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ThesisItem Open Access Isolation and Screening of Nitrogen Fixing Paenibacillus spp. for Growth Promotional Traits and Their Effect on Growth and Yield of Maize (Zea mays L.)(University of Agricultural Science, Dharwad, 2017-01) Basavesha K.N.; Savalgi, V.P.ThesisItem Open Access Isolation, Characterization and Screening of Gluconacetobacter diazotrophicus From Various Crops and Their Influence on Growth and Yield of Maize (Zea Mays L.)(University of Agricultural Science, Dharwad, 2017-05) Rao, Hema C.; Savalgi, V.P.A total of 113 isolates of Gluconacetobacter diazotrophicus isolated from sugarcane, maize, pineapple and carrot roots through survey from Dharwad, Belagavi, Uttara Kannada, Haveri and Shivamogga districts of Karnataka during 2014, were characterized morphologically, physiologically and biochemically and subjected for in vitro nitrogen fixation which ranged between 14.01 to 147.1 µg mg-1. The highest N2 was fixed by the native isolate NFGS25 (147.1 µg mg-1). Twenty best isolates were screened for further functional characterization viz., P and Zn solubilization, phytohormone production, antagonistic activity and endoglucanase activity. The zone of solubilization of P and Zn (3.1 and 2.5 cm), IAA and GA production (11.54 and 10.1 µg ml-1) and endoglucanase activity (86 µg ml-1) were maximum in NFGS25. The highest per cent inhibition was exhibited by NFGS25 against Rhizoctonia bataticola (83.33%) and Exserohilium maydis (88.86%). Five best isolates (NFGS25, NFGS26, NFGM5, NFGM6 and NFGP7) were selected for screening of growth parameters and N uptake of maize in the pot trial. The results revealed that plant height, stem girth, root length, chlorophyll content, dry weight, per cent N and N uptake were significantly highest in the treatment inoculated with NFGM5 at 75 % RDN followed by NFGS25 at 75% RDN. The population of G. diazotrophicus was maximum in the treatment NFGM5 at 75 % RDN (7 x 104 cfu ml-1). Based on their performance in pot trial, two efficient isolates (NFGM5 and NFGS25) were selected for field trial and confirmed as G. diazotrophicus isolates by molecular characterization. The results of the field experiment conducted during kharif 2016 at MARS, Dharwad, revealed that all the growth and yield parameters (number of cobs, cob weight, grain yield per plant and test weight) were significantly highest in the treatment NFGM5 at 75% RDN followed by NFGS25 at 75% RDN. The maximum grain yield of 183.33 q ha-1 (15% higher than control) was observed in the treatment NFGM5 at 75 % RDN which was better than the reference strain Azospirillum ACD15 with 75% RDN. Thus it is evident that the inoculation of native isolates of G. diazotrophicus (NFGM5 and NFGS25) at 75% RDN promotes better growth, grain yield and saves nitrogenous fertilizer up to 25% of recommended dose in field condition.ThesisItem Open Access ISOLATION, CHARACTERIZATION, SCREENING AND SELECTION OF AN EFFICIENT AM FUNGUS FOR ASHWAGANDHA (Withania somnifera L.)(University of Agricultural Sciences, Dharwad, 2014-11) NAGARAJ S. B.; Dr. M. N. SREENIVASAAshwagandha (Withania somnifera L.) has tremendous medicinal values. The ashwagandha berries and leaves are locally applied to tumors, tubercular glands, carbuncles and ulcers. The main active constituents are alkaloids and steroidal lactones. These include tropine and cuscohygrine. In Ayurveda, Withania is widely claimed to have potent aphrodisiac, sedative, rejuvenative and life prolonging properties. The present study emphasized the isolation, characterization, screening and selection of arbuscular mycorrhizal (AM) fungi from ashwagandha growing areas of Karnataka. The efficient AM fungal isolates were tested at different phosphorus levels, Mycorrhizal dependency and mycorrhizal inoculation effect on ashwagandha was also worked out. Further, an attempt was made to produce manoxinic culture of AM fungus in artificial media. Additionally, the growth promoting ability of efficient AM fungal isolate was evaluated in pot as well as in field condition. The results of this study clearly brought out AM-G-9a to be an efficient isolate of AM fungus for ashwagandha as it might have a better adoptability to the local conditions. The inoculation of this isolate resulted better plant nutrition, alkaloid content, growth and yield of ashwagandha in both pot and field condition. The shelf life of soil based inoculum was 3 months at room temperature while it was 5 months under refrigerated condition.ThesisItem Open Access Bioprospecting of pectinase enzymes from fungi(UAS Dharwad, 2013) Dushyantha D. K.; K. S. JagadeeshThe present study was conducted to isolate, and characterize fungi from diverse natural sources capable of producing pectinases. Based on the preliminary screening of 100 isolates, 48 isolates were found positive for pectinase production. Out of them, isolates RBF96 (3.25), RC27 (3.21) and DLP51 (3.21) possessed the highest potency index values compared to the reference strain Aspergillus niger MTCC1344 (2.99). Following the PCR based molecular methods, the isolates were identified as Aspergillus niger, Aspergillus oryzae and Penicillium citrinum respectively. Solid state fermentation (SSF) was carried out using various locally available agro-wastes as the carbon source. Citrus peel was identified as the most suitable substrate. The particle size of 2 mm, moisture level of 65 per cent, incubation temperature of 35oC, inoculum level of 1 x 107 spores g-1 and the incubation time of six days were found optimum. After purification the maximum specific activity of 83.06 U μg-1 protein was obtained for polygalacturonase (PG) by A. niger RBF96 with seven fold increase in purification. Similarly, about six folds and five folds increases in purification were obtained for pectin lyase (PL) and pectin esterase (PE) in P. citrinum DLP51 and A. oryzae RC27 respectively. The molecular weights of PG, PL and PE, as determined by SDSPAGE, were 45 kDa, 37 kDa and 31 kDa respectively. The purified PG showed maximum stability at pH 4.5, whereas, PL and PE showed at pH 6.5 and 5.0 respectively. The temperature of 50oC was found optimum. The orange pulp treated with (PG+PL+PE) at 100 U resulted in the highest juice yield (90.17%) and the highest clarity (96.28%). Similarly, in guava this treatment resulted in the highest juice yield (66.75%) and maximum clarity (37.57%). The organoleptic evaluation proved that pectinases could be used to improve the sensorial attributes of fruit juices.ThesisItem Open Access Studies on Ping Pigmented Facultative Methylotrophs of Major Chilli Growing Areas of North Karnataka(UAS Dharwad, 2013) Pesal Savitha; M. N. SreenivasaInvestigations were carried out to study the plant growth promotional ability and management of anthracnose by native pink pigmented facultative methylotrophs (PPFMs) of major chilli (Capsicum annuum L.) growing areas of North Karnataka. As many as 200 PPFM isolates were isolated and subjected to rapid screening for antibiosis against Colletotrichum capsici (C. capsici) and qualitative assay for production of Indole acetic acid (IAA). They were characterized based on morphological and biochemical characteristics. Selected isolates were further screened for beneficial characters. Highest IAA and GA production was recorded in PPFM6 (19.77 and 128.28 μg/ml of culture filtrate respectively) and highest zeatin production was recorded in PPFM170 (2.54 ıg/ml). Some of the isolates were positive for phosphate solubilization and antagonistic against Sclerotium rolfsii and Fusarium oxysporum. These antagonistic isolates produced catechol type of siderophores whereas production of HCN and volatile antibiotics was not displayed. Based on in vitro studies, six promising isolates were selected for pot culture experiments. The pathogen (C. capsici) was challenge inoculated and ISR activity was estimated. The defence molecules tested were significantly influenced by PPFM isolates. The peroxidase and PALase activity was increased from one DAI up to 7 DAI whereas chitinase, PPO and phenol contents increased up to 5 DAI, after which gradual decrease was noticed. Inoculation of PPFM isolates to chilli crop significantly improved plant height, dry matter and chlorophyll content. Significantly lesser disease incidence, more yield and more capsaicin content was observed with application of carbendazim followed by inoculation of PPFM6 + PPFM170 + PPFM35 and PPFM6 both under pot culture and field conditions. The results of the present study clearly indicated that PPFM6 is a very good plant growth promoter and have biocontrol efficiency against C. capsici.ThesisItem Open Access Functional Analysis and Molecular Characterization of Salt-Tolerant Microorganisms of Agricultural Importance(UAS Dharwad, 2012) Mahantesh G. Mudenoor; A.R. AlagawadiSalt-tolerant agriculturally important microorganisms (AIMs) isolated from stressecosystems of Karnataka were identified and assessed for their functional properties under salt stress. The biochemical basis for salt tolerance was also analyzed under salt stress. Besides, their plant growth promotional activities were assessed at different soil ECe levels in wheat under green house as well as field conditions. Plant growth promotional traits (N2 fixation, P-solubilization, phytohormone production) of each isolate were found to be affected with increased addition of NaCl to the growth medium. The amount of N2 fixed by the isolates, in general, was found to decrease with increase in NaCl concentration. While, the amount of Pi released from TCP was found to increase with increase in NaCl concentrations up to 7.5 per cent and declined thereafter. It was observed that, all the isolates were found to accumulate higher amounts of compatible solutes (proline and glycine betaine) and sugars in the presence of increasing NaCl concentrations as compared to control (without added salts), besides maintaining their intracellular Na to K ratio between 0 to 0.5. The field performance of salt-tolerant AIMs was assessed in wheat (cv. DWR-162) under gradient of soil salinity. In general, the inoculation treatments significantly enhanced both growth and yield parameters over un-inoculated control (UIC) at all the soil ECe levels tested. Field efficacy of salt tolerant AIMs gave 6.5 to 27.8% increase in dry matter production and 13.9 to 80.5% increase in grain yield of wheat over UIC at soil EC of 2.4 - 14.3 dS/m. It was also observed that, activity of antioxidative enzymes (AOE) viz., catalase, superoxide dismutase and peroxidase were decreased with inoculation but increased with soil salinity. Inoculated wheat plants recorded significantly lower proline and polyols content but higher glycine-betaine and total sugar contents as compared to UIC plants. Similarly, inoculated plants also recorded significantly higher potassium content and K to Na ratio but lower Na content as compared to UIC plants. It was also observed that, inoculation of salt-tolerant AIMs recorded significantly higher microbial biomass (C and N) and soil enzyme activities over UIC. In conclusion, these data suggest that the salt-tolerant functional groups of AIMs tested in the present study can play an important role in conferring salt tolerance in crops.ThesisItem Open Access Plant Growth Promoting rhizobacteria-Induced Systemic Resistance Against ToLCV Disease in Tomato (Lycopersicon esculentum Mill.)(UAS Dharwad, 2012) Shefali Mishra; K.S. JagadeeshFifty rhizobacterial isolates were screened against ToLCV disease in tomato. Based on the disease severity control, ten isolates were selected for further studies. The presence of ToLCV in affected leaves was confirmed by both transmission electron microscopy and sequencing of viral coat protein specific gene. The mechanism of virus control was elucidated. All the isolates induced systemic resistance in tomato plants. These plants also showed the highest reduction in the insect population. The green house experiment revealed Pseudomonas 206(4) as the most promising isolate. Semi quantitative PCR analysis revealed lower viral load accumulation in the plants inoculated with Pseudomonas 206(4). The plants inoculated with this isolate also recorded maximum plant height, total biomass, chlorophyll content and fruit yield over the diseased control. Based on biocontrol potential and plant growth promotion, three efficient isolates were further evaluated in combination with chitosan both under glasshouse and field conditions. The rhizobacterial consortia + chitosan emerged as the best treatment in disease severity control, plant growth promotion as well as in increasing the yield both under glasshouse and field conditions in two consecutive seasons. The histopathological studies revealed more anatomical destruction in diseased plants as compared to rhizobacteria + chitosan treated plants. The root colonization ability of the most efficient PGPR strain 206(4) was assessed by SEM, which indicated that the roots inoculated with this isolate in combination with chitosan showed higher population than the isolate without chitosan. The subcellular changes due to ToLCV and rhizobacterial+chitosan inoculation, as studied by TEM , revealed complete loss of subcellular architecture in diseased cells and the cells appeared somewhat closer to normal cells in the treated leaves. Thus, the rhizobacterial consortia and chitosan, significantly reduced ToLCV-induced symptom severity and yield losses, and can be recommended as a component of integrated disease management systems.ThesisItem Open Access Studies on Bioethanol Production From Selected Agro-Residues(UAS Dharwad, 2011) T.H. Shankarappa; Geeta G. ShirnalliThe present study was conducted on bioethanol production from selected agroresidues with the objectives of achieving higher recovery of fermentable sugars through delignification and saccharification followed by fermentation of the derived sugars to bioethanol. Five agro-residues namely sugarcane bagasse, sugarcane tops, sugarcane trash, corn husk and corn stover of particle sizes 0.5, 1.0 and 10.0 mm were delignified by various pre-treatment methods such as alkali, autohydrolysis and lignolytic fungi. The treatment combination of 3.0% NaOH with 1210C temperature for one h on particle size 0.5 mm resulted in the highest recovery of cellulose and hemicelluloses in sugarcane bagasse (0.813 g/g), sugarcane tops (0.786 g/g), sugarcane trash (0.806 g/g), corn husk (0.806 g/g) and corn stover (0.806 g/g). The delignified substrates were further saccharified using cellulolytic fungi and their crude enzymes and commercial cellulase enzymes. Among these, the commercial cellulase enzyme with 15 U/g along with b-glucosidase (10 U/g ) and Xylanase (5 U/g) enzymes at 5% substrate produced significantly highest reducing sugars with per cent saccharification in sugarcane bagasse (93.17%), sugarcane tops (90.33%), sugarcane trash (84.61%), corn husk (80.61%) and in corn stover (80.80%) in 12 h of incubation period. The pre-treated substrates were fermented to bioethanol by using six microorganisms. The combined inoculation of Saccharomyces cerevisiae and Candida shihatae on pre-treated substrates resulted in the highest ethanol yield in all the substrates, sugarcane bagasse (278.40 mg/g) followed by sugarcane tops (262.75 mg/g), sugarcane trash (241.42 mg/g), corn stover (239.82 mg/g) and corn husk (232.36 mg/g). The scaled up study on bioethanol production with sugarcane bagasse under optimized conditions produced 223 g bioethanol from one kg pre-treated substrate. Thus, it can be concluded that the substrates need to be alkali pretreated followed by treatment with commercial enzymes and further conversion to ethanol. The combined inoculation of the yeast cultures yields maximum ethanol.ThesisItem Open Access Isolation, Characterization and Development of Bacillus thuringiensis Formulations Against Diamond Back Moth (Plutella xylostella L.)(UAS Dharwad, 2011) Geeta D. Goudar; A.R. AlagawadiTotal of 80 B. thuringiensis isolates were obtained from 396 samples collected from the Western Ghats of Karnataka, out of which 44, 27 and 9 isolates were obtained from the samples collected in Uttara Kannada, Dakshina Kannada and Udupi districts respectively. All the isolates were subjected for morphological, biochemical and molecular characterization. Sixty five per cent of the isolates showed spherical type of crystals and only 3.75 per cent of the isolates had bipyramidal type of crystals. All the 80 isolates showed positive reaction for nitrate reduction, catalase production, voges-proskauer reaction and oxidase test, but all were negative for acid and gas production from glucose, arginine hydrolysis, chitinase activity and esterase activity. Rep-PCR fingerprinting of 80 isolates indicated that, the isolates grouped into two major clusters having seven sub-clusters. Bioassay against third instar larvae of Plutella xylostella showed 100 per cent mortality by the isolates UK-13C and UK- 762D. Efficacy of six different concentrations of crude protein of selected Bt isolates against DBM showed the mortality ranged from zero to one hundred per cent. The isolate UK-13C having LC50 value close to that of the reference strain HD1 and least LC99 was recorded by UK-762D. Thirteen efficient isolates showed good growth in presence of chemical pesticides Indoxacarb, Novaluron, Chlorofenapyr and Spinosad and no growth in the presence of Thiodicarb, Profenofos, Captan and Mancozeb. Three isolates and reference strain HD1 with least LC99 value were selected for preparation of wettable powder (WP) and water based flowable (WBF) formulations and were tested for their efficacy against DBM under shade house and field condition using cabbage as a test crop. In both shade house and field condition, Dipel recorded the highest mortality of insect larvae followed by HD1 WBF @ double the LC99 and UK-762D WBF @ double the LC99.
