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rssN 2229-3469 Bioscience DiscoveryVol02, No. 1, Jan. 201L MORPHOLOGICAL, CUUIURAL AND BIOCHEMICAL CHARACTERISTICS OF RIIIZOBIUM JAPONICUM SYN AND BMDYRHIZOBIUM TAPONICUM OF' SOYBEAN I Dqnd Khansole 6 S1 Post Graduate Department of Botany and Chemistryr N. F.S. Science College, Nanded-431605 (M.S.) Received: 10 July 2010; Revised: 22october 2010 Gachande ABSTRACT , Rhizobium joponicum syn. and Brodyrhizobium japonicum was isolated from root nodules of Soybean (Glycine max L.) on yeast extract manitol agar (YEMA) medium and its morphological, cultural and biochemical-characteristics were studied. lt was observed that the colonies were circular, light pink, convex, entire and opac. The bacterium was rod shaped, aerobic, non-spore forming and motile. lt showed negative chemical reaction for indole, methyl red, Voges-Proskaur, hydrogen sulphide production, utilization of carbohydrates and gelatin hydrolysis. While it showed positive reaction for citrate utilization, catalase dnd ammonia production from peptone and urea. Key words: Soybean, Rhizobium japonicum syn. Bradyrhizobium ioponicum. |NTRODUC,I|oN ! , (Purohit & Kumar 1998). Therefore the attempt has Rhizobio are well known for their been made to study the morphological and ' capacity to establish a symbiosis with legumes. liochemical characters of the bacterium. Legumes are unique plants which have the ability to work with certain bacteria i.e. Rhizoibio to MATERIATANDMETHODS The mature root nodules from Soybean gather available nitrogen from the soil atmosphere and convert it to usable ammonia plantwerecollectedfromfieldwashedthoroughly They under tap water and surface sterilized with 0.1 % inhabit root lnodules, where they reduce mercuric chloride. Surface sterilized root nodules atmospheric nitrogen and make it available to were crushed in small quantity of sterile distilted the plant. Biological nitrogen fixation is a water.Rhizobialsuspensionwasinoculatedonpre component of sustainable agriculture and sterilized YEMA medium and incubated at 26 t3-eC Rhizobialinoculantshavebeenappliedfrequently temperature for 10 days. The isolates were maintained on slopes of YEMA medium as as bio-fertilizers. Each major legume group is nodulated described by Graham and Parker (1964) these by different species of Rhizobium. Soybeans are isolates were used to study the morphological, nodulated by Rhizobium joponicum (Krichner cultural and biochemical characteristics. Seven nitrogen and make it available to the plant. 1896; Buchanan 1926). Fred and his associates days old culture was used for Gram's reaction, (1932) recognized eight cross inoculants group morphology and colony characters. The pattern of in legumes. The genus Rhizobium was erected by growth was observed in motility agar medium. Frank (1890) based on its characters to form Growth on Hofer's alkaline medium was examined nodules on roots of legume plants. This property in YEMA broth with bromothymol blue at PH L0.00 is the only valid test in the identification of the (Hofer 1935). organism. Apart from it some diagnostic features Production of indole was noticed in of Rhizobium could be conveniently not only to inoculated tryptophan broth after 7 days of determine and identify the organism but also incubationbyaddingcovac'sreagent.Thereduction delinate different species (Graham and Parker ;il;;i ;"d-rnJ vog"r- proskauer reaction was 1.964; Vincent L97O; Gaur 1975; Mahana 1981) in glucose phosphate broth by adding examined Rhizobium iaponicum syn. Bradyrhizobium- methyl reA a'nA a-napthol solution with KOH ioponicum is associated with the root nodules of respectively. - ',,Citrate utilization was observed by .,,. Soybean and fixes 100 kg nitrogen/ha/year .i-r. ,","..u,o *.rn ,rorornrro, L Gachonde B D ond Khqnsole G S blue in basal medium. Liquefaction of gelatin was tested in LT/o gelatin in agar medium after 14 days of lncubation (Polhman 1931). Hydrolysis of starch was examined by streaking the organism on starch with nutrient agar and iodine solution. The production of ammonia by the bacterium was eiamined from the inoculated tube containing peptone water with potassium nitrate and Nessler's reagent. Production of hydrogen sulphide gas examine_d by method described by Zobell and Felt ham 1934. Production of ammonia:from urea was examined by Christensen urea agar with phenol red as an indicator. Fermentation of carbohydrates was tested by adding 1.0% Andrade's,indicator in the basal medium containing peptone water and 2% sugar. ln case of effect of PH and temperature, yeast extract manitol broth tubes were incubated foi three days and growth was recorded in terms of turbidity. The Gram's staining technique was followed as suggested by Graham and Parker 1964. Catalase activity was observed by stirreing the culture in a drop of hydrogen peroxide (10% by W/V), while oxidase activity was tested according to Kovaks (1956). Table 1: cultural/Morphological and staining characten ol Rhhobium japonicum. Charapters Result Shape Circu lar Size of colony .i 3.1 mm Colour/Pigmentation Whitish pink and glistering Elevation Convex Margin Regular/entire Opacity Opaque Mdtility Motile Bacterium shape Rod shaped Oxygen demand Aerobic Spore formation Non spore forming Gram's nature Gram - ve RESULTSAND DISCUSSION M o rp hol og i ca I Ch a ra cte rs show any growth on Hofer's alkaline medium at PH 10.0 with slight growth and evolution of gas and acid production. According to Ditmer (1930) The Rhizobium japonicum (Krichner and those Rhizobia, which produce acid are Buchanan) Syn. Brodyrhizobium japonicum was Gram negative, aerobic, non-spore forming and motile rods. ln general, the colonies we?e circular, convex, whitish pink and glistering with entire margin. considered to be advanced type and those, which Biochemicol Chorocter The bacterium showed well-marked growth on YEMA medium at PH 7.0.However poorgrowth was observed on Hofer's medium. Mahana et al. (2000), reported that the Rhizobium isolated from Vigno mungo L. showed marked variations in growth with respect to time period on YEMA while they do not produce alkali, are ancient type. The bacterium showed positive test for citrate, production of ammonia and catalase activity. Nitrate is reduced to nitrite producing ammonia. Neal and Walker (1935) suggested rapid nitrate utilization by slow growing root nodule bacteria. Mahana et ol. 2000 -reported catalase activity in some isolates from Vigna mungo.The bacterium is negative for MR-VP and indole reaction. Similarly, Grahm and Parker (1964) did not observe MR reduction in all the isolates of seven rhizobia groups. While Basak Bioscience DiscoveryVol02, No. L, lan.20LL and Goyal (1980) also reported that none of ISSN 2229-3469 the rhizobial. isolates indole. of of seven groups produces Table2: Biodremical Characters of Rhizobium japonkum 5r.No Test Remark 1 Production of indole from tryptophan Methyl red test -Ve 2 3 Voges-Proskaur test -Ve 4 +Ve 6 Ctrate utilization as source of carbon Hydrolysii of gelatin Production of ammonia from peptone 7 Production of hydrogen peroxide -Ve 8 Production of ammonia from urea rVe 9 Uti 10 PH 7.0 11 Eltect of PH on giowth'of Rhizobium Effect of temperature on growth of Rhizobium t2 Catalase test +Ve 13 Hydrolysis of starch -Ve 5 -Ve -Ve +Ve lization of carbohydrates -Ve Room temp; Biochemiral teets fcr R* fuedion otrr**b &oss pg*oe lilrrdlGf€&cde ldbftyleeadtert Gochande B D and Khonsole G S TITERATURECITED Basak M K and Goyal S K. 1980. Studies on the biology of tree legumes-Rh2obium symbiosis: Nodulation pattern and cross inoculation trials wjth tree legumes and cultivated legumes. Annals of Arid Zone.L9, 427-43t. Ditmer Fred E E E. 1930. The origin of cultivated Phoseolus. Burtt Apptied Bot Genet Plont Breed 23: 309-406. B, Baldwin I L and McCoy E. 1932. Root nodule bacteria and leguminous plants. Studies Science University of Wisconsin 5 Madison. Frank B. 1890. Uber die Pilzsymbiose der leguminosen. Landw Jb 19:523-640. GaurYD.1975-Studiesonrootnodulebacteria of CicerorietinumL.(Chickpea).ph.D.Thesis,University of Rajsthan, Jaipur, lndia. Graham P H and Parker C A. 1964. Diagnostic features in the characterization of the root nodule bacteria of legumes. Plant ond soil2O:283-395 Hoftir A W. 1935. Methods for distinguishing between legume bacteria and their most common contaminant. J. Am. Soc. Agron 27:228-23O. Krichnerand Buchanan. 1926. Rhizobium joponicum syn. Brodyrhizobium joponicum.lnt. J. Syst. Bocteriol. 3O:335 742. ffahana V K. 1981. Studies on the causal organismsl,origin, development and biology of root nodules oftreelegumes,Ph.D.thesisUniversityofRajsthan,,Jaipur,lndia. Mahna S K, Rekha Garg and Parvateesam M. 2000. Cultural and Biochemical Characteristics of root nodule bacteria from induced mutants of Vigno mungo L. Seed Pathology, Printwell publ, Jaipur. pp. 4L7-421. Neal O R and Walker R H. 1935. Physiological studies on Rhizobium tV. Utitization of Carbonaceous materials. J. Bacterial 30:173-187. Pohlman G G. 1931. Changes produced in nitrogenous compounds by Rhizobium metilotiond Rhizobium jo po nicu Purohit m. Soit Sci. 31: 385-406. S S and Kumar A. 1998. Plant Physiology, Agro Botanical Publishers (lncual/Agro Botonica Bikone.289. Vincent J M. 1970. A Monuol for the procticat study of the root nodule Bqcterio.lBp Hand Book and Blackwell Scientific Publications, Oxford. Zobell C E and Faltham C B. 1934. A comparison of lead and iron as detectors of hydrogen sulphide produced by bacteria. J. Boct.28:!69-L76.