Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502 Vol. 3(IVC-2014), 59-63 (2014) Res. J. Recent. Sci. International Science Congress Association 59 Azotobacter species as a Natural Plant Hormone SynthesizerP.S. VikheDepartment of Botany, P.V.P. College of Arts Science and Commerce, Pravaranagar (Loni), Arts Science and Commerce College Kolhar Available online at: www.isca.in, www.isca.me Received 30th June 2014, revised 15th September 2014, accepted 24th September 2014 AbstractAzotobacter plays an important role in increasing the development and physical condition of plants.Azotobacter as non symbiotic nitrogen fixing bacteria was isolated from six different rhizospheric soil samples were collected from the ten different villages of Pravara area. Isolation of Azotobacter spp was carried out using Ashbys liquid and were purified further. Each Azotobacter spp culture were recognized through microscope and biochemically tests were carried out and as per consequences obtain the spp were in, A. vinelandii, A.insignis, A. chroococcum , A.agilis. A. beijerinckia, A.macrocytogenes.Results strongly supports that the Azotobacter spp in combinations C+M+I and B+V+A are significantly effective to improve the crop yield. Their efficiency equal to the recommended dose of fertilizers. Highest gibberllic acid in production AzT, kinetin in A, NAA in A1 3 andIAA. This supported that mutual association of Azotobacter spp. lead to synthesis of growth hormone, nitrogen, phosphate uptake and modification in rhizospheric interactions with respect to wheat varieties. Results strongly supports that the Azotobacter spp combinations C+M+I and B+V+A are significantly effective to improve the crop yield. Their efficiency equal to the recommended dose of fertilizers. The Azotobacter spp are capable of producing two phytohormones. A. chroococcum is found in the rhizospheric which posses the ability to enhance plant growth when applied to roots applied on tubers seed dressed are functioning as a plant growth promoting rhizobacteria. It was further experimentally proved that plant response to inoculation with PGPR enhance nitrogen and parameters such as plant dry weight, development, morphology of root system, grain yield, protein and mineral nutrient content. They further observed that plant response to inoculation with PGPR enhance nitrogen and parameters such as plant dry weight, development, morphology of root system, grain yield, protein and mineral nutrient content.Keywords: Azotobacterspp, PGPR, Rhizospheric soil, HPLC, microbial population. Introduction Three main groups of compounds are auxins, gibberellins (GAs) and cytokinins. Auxins, cytokinins and gibberellins are activators of growth and developmental processes. Generally, there is no direct correlation between the absolute concentrations of endogenous phytohormones their role in plants. Azotobacter is free living bacteria and is widely distributed in different types of soils. Azotobacter is a nitrogen fixer so it enhances the plant development, produces plant growth regulators and increases mineral phosphates solubility by producing hydrogen cyanide, siderophore. Azotobacter has antifungal activity it produces antibiotics. Response of Azotobacter varies with wheat genotypes. Such variation is generally attributed to variability in root characters, vernalization, photoperiodic response, tillering, nutrient uptake, photosynthesis duration and grain yield1,2 The response of Azotobacter inoculation with varieties led to low output yieldand to draught tolerant genotypes led to increase in grain. So, proper selection of Azotobacter strains and wheat variety is most important. Therefore research should be intensified to establish an effective, associative/ endophytic system in wheat by identifying effective bio-inoculants strains and respective wheat genotypes for better understanding and use of PCR based molecular markers to identify genotype-microbe interactions Mutant strains of Azotobacter shows higher increase in yield of grain, straw and root biomass. Inoculation of wheat varieties with mutant of Azotobacter chroococcum led to greater uptake of NPK. Survival rate of mutant in the rhizosphere is also higher than normal strains. Review of literature: Azotobacter as a Plant Growth Promoting Rhizobacteria (PGPR) Apart from nitrogen fixation, Azotobacter synthesizes auxins, cytokinins and gibberellins like substances. Three gibberellin like substances were detected in an Azotobacterchroococcum strain. The amount was in between 0.1 to 1g/ml in 14 days old culture. These hormones are originating from rhizosphere of root surface and affect the growth of plants. Hormones enhance phosphate solublizationinfluence nutrient uptake by increasing phosphotase activity, increases water and mineral uptake, production of Amino acids as well as Vitamin which are biologically active substances. Apart from phytohormone synthesis, Azotobacter also produces certain extra cellular substances like vit.B-12, thiamine, riboflavin, pyridoxine, biotin, cyanocobelamaine, panthothenic acid and folic acid. Mutant strains of Azotobacter shows higher increase in yield of grain, straw and root biomass. Inoculation of wheat varieties with mutant of Azotobacter chroococcum led to greater uptake of NPK. Survival rate of mutant in the rhizosphere is also higher than normal strains. It has been Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(IVC-2014), 59-63 (2014) Res. J. Recent. Sci. International Science Congress Association 60 proved that some mutants of Azotobacter can fix nitrogen if surplus amount of ammonia is present which is interrelated to the industrial application of Azotobacter. The mutant of Azotobacter plays animportant role in because hinders mobilization in alginate beads and it also give the opportunity to generate ammonia (plant manure) and production of antibiotics proved that when maize crop is inoculated with Azotobacternitrogen fixation, production of phytohormones increased the uptake of food and also one of the reason to increase crop production and yield10,11. It have been recommended that oak seedlings inoculated with Azotobacter results in alternative growth responses 12. It have been stated that after inoculation of azotobacterin to barley (Hordeom vulgare) leads to growth of plant height , dry matter content, soil nitrogen content in the lands which are having the deficiency of nitrogen in soil13. Ithas also been observed that Rhizoctonia solani growth is inhibitedby producing an antifungal antibiotics which inhibitsso itmainly acts as a Plant Growth Promoting Rhizobacteria (PGPR). Material and Methods Collection of soil samples: Soil samples of ten different wheat fields were collected from different sites in Pravara area. These soil samples are air dried, cleaned and sieved to fine particles Isolation of Azotobacter spp: 1g soil samples were inoculated in sterile Ashby’s medium and incubated for 8 days at 25C C. Azotobacter spp. were identified with microscopic observations on pigment basis and purified by transfer and retransfer on the same medium14. Fertilizer dose: As per recommended dose of NPK (40:50:100 kg/ha.) chemical fertilizers like urea and single super phosphate, were worked out as per treatment and plot size. Mutation: Four days mix culture of Azotobacter spp.were used for mutation treatment. 20 ml cultures were poured in separate sterilized petriplates in laminar air flow in aseptic condition. The plates were exposed to U.V radiation for 20 min. 5ml treated culture broth was taken out from respective cultural plates after 5 minutes interval and poured in sterile Ashbys liquid medium and incubated for 7 days. Mutants were identified on pigment basis and their effect was studied by seed dressing in the field experiment15,16. Field study: Field experiment was carried out to study the effectiveness of Azotobacter alone and in mix combination treatment with graded levels of nitrogen on growth and yield of wheat Experiment Design: Two wild and mutated combination of C+M+I and B+V+A were selected for genotypical study. These effective formulations were freshly grown on Ashbys medium for 7 days. Four local varieties HD 2189, Lok 1, 496 and Trimbak were selected for field experiment. Seeds were sterilized with 0.1% HgCl for 5 min and consecutively washed with sterilized distilled water. After washing seeds were dressed with selected formulations of culture broth and dried in shade. Experimental plots were laid down in randomized block design with 6 treatments and 3 replicates and sowing was done at 22.5 cm row spacing. The effect of selective formulation was studied with different parameters viz % germination, number of tillers, height of tillers, fresh weight, dry weight, number of leaves, leaf area, chlorophyll content, spike length, spike weight, grain weight nitrogen and phosphorus uptakeHarvesting of Crop: Crop was harvested at 120 days after sowing. The straw and grain production of wheat were recorded Total dry matter: Plant samples were collected from each treatment at 30, 60 and 90 days after sowing. Samples were rolled in brown papers, kept in oven at 60C for12 hrs for drying and dry weight was recorded. The records acquired from field trials were subjected to statistical analysis by using standard means. Statistical analysis: From field experiments it observations noticed were subjected to statistical analysis by using standard methods17The standard error from the treatments and the critical difference at the 5 % level of significance were worked out and used for the comparison between treatments.Results and Discussion From the results it was observed that int able1,2 at 60 and 90 days maximum plant height, tillers, number of leaves, leaf area, fresh weight and dry weight was observed in mutated non mutated treatment. This supports that enhancement is due to mutualistic interactions of strains and synthesis of growth promoting hormones in combinations of strains. The results also indicates that effectiveness was dependant on the stages of crop growth. ConclusionFrom the experimental finding it was proved that C+M+I treatment proved to be significant for all wheat varities followed by B+V+A. Maximum average grain yield per plot recorded in Trimbak (1.0897kg) followed by nonmutated C+M+I, B+V+A, RDF, control treatments. This supported that mutual association of Azotobacter spp leads to synthesis of growth harmones, nitrogen uptake and increase in growth parameters. Therefore present investigation strongly recommended that one has to investigate suitable Azotobacter spp.combination with respect to locally adapted climatic conditions and wheat varities to increase growth yield and economy of farmer. Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(IVC-2014), 59-63 (2014) Res. J. Recent. Sci. International Science Congress Association 61 Table-1 (Average plant height (cm), Average number of leaves, Average number tillers of Average leaf area, Average dry weight, Average grain yield) at 60,90 Days Varities Treatment Average plant height Replicate Mean(cm) Average number of leaves Replicate Mean Average leaf area Replicate Mean(cm)Average number of tillers Replicate Mean Average dry weight Replicate Mean(gm) Average spike weight Replicate Mean(gm HD 2189 Az-T 73.80 7.4 18.4 30.00 2.40 4.77 Az-T 69.90 6.5 16.0 18.00 2.05 4.30 Az-M 70.87 7.1 23.8 29.67 2.15 5.00 Az-M 68.40 6.3 17.7 23.00 2.05 3.40 RDF 65.47 5.6 17.7 16.33 1.85 3.73 CONTROL 64.13 5.2 15.6 12.00 1.60 2.97 Varities Treatment Average seed Replicate Mean Average number of leaves Replicate Mean Average leaf area Replicate Mean(cm)Average number of tillers Replicate Mean Average dry weight Replicate Mean(gm) Average spike weight Replicate Mean(gm LOK1 Az-T72.47 7.0 25.9 23.00 2.95 4.30 Az-T69.07 5.6 11.4 18.33 2.35 3.30 Az-M77.50 6.7 13.3 20.00 2.55 3.63 Az-M69.67 5.6 11.2 18.07 2.30 2.97 RDF 67.67 5.6 13.3 19.00 2.10 2.87 CONTROL 61.07 4.5 10.00 14.67 1.65 2.63 Varities Treatment Average plant height Replicate Mean(cm) Average number of leaves Replicate Mean Average leaf area Replicate Mean(cm)Average number of tillers Replicate Mean Average dry weight Replicate Mean(gm) Average spike weight Replicate Mean (gm TRIMBAK Az-T 1 73.67 7.7 14.2 21.67 2.36 4.50 Az-T 2 70.87 5.8 12.8 17.00 1.60 4.17 Az-M 3 72.47 5.9 14.3 16.00 2.20 4.73 Az-M 4 65.67 5.5 12.4 14.67 1.85 3.17 RDF 50.23 5.5 11.8 15.00 1.80 3.10 CONTROL 40.33 4.5 9.4 14.00 1.70 3.07 Varities Treatment Average seed Replicate Mean Average number of leaves Replicate Mean Average leaf area Replicate Mean(cm)Average number of tillers Replicate Mean Average dry weight Replicate Mean(gm) Average spike weight Replicate Mean(gm LOK1 Az-T 1 72.47 7.0 25.9 23.00 2.95 4.30 Az-T 2 69.07 5.6 11.4 18.33 2.35 3.30 Az-M 3 77.50 6.7 13.3 20.00 2.55 3.63 Az-M 4 69.67 5.6 11.2 18.07 2.30 2.97 RDF 67.67 5.6 13.3 19.00 2.10 2.87 CONTROL 61.07 4.5 10.00 14.67 1.65 2.63 Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(IVC-2014), 59-63 (2014) Res. J. Recent. Sci. International Science Congress Association 62 Varities Treatment Average plant height Replicate Mean(cm) Average number of leaves Replicate Mean Average leaf area Replicate Mean(cm)Average number of tillers Replicate Mean Average dry weight Replicate Mean(gm) Average spike weight Replicate Mean(gm TRIMBAK Az-T 1 73.67 7.7 14.2 21.67 2.36 4.50 Az-T 2 70.87 5.8 12.8 17.00 1.60 4.17 Az-M 3 72.47 5.9 14.3 16.00 2.20 4.73 Az-M 4 65.67 5.5 12.4 14.67 1.85 3.17 RDF 50.23 5.5 11.8 15.00 1.80 3.10 CONTROL 40.33 4.5 9.4 14.00 1.70 3.07 Varities Treatment Average plant height Replicate Mean(cm) Average number of leaves Replicate Mean Average leaf area Replicate Mean(cm)Average number of tillers Replicate Mean Average dry weight Replicate Mean(gm) Average spike weight Replicate Mean(gm 496 Az-T 1 73.13 7.4 18.2 24.00 2.50 5.13 Az-T 2 72.07 6.4 11 13.33 2.00 3.67 Az-M 3 76.53 6.3 17.1 16.33 2.30 4.20 Az-M 4 71.53 6.0 11.0 12.67 2.00 3.60 RDF 70.93 4.4 9.6 12.00 1.85 3.17 CONTROL 60.20 4.0 14.2 11.00 1.55 2.93 References 1.Yadav K.S., Singh D.P., Suneja S., Narula N. and Lakshminarayana K., Effect of Azotobacter chroococcum on yield and nitrogen economy in wheat Triticum aestivumunder field condition, Environment Ecol,18, 109-113 (2000) 2.Behl R.K., Sharma H., Kumar V. and Narula N., Interaction amongst mycorrhiza, Azotobacter chroococcum and root characteristic of wheat varities, Journal Agronomy andcrop science, (189), 151-155 (2003) 3.Narula N., Behl R.K., Dudi H.R., Suneja S. and Lakshminarayana K., Response of wheat genotypes to inoculation under rain fed conditions, Rachis ICARDA), (17) 66-67 (1998)4.Barea J.M. and Brown M.E., Effect on plant growth produced by A paspali related to the synthesis of plant growth regulating substance, J Appl. Bacteriol, (37) 583-593 (1974)5.Brown and Burlingham S.K., Production of plant growth substances, J.Gen Microbiology,53, 135-144, (1968) 6.Ramos A., Barea J.M, Callaov, A phosphate dissolving and nitrogen fixing microorganism and its possible influence on soil fertility, Agrochimica, 16 345-350 chroococcum to crop plants, Indian J. Microbial,30(2), 221-224 (1972)7.Hoflich G., Wichc W. and Kuhn G., Plant growth stimulation by inoculation with symbiotic and associative rhizosphere microorganism. Expereientia, 50: 897- 905, Indian Society of Soil Science,46(3), 379-383 (1994)8.Bashan and Levanony H., Alterations in membrane potential and in proton efflux in plant roots induced by Azospirillum brasilense, Plant and Soil,137, 99-103 (1991)9.Terzaghi B.E., J. General Microbiol, 271–274 (1980) 10.Giacomodonato M.N., Pettinari M.J., Souto G.I., Mendez B.S. and Lopez N.I., A PCR based method for screening of bacterial strains with antifungal activity in suppressive soyabean rhizosphere, World journal of Microbiology, (2001) 11.Gonzalez-lopes J.M., V. Martinez-Toledo, Reina S. and Salmeron V., J. Technol And Environ Chem., (33), 69-72 (1991)12.Pendey R.K., Bahl R.K., R.T Rao P., J. Indian Forest, Dehra Dun, (112), 75-79 (1986) 13.Monib M., Abd-El-Malek Y., Hosny I. and Fayez M., J. microbial, (134), 243-248 (1979) 14.Mishustin E.N. and Shilnikova V.K., Free – living nitrogen fixing bacteria of the genus Azotobacter,In soil Biology, Reviews of Research, UNESCO Publication., Paris., 82-109 (1969)15.Gupta B.M. and Kleczkowska, A study of some mutation in a strain of Rhizobium trifolii, J. Gen. Microbiol, (35), 371-380 (1964) 16.Bose P. and Venkatraman G.S., Ultra violet light induced mutations in Rhizobium legumnosaram, Ind. J. Micro bio., (12), 99-86 (1972)17.Panse and Sukhatme, Statistical methods for agricultural workers, New Delhi, 1, 22 (1978) Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(IVC-2014), 59-63 (2014) Res. J. Recent. Sci. International Science Congress Association 63 Table-2 At 90 DaysVarities Treatment Average plant height Replicate Mean Average number of leaves Replicate Mean Average leaf area Replicate Mean(cm)Average number of tillers Replicate Mean Average dry weight Replicate Mean(gm) Average spike weight Replicate Mean(gm Average grain weight Replicate Mean(gm HD2189 Az-T84.0 7.4 21.7 30.00 2.80 6.10 0.978 Az-T82.13 6.6 11.8 18.00 2.35 6.00 0.897 Az-M92.60 7.1 21.2 29.67 2.60 6.90 0.947 Az-M75.89 6.3 14.6 23.00 2.20 5.20 0.892 RDF 75.1 5.6 13.5 16.33 2.00 5.00 0.793 CONTROL 67.33 5.2 12.5 12.00 1.70 4.80 0.752 Varities Treatment Average plant height Replicate Mean(cm) Average number of leaves Replicate Mean Average leaf area Replicate Mean(cm)Average number of tillers Replicate Mean Average dry weight Replicate Mean(gm) Average spike weight Replicate Mean(gm Average grain weight Replicate Mean(gm LOK1 Az-T 89.73 7.0 24.5 23.00 2.65 6.30 0.967 Az-T 83.13 6.0 17.5 18.33 2.45 5.33 0.811 Az-M 86.60 6.3 24.0 20.00 2.70 6.20 0.888 Az-M 73.50 5.6 17.1 18.07 2.25 5.20 0.824 RDF 67.53 5.0 14.4 19.00 2.05 5.10 0.726 CONTROL 65.37 4.5 10.5 14.67 1.90 4.00 0.701 Varities Treatment Average plant height Replicate Mean(cm) Average number of leaves Replicate Mean Average leaf area Replicate Mean(cm)Average number of tillers Replicate Mean Average dry weight Replicate Mean(gm) Average spike weight Replicate Mean(gm Average grain weight Replicate Mean(gm TRIMBAK Az-T 1 88.40 6.8 19.5 21.67 2.40 7.10 1.087 Az-T 2 73.00 5.8 16.8 17.00 1.95 5.50 o.898 Az-M 3 85.93 5.9 16.0 16.00 2.20 6.00 0.965 Az-M 4 88.53 5.5 14.7 14.67 2.00 5.20 0.829 RDF 78.37 5.0 12.5 15.00 1.90 5.00 0.819 CONTROL 70.33 4.5 15.5 14.00 1.65 4.70 0.780 Treatment Average seed Replicate Mean Average number of leaves Replicate Mean Average leaf area Replicate Mean(cm)Average number of tillers Replicate Mean Average dry weight Replicate Mean(gm) Average spike weight Replicate Mean(gm Average grain weight Replicate Mean(gm) 496 Az-T 1 80.47 8.1 19.6 24.00 2.60 6.90 0.839 Az-T 2 72.80 6.0 13.6 13.33 2.05 5.50 0.781 Az-M 3 83.13 6.7 16.7 16.33 2.35 6.50 0.844 Az-M 4 75.13 5.2 11.3 12.67 2.05 5.00 0.746 RDF 69.33 5.0 11.8 12.00 2.00 4.50 0.655 CONTROL 66.33 4.6 14.1 11.00 1.70 4.00 0.599