. The present study on “Assessment of soil temperature and soil parameters on the population and functional efficiency of Pseudomonas fluorescens in the rhizosphere of Pokkali rice (Oryza sativa L.)”, was conducted in Rice Research Station, Vyttila, under Kerala Agricultural University during 2016 to 2018. Two plots (40 m2) were used for the study. One of the plots was maintained as control (without application of Pseudomonas fluorescens) and another plot with rice was treated with P. fluorescens. P. fluorescens (KAU) was applied as seed treatment (10g kg-1) just before sowing and soil application (2.5 kg ha-1) at 1 week after dismantling. The main objectives were to study the effect of soil temperature and soil parameters on the population and functional efficiency of P. fluorescens / fluorescent Pseudomonas in Pokkali rice. The rhizosphere soils of Pokkali rice were collected at monthly interval from June, 2017 to October, 2017. The population of fluorescent Pseudomonas sp. in the rhizosphere soils of Pokkali rice were recorded at monthly interval. The population was absent before the start of the experiment in treated plot. However, the highest population 3.3x102 cfu g-l) was recorded at 90 DAS (August, 2017). However, the lowest population was recorded at 60 DAS (July, 2017). The population was not found at 30 DAS (June, 2017), 120 DAS (September, 2017) and 150 DAS (October, 2017). In the case of control plot, population of fluorescent Pseudomonas was absent before the start of experiment. At 30 DAS (June, 2017) population of fluorescent Pseudomonas was 3.3 x 102 cfu g-1. However, no fluorescent Pseudomonas were found at 60 DAS (July, 2017), 90 DAS (August, 2017), 120 DAS (September, 2017) and 150 DAS (October, 2017). Out of the six isolates (VPJU, VPJL, VPAU1, VPAU2, VPAU3 and VPAU4) of fluorescent Pseudomonas obtained, all the isolates produced IAA and ammonia with varied intensity. Three isolates (VPAU1, VPAU3 and VPAU4) produced HCN and none of the isolates showed siderophore production. The correlation studies between soil temperature, soil pH, EC and C02 evolution with population, revealed that the population of fluorescent Pseudomonas was affected by soil pH and soil EC. However, soil temperature and C02 evolution did not affect the population of fluorescent Pseudomonas. The functional efficiency of the fluorescent Pseudomonas were correlated with soil temperature, soil pH, EC and C02 evolution It was found that soil temperature, soil pH, EC and C02 evolution did not affect the functional efficiency of fluorescent Pseudomonas. All the six isolates obtained m the study were identified and confirmed through 168 rDNA sequencing. The isolates VPAJU, VPAU1 and VPAU2 were identified as Pseudomonas sp. and VPAJL, VPAU3 and VPAU4 isolates were found to be Pseudomonas aeruginosa. The present studies indicated that the inoculated P.fluorescens did not survive in Pokkali fields. The population of fluorescent Pseudomonas showed negative correlation with soil pH and EC. However, soil temperature and C02 evolution did not had any effect on population. The functional efficiency of fluorescent Pseudomonas was not affected by soil temperature, soil pH, EC and C02 evolution. However, further studies on influence of soil edaphic factors and weather variables on host plant and P. fluorescens needs to be studied and develop a suitable PGPR for Pokkali rice.