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Earthern Pot Culture Method To Check The Stability Of Marine Azotobacter Sp In The Soil By Assessing The Growth Of Green Gram

INTRODUCTION
Nitrogen is a fundamentally important element in biologically mediated production and nutrient cycling processes. N2 containing constituents of organic molecules often confer bioactivity to these molecules. Major cellular, structural, and functional constituents have essential and often highly specific requirements for N2.Nitrogen fixation is the reduction of N2 (atmospheric nitrogen) to NH3 (ammonia). Free living prokaryotes with the ability to fix atmospheric dinitrogen (diazotrophs) are ubiquitous in soil. But our knowledge of their ecological importance and their diversity remains incomplete. In natural ecosystems, biological N2 fixation is most important source of N. The capacity for nitrogen fixation is widespread among bacteria and archaea. The estimated contribution of free-living N-fixing prokaryotes to the N input of soil ranges from 0-60 kg/ha /year (Burgmann et al., 2003). Dinitrogen (N2)-fixing microorganisms (diazotrophs) play important roles in ocean biogeochemistry and plankton productivity (Church et al., 2005). Nitrogenase catalyzes the reduction of nitrogen gas to ammonium in an ATP-and reductant dependent reaction. It is one of the best characterized metalloenzyme and is an excellent model for elucidating metalloprotein assembly. Nitrogenase is composed of two oxygen-labile metallo protein; dinitrogenase and dinitrogenase reductase

Nitrogen fixation can be an important source of nitrogen for biological productivity in the marine environment. Biological nitrogen fixation is catalyzed by the enzyme nitrogenase, which is possessed by diverse microorganisms representing virtually all phylogenetic groups. Interest in nitrogen fixation in the sea has usually been focused on rates of nitrogen fixation, but information on the types of species present with the capability for nitrogen fixation can be important for predicting nitrogen fixation rates in situ (Zehr et al., 1998).Understanding how fixed N regulates nitrogenase availability is necessary for devising strategies to increase the amount of ammonium synthesized by nitrogen fixing bacteria with the potential to be used in agriculture (Kennedy et al., 2004).

Azotobacter is used as a biofertilizer in the cultivation of most crops. Azotobacter is an obligate aerobic diazotrophic soil-dwelling organism with a wide variety of metabolic capabilities, which include the ability to fix atmospheric nitrogen by converting it to ammonia. Azotobacter naturally, fixes atmospheric nitrogen in the rhizosphere. There are different strains of Azotobacter each has varied chemical, biological and other characters. However, some strains have higher nitrogen fixing ability than others (Burgmann et al., 2003). Besides, nitrogen fixation, Azotobacter also produces, Thiamin, Riboflavin, indol acetic acid and gibberellins.

There is firm evidence that indole -3-acetic acid ,gibberellins (Brown, et al 1976) , and cytokinins all produced by plants and essential to their growth and development, are produced also by various bacteria which live in association with plants. There is also evidence that the growth hormones produced by the bacteria can in some instances increase growth rates and improve yields of the host plants (Brown et al., 1976). When Azotobacter is applied to seeds, seed germination is improved to a considerable extent, so also it controls plant diseases due to above substances produced by Azotobacter (Kader et al., 2002.)

MATERIALS AND METHODS
Sample collection
Samples were collected in different locations of Rameshwaram marine region at the depth of 1–5 m. The randomly collected samples in the sterile plastic bags (soil sample) and water sampling bottles (water sample) bottles were kept in an ice-cold box and transported safely to the lab for further analysis with in 12 hrs. The sample with media tubes were packed and transported safely to the laboratory.

Isolation of Azotobacter from water and sediment samples (Mary et al., 1985)
Different selective media were used for the isolation of Azotobacter sp from marine source as described previously. Azotobacter strains used for this study were maintained and cultured in Burk medium as previously described (Joerger et al., 1988). As the isolates are of marine origin, the media were prepared by the 3.5% sodium chloride (NaCl). Media used for the isolation of nitrogen fixing organism (Azotobacter) from marine sources were Jensen’s agar medium, Azotobacter agar medium, Burk’s Medium and marine agar medium. Followed by Gram staining, catalase test, starch hydrolysis test were also carried out. (Bagwell et al., 1988)

Pot culture method
The broth containing active culture of Azotobacter sp was selected. Three marine strains, three soil strains, three standard Azotobacter sp procured from MTCC, (Chandigarh, India were used. Forty healthy seeds of green gram were mixed with 3ml of Azotobacter inoculum and 3ml of cooled rice porridge. The seeds were dried and sown in each pot. The control is devoid of the inoculum. The pots were watered regularly, at an interval of 5 days the length of the root and shoot were observed and recorded. After 15 days interval leaves were collected for estimating the chlorophyll content by spectrophotometric method. Spectrophotometric analysis of chlorophyll pigments were developed in the 1930's and 1940's (Weber et al, 1986). Chlorophyll is extracted in 80% acetone and the absorption at 663nm and 645nm are read in a spectrophotometer.

RESULTS AND DISCUSSION
Totally 50 samples were collected in marine region of both water and sediments in the intervals of approximately 20 days .Out of 35 marine water samples collected, all the 35 samples were showing the presence of Azotobacter, but only 13 marine sediments out of 15 were showing the presence of Azotobacter. These samples were processed through the commonly used procedures such as selective media, Gram’s staining, and Phase contrast observation for motility, starch hydrolysis test and Catalase test for identification of free- living diazotrophic organism i.e Azotobacter from the above samples, and that can be processed. The colony morphology of Azotobacter strains were varying during the isolation in the selective media. The colonies were very clear, large, mucoid, watery due drops like initially i.e. from the marine source. Other testes show that Azotobacter sp is gram negative, motile, catalases and starch hydrolysis positive. All the isolated Azotobacter strains were numbered for the easy identification and convenience.

Result of pot culture experiment

The main objective of the pot culture study is to examine the influence of Azotobacter on green gram, 5 days interval after sowing various characteristics of growth such as percentage of germination, shoot and root length was measured results were noted % of germination range from 78 to 93 and for root and shoot length values range from 7 to 15 cms. 3 marine strains (400,408,409),3 soil strains,(1,2,6) and 3 standard strains (2452,2632,2633)were used for the experiment. Approximately (10 × 107 CFU/pot) broth inoculum was introduced in all the 18 pot (original and duplicate) except the control pot.
Significant differences were observed. The plants inoculated with Azotobacter sp were taller than that of control pot. Marine Strain 400 and soil strain 6 shows remarkable effect on the shoot length of plant and Marine Strain 408 and soil strain 1 shows remarkable effect on the root length of the plant than others on 15th day. The plant leaf was analysized to check the differences in chlorophyll content. Significant difference was observed results were .The OD value shows at 645 nm ranges from 0.028 to 0. 058 and OD value at 663 nm ranges from 0.090 to 0.109

Azotobacter is a heterotroph bacterium of aerobic character having the capability of fixation of dinitrogen as nonsymbiont. However; some strains have higher nitrogen fixing ability than others (Burgmann et al., 2003). Besides, nitrogen fixation, Azotobacter also produces, Thiamin, Riboflavin, indole acetic acid and gibberellins. When Azotobacter is applied to seeds, seed germination is improved to a considerable extent, so also it controls plant diseases due to above substances produced by Azotobacter (Kader et al., 2002.)

JOURNAL REFERENCES

Ahmand.F,Ahmand.I,Khan.M.S, IAA production by the indigenous isolates of Azotobacter & Fluorescent Pseudomonas in the presence &absence of Tryptophan. Department of Agriculture microbiology. vol.29, p.29-34. 2004

Bagwell, C.E., Piceno, Y.M., Lucas, A.M. and Lovell, C.R., 1988. Physiological diversity of the rhizosphere diazotroph assemblages of selected salt marsh grasses. Appl. Environ. Microbiol., 64(11): 4276-4282.

Brown . M .E and S.W . Burlingham , 1976 : Production of plant growth substances by Azotobacter chroococum J. Gen micro 53: 135-144.

Bürgmann H, Manuel Pesaro, Franco Widmer and Josef Zeyer strategy for optimizing quality and quantity of DNA extracted from soil. Bacteriological Reviews, vol.36, No.2 p.295-341. 2003

Church M J, Cindy M. Short, Bethany D. Jenkins, David M. Karl, and Jonathan P. Zehr, Temporal Patterns of Nitrogenase Gene (NifH) Expression in the Oligotrophic North Pacific Ocean’ Ocean Sciences Department, Environmental Microbiology, Vol 134, No.1 p.155-193, 1999.

Gerhard.P.,R.G.E.Murray,R.N.Costilow,E.W.Nester,W.A.Wood,N.R.Krieg,andG.B.phillips.1981.Manual of method for generalbacteriology.American SOCIETY FOR microbiology, Washington, D.C.

Kader.M.A, Mian.M.H, Hoque.M.S. Effect or Azotobacter inoculant on the yield and nitrogen uptake by wheat. Department of soil science, Bangladesh Agricultural University, Mymensigh, Bangladesh. Vol 2(4) p 251-261,2002.

Kennedy.C, R.K.Poole, M.G.Yates, and M.J.S.Kelly. Cloning and Mutagenesis of Genes Encoding the Cytochrome bd Terminal Oxidase Complex in Azotobacter vinelandii: Mutants Deficient in the Cytochrome d Complex are unable To Fix Nitrogen in Air.Journal of Bacteriology, Vol.172,No.10 p.6010-6019 . 1990

Mary. L. G and rita r. colwell, Enumeration ,isolation and characterization of N2 fixing bacteria from sea water . Department of microbiology, University of Maryland. Vol 50 no .2. 1985

Zehr .J. P ,Mark T. Mellon, and Sabino Zani. New Nitrogen-Fixing Microorganisms Detected in Oligotrophic Oceans by Amplification of Nitrogenase (NifH) Genes. 1998.

Zehr .J. P. Sarah Braun, Yibu Chen and Mark Mellon Nitrogen fixation in the marine environment: relating genetic potential to nitrogenase activity. Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA . 1999.

BOOK REFERENCES
1. Subba Rao .N. S., soil Microbiology and plant growth 4th edition.
2. Tate R. L., 1995 soil Microbiology. Jhon Willey and sons, Inc.
3. Atlas R. and Bartha. R, 1998. Microbiol Ecology fundamentals and applications 4th edition Benjamin Cummings. Menlopark. Ca.694pp
4. Campbell. N. 1993 Biology 3rd edition. Benjamin Cummings, Redwood City, Ca 1190
5. Jan Dirk Van Elsas, Jack T. Trevors, Elizabeth, M. H. Wllington 1997, Modern Microbiology.
6. N. S. Subba Rao, soil Microbiology and plant growth 4th edition.
7. Robert L. Tate, 1995 soil Microbiology. Jhon Willey and sons, Inc.
8. S.SADASIVAM AND A.MANIKAM , 2004 Biochemical methods. 2nd Edi, Centre for plant molecular biology, TNAU.

About the Author

Karthick.A and Jayashree.V.S,
Department of Microbiology,
Dr. GRD College of Science,
Coimbatore-14.

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