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ThesisItem Open 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, DBlack 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.ThesisItem Open Access Evaluation of bioinoculant consortia for organic cultivation of ginger(Department of Agricultural Microbiology, College of Horticulture, Vellanikkara, 2015) Haritha, T R; KAU; Surendra Gopal, KGinger is one of the major spice crops of Kerala. Several constraints hinder its production and the major one is its succeptibility to various diseases. This has led to the use of high doses of chemical pesticides. Ginger is also a highly nutrient exhausting crop, which demands use of high doses of fertilizers. Although, chemical fertilizers and pesticides are highly effective, their continuous use has led to many environmental problems. Alternative approaches are therefore needed to minimize the use of chemical fertilizers and agrochemicals, since ginger is directly consumed. Emphasis should be given for the organic cultivation of ginger. The role of bioinoculants assumes special significance in this context. The magnitude of plant growth promoting activities is reported to be better in the case of consortia or mixed cultures than single strain. Therefore, bioinoculants formulation consisting of biofertilizer and biocontrol agent would be a novel technology which will provide nutrients as well as manage diseases. The literature on the use of consortia of biofertilizers and biocontrol agents are scanty. Hence, a study was undertaken on “Evaluation of bioinoculant consortia for organic cultivation of ginger’’ with an objective to evaluate and find a suitable consortia of bioinoculants for ginger cultivation. Azospirillum lipoferum, phosphate solubilizing bacteria (PSB), potash solubilizing bacteria (KSB), Pseudomonas fluorescens and Trichoderma viride cultures of KAU were used for the study. When tested for their compatibility with each other, it was found that Azospirillum lipoferum, PSB, KSB, Pseudomonas fluorescens were mutually compatible with each other. However, Pseudomonas fluorescens and Trichoderma viride were incompatible. Based on the compatibility test, consortia consisting of biofertilizers alone and biofertilizer + biocontrol agents were selected for the field evaluation. The consortia KAU-AZO +KAU- PSB + KAU-KSB, KAU-AZO + KAU-PSB + KAU-KSB + KAU-PF and KAU-AZO + KAU-PSB + KAU-KSB + KAU-TV were selected. These consortia were compared with individual bioinoculants, vii PGPR Mix I, PGPR Mix II, Organic adhoc package (KAU, 2009) and POP recommendation (KAU, 2011). Based on the overall biometric and yield parameters, T11 (Organic adhoc package, KAU, 2009) was found to be best among all the treatments evaluated. Among the consortia, days taken for germination was minimum (17.33) in the case of T6 (KAU-AZO+KAU-PSB+ KAU-KSB) while both T7 (KAUAZO+ KAU-PSB+ KAU-KSB +KAU-PF) and T8 (KAU-AZO+KAU-PSB+ KAUKSB +KAU-TV) recorded the highest per cent (91.67) germination. Plant height, number of tillers, and yield were maximum in T8 (KAU-AZO+KAU-PSB+ KAUKSB +KAU-TV). With regard to disease and pest incidence, T8 (KAU-AZO + KAU-PSB + KAU-KSB + KAU-TV) recorded minimum per cent rhizome rot (5.23%) incidence. However, T7 (KAU-AZO+KAU-PSB+ KAU-KSB +KAU-PF) recorded the minimum per cent incidence (5.21) of Rhizoctonia leaf blight and shoot-borer (4.17 %). The T6 treatment (KAU-AZO+KAU-PSB+ KAU-KSB) was the least succeptible (6.25 %) to rhizome maggots. At the time of harvest, T7 (KAU-AZO+KAU-PSB+KAU-KSB +KAU-PF) and T8 (KAU-AZO+KAU-PSB+ KAU-KSB +KAU-TV) recorded the lowest pH (5.30) and T8 (KAU-AZO + KAU-PSB+ KAU-KSB+ KAU-TV) registered highest available nitrogen (188.68 kg/ha) among the consortia. However, highest organic carbon (1.55 %) and available phosphorus (37.44 kg/ha) was recorded in T7 (KAU-AZO+KAU-PSB+KAU-KSB+ KAU-PF). All the consortial treatments were on par with each other with respect to available potassium content in soil. Population of inoculated individual and consortial isolates in soil indicated a decreasing trend till the time of harvest. The population decreased from 108 cfu/ml to 104 cfu/ml in the case of bacteria and 106 to 10 3 cfu/ml in the case of fungus. The Benefit:Cost ratio was maximum (1.65) in the case of T8 (KAU-AZO + KAU-PSB + KAU-KSB + KAU-TV). viii The present studies clearly indicated that consortia inoculated plants performed better than the individual isolates. The consortia of bioinoculants treated plants were on par, but T8 (KAU-AZO + KAU-PSB + KAU-KSB + KAUTV) was the most promising treatment among the consortia. Therefore, it can be concluded that bioinoculant consortia consisting of bioagents for nutrient fixation /solubilization (N, P, K) and fungicidal effect would be a novel technology in present-day agriculture.ThesisItem Open Access Isolation characterization and evaluation of soil microorganisms for bioremediation of chlorpyrifos(Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2014) Karolin, K P; KAU; Meenakumari, K SThe present study on “Isolation, characterization and evaluation of soil microorganisms for bioremediation of chlorpyrifos”, was conducted in the Department of Agricultural Microbiology at College of Agriculture, Vellayani during 2012-14, with the objective of isolation, characterization and evaluation of microorganisms for chlorpyrifos degradation, development of consortia and evaluation of bioremediation potential against chlorpyrifos in vivo. Microorganisms capable of degradation of chlorpyrifos were isolated by enrichment culture technique from identified locations with high residue levels of chlorpyrifos. In all, nineteen isolates comprising eleven bacteria, seven fungi and one actinomycete obtained were subjected to a preliminary screening based on the ability of isolates to utilize 50,100,200,400 and 800 ppm concentrations of chlorpyrifos at intervals of 7, 15, 20, 25, 30 DAI. The six isolates selected (M5, M6, M7, M10, M12, M17) were further evaluated for their ability to degrade different concentrations of chlorpyrifos based on population build up , analysis of chlorpyrifos residue and chloride released into the medium. The fungal isolates, M5, M6, M7 and M17 which recorded significant growth in terms of viable count, maximum reduction in chlorpyrifos residue and release of chloride were selected and subjected to morphological and molecular characterization. The isolates M5, M6, M7 and M17 were identified as Isaria farinosa, Aspergillus fumigatus, Trichoderma viride and Penicillium griseofulvum respectively. In order to develop a consortium, the compatibility of the selected fungal isolates - M5, M6, M7 and M17 was tested by co-culturing in liquid MSM and by dual culture technique. All the fungal isolates were compatible and no inhibition could be recorded. A consortium of the four fungal isolates was prepared in liquid formulation and its ability to degrade different concentrations of chlorpyrifos was studied under in vitro conditions on 25th day of inoculation. The percentage degradation of chlorpyrifos by the isolates increased with increase in concentrations, but showed a decline at 800 ppm. The percentage degradation of chlorpyrifos was higher in consortium compared to individual isolates under in vitro conditions. The developed liquid consortium was evaluated in sterilized soil spiked with 100 and 400 ppm concentration of chlorpyrifos with cowpea as the test crop. Significant reduction in all biometric characters was observed due to spiking with chlorpyrifos at 100 and 400 ppm concentrations. Application of consortium in soil spiked with chlorpyrifos enhanced all the biometric characters and reduced the residue of chlorpyrifos. The study also established efficient colonization of the chlorpyrifos degraders present in the consortium in the rhizosphere of cowpea.ThesisItem Open Access Utilization of free living diazotrophic bacteria from Wayanad as a biofertilizer(College of Horticulture, Vellanikkara, 2015) Fathima, Shaheena K M; KAU; Girija, DAgriculture is heavily dependent on the use of chemical fertilizers. However, excessive and unbalanced use of these inputs may lead to environmentalpollution and health hazards. Biofertilizers are biological fertilizers which play a key role in maintaining productivityand sustainability of soil, thereby protecting the environment. Hence, a study was undertaken to isolate, screen and evaluate free living diazotrophic bacteria to develop an efficient biofertilizer. Rhizosphere soils and plant samples were collected from ten healthy black pepper gardens of different locations of Wayanad district. A total of 43 free living diazotrophic bacteria were isolated on three different N-free media (Jensen’s agar, Beijerinckia agar and Ashby’s agar). Maximum population of nitrogen fixers was obtained on Jensen’s agar. Twenty predominant isolates were selected, including eleven isolates from rhizosphere soil and nine isolates from phylloplane, based on their growth characteristics on N-free media for further evaluation. Twenty selected isolates were screened under in vitro condition for growth, nitrogen fixation and acid tolerance. As pH was lowered from 7.0 to 5.0, a reduction in growth and nitrogen fixation was observed. However, among the isolates, two from the rhizosphere (NKdS and NPS-1) and two from phylloplane (NKPV-2 and NPPV) performed better. Twenty selected isolates were screened in vitro for plant growth promoting activities like production of IAA, HCN, siderophore and ammonia.The antagonistic activity of all the twenty isolates was tested against three soil borne plant pathogens Fusarium oxysporum, Rhizoctonia solaniand Ralstonia solanacearum. Isolates NkdS, NPPV and NKS-1 were found to exhibit antagonistic activityagainst Rhizoctonia solani.Six isolatesinhibited the growth ofRalstonia solanacearum. None of the isolatesrecorded antagonistic activity against Fusarium oxysporum. Four most promising diazotrophs including, two rhizosphere isolates (NKdS and NPS-1) and two phylloplane isolates (NPPV and NKPV-2) were selectedfor in xxx planta evaluation, based on nitrogen fixation ability, tolerance to acidic pHand PGPR activities. 16S rDNA sequence analysis was carried out and promising isolates were identified asMicrobacterium sp. (NKdS and NPPV); Cellulosimicrobium sp. (NPS-1) and Brevundimonas sp. (NKPV-2). In planta evaluation was carried out with tomato as the test crop. These isolates were applied alone, in combination with 75% N and with 75% N + 50% FYM. Diazotrophs were inoculated as seed treatment, seedling dip and soil/foliar application. Seeds treated withKAU Azotobacter formulation triggered faster germination compared to all other treatments. However, after 7 days of sowing, maximum number of seedling emergence (90.24%) was recorded in seeds treated with Cellulosimicrobium sp. Plant height, number of leaves, fresh weight and volume of root, fresh weight and dry weight of plants,yield per plant, average fruit weight and number of fruits were observed to be maximum in treatment T12 (Microbacterium sp.+ 75% N + 50% FYM). T11 (Cellulosimicrobium sp. +75% N + 50% FYM) recorded maximum girth of stem, leaf area and minimum days to flowering. Total nitrogen content of index leaf and plant was maximum in T12 (Microbacterium sp. + 75% N + 50% FYM). The same treatment recorded maximum nitrogen content in soil at 60DAS. At harvest, T11 (Cellulosimicrobium sp. + 75% N + 50% FYM) recorded highest nitrogen content in soil. Population build up of bioinoculants in soil was higher in presence of FYM.Endophytic colonization was higher in root, in case of soil application of bioinoculants and higher in leaves, in case of foliar spray. The study revealed that novel, free living diazotrophic bacteria like Microbacterium and Cellulosimicrobium could be exploited as biofertilizer formeeting 25% of the nitrogen requirement. Results also indicated the importance of application of organic manure along with the isolates, for their best performanceThesisItem Open Access Screening of acid-tolerant consortia of Azospirillum and Phosphate solubilizing bacteria from Lateritic soils(College of Horticulture, Vellanikkara, 2014) Athulya, M M; KAU; Surendra, Gopal KA study was undertaken on “Screening of acid-tolerant consortia of Azospirillum and phosphate solubilizing bacteria from lateritic soils”. The main objective was to develop an acid-tolerant and efficient consortium of Azospirillum and phosphate solubilizing bacteria (PSB) for acidic soils of Kerala. Lateritic soils with acidic pH were collected from ten different locations of Thrissur district for the isolation of Azospirillum and phosphate solubilizing bacteria. The pH of the soil ranged from 5.22 (Vellanikkara) to 6.51 (Madakkathara). The highest population of Azospirillum was recorded in soils of Madakkathara (MD) and Mulayam (MU) (1.1x105 MPN g-1). However, Chelakkara (CH), Elanad (EL) and Perumpilavu (PV) recorded least population (0.9x104 MPN g-1). Highest population of PSB was obtained from Mulayam (12.7x106 cfu g-1) and least PSB population was obtained from Perumpilavu (PV) (1.0x105 cfu g-1). The population of both Azospirillum and phosphate solubilizing bacteria were higher in rhizosphere soil than non-rhizosphere soil. A total of six isolates of Azospirillum and 35 isolates of PSB were obtained. The isolates AND-4 (Azospirillum from Nadavarambu) and AMU-2 (Azospirillum from Mulayam) were acid-tolerant upto pH 4.5. Similarly, PMD-7 (PSB from Madakkathara) and POL-1 (PSB from Ollur) were also tolerant upto pH 4.5. The AND-4 isolate of Azospirillum sp. fixed highest amount of nitrogen (17.94 mg of N g-1 of malate) followed by AMU-2 isolate (14.58 mg of N g-1 of malate). The isolate AMU-2 showed maximum IAA production (51.95 µg ml-1) followed by AND-4 (30.53 µg ml-1). The PMD-7 was the most efficient P-solubilizer (207.22 µg ml-1) followed by POL-1(187.78 µg ml-1). The PMD-7 isolate produced maximum IAA (33.07 µg ml-1). Sixteen combinations of Azospirillum and PSB were compatible. The three consortia selected based on acid tolerance, efficiency and compatibility were AND-4 (Azospirillum sp.) + PMD-7 (Acinetobacter sp.), AMU-2 (Azospirillum sp.) + PMD-7 (Acinetobacter sp.) and AND-4 (Azospirillum sp.) + POL-1 (Bacillus megaterium). Among the consortia, Azospirillum sp. + Bacillus megaterium (T4) was found to be the most efficient in enhancing the growth of okra under acidic pH based on biometric and yield parameters. However, the POP recommendation (T1) recorded maximum plant height, maximum number of fruits, fruit yield and minimum days for flowering. The population of Azospirillum and PSB increased at the time of harvest in all the treatments except in T7 (Absolute control). The population of Azospirillum was highest in T3 (Azospirillum sp. + Acinetobacter sp.) (23.38x103 MPN g-1) and minimum in T7 (Absolute control) (7.73x103 MPN g-1). The population of PSB was highest in T3 (Azospirillum sp. + Acinetobacter sp.) (6.43x105 cfu g-1) and minimum in T7 (Absolute control) (2.08x105 cfu g-1). The maximum nitrogen uptake was recorded in T1 (KAU POP recommendation) (0.99 g plant-1). Among the consortia, T4 (Azospirillum sp. + Bacillus megaterium) (0.81 g plant-1) and T3 (Azospirillum sp. + Acinetobacter sp.) (0.80 g plant-1) recorded highest nitrogen uptake. The maximum uptake of phosphorus was noticed in T1 (KAU POP recommendation) (0.41 g plant-1) followed by T4 (Azospirillum sp. + Bacillus megaterium) (0.36 g plant-1). The most efficient isolates AND-4 and AMU-2 were identified as Azospirillum sp. The most efficient PSB isolates POL-1 and PMD-7 were identified as Bacillus megaterium and Acinetobacter sp. respectively. The present studies clearly showed that the acid-tolerant and efficient consortium of Azospirillum + Bacillus megaterium (T4 treatment) performed better under pot culture studies, which was on par with chemical fertilizer. However, further studies are needed to evaluate its efficiency under field conditions.