Original Article Isolation and Identification of Root Nodule Bacteria of Mung Bean (Vigna radiata L.) for Biofertilizer Production

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1 Available online at International Journal of Research in Pure and Applied Microbiology Universal Research Publications. All rights reserved ISSN Original Article Isolation and Identification of Root Nodule Bacteria of Mung Bean (Vigna radiata L.) for Biofertilizer Production Shraddha Bhatt* 1, Dr.R.V.Vyas 1, H. N. Shelat 1 and Sneha J. Mistry Address: 1 Anand Agricultural University, Department of Microbiology, Anand , Gujarat, India, *Corresponding author: shraddha.bhatt761@gmail.com Received 20 October 2013; accepted 05 November 2013 Abstract Nine different isolates of Rhizobium were isolated from Mung bean root nodules, cultivated in different Mung bean agricultural fields in Gujarat. Background: Present investigation nodules bearing a smooth surface of varying sizes were well established on top as well as lateral portions of roots. Some of the nodules were found to be have rough surface and the colour of the nodules ranged from pink to brown. Good growth of all the isolates was observed on congored yeast extract mannitol agar (YEMA) medium but was unable to produce chromo genesis on congored medium. All the isolated isolates of Rhizobium failed to show a growth on glucose peptone agar medium as well as Hofer s alkaline broth. All test isolates gave a negative test on ketolactose medium. The isolated isolates were found to be Gram negative in nature. In case of casein hydrolysis and starch hydrolysis all the isolates gave negative results. All test isolates reduced nitrate to nitrite and none test isolates produced H 2 S. Isolate AAU-6 produced a maximum gum of 122 mg while isolate AAU-9 produced a minimum of 90 mg. All isolates showed varied on growth characteristic in 2% NaCl while glucose consumption and utilization of nitrogenous compound varied from poor to very good. Conclusion: On the basis of result obtained in the present observation it can be concluded that isolate AAU-6 and AAU-7 were found to be the unsurpassed isolate of Rhizobium and can be exploited as biofertilizers for better yield of Mung bean Universal Research Publications. All rights reserved Keywords: Mung bean, Rhizobium, root nodules, biochemical characterization INTRODUCTION: Chemical nitrogen fertilizers will continue to serve for increasing grain production until a predictable future, but efforts should also be oriented towards augmenting biological nitrogen fixation which will mediated by microorganisms. In developing countries like India the construction of new nitrogen fertilizers plant is not only expensive but time consuming. Therefore it is essential for us to evolve and adopt a strategy of integrated nutrient supply by using a judicious combination of chemical fertilizer, organic manures and biofertilizers. Biologically active product more appropriately called as microbial inoculants contains active strength of selective microorganisms like bacteria, algae, fungi; alone or in combination helps in increasing crop productivity by biological nitrogen fixation. The rhizobia are a group of Gram-negative bacteria that form species-specific symbioses with legume plant, traditionally thought to include only members of the α-subdivision of proteobacteria and more recently found to include members of the β-proteobacteria. Nitrogen fixation, the reduction of atmospheric dinitrogen (N 2 ) to ammonia (NH 3 ), by rhizobia only occurs during symbiosis and provides a significant proportion of available nitrogen in the biosphere (Figure 1). Symbiotic nitrogen fixation is therefore of great ecological and socio-economic importance. Sustainable agricultural methods successfully exploit the rhizobia-legume symbiosis as a natural fertiliser by cultivating legume crops in rotation with non-symbiotic crops. In many soils across India the nodule s producing bacteria are absent or are present in non-adequate number or quality to meet the nitrogen requirement of legumes. To meet the requirement of nitrogen fixation it is necessary to inoculate the seeds with highly effective rhizobia (bacteria) cultures. Different legumes require different rhizobia in order to produce an effective symbiosis. Inoculants are prepared in laboratory by culturing nodule producing bacteria and are generally mixed with a suitable carrier material such as peat or lignite to make inoculants. The process of adding these inoculants to seed is called inoculation. Rhizobia are soil bacteria which can infect the roots of legumes to form effective nitrogen fixing nodules. In many soils rhizobia which are already present are either not in sufficient number or quality, or are not compatible with the farmer s legume crops to form effective symbiosis. In those soils, it is necessary to inoculate legume with rhizobia to 127

2 increase the amount of nitrogen fixed by the crop and increase the farmer s yield. By proper inoculation with rhizobia, the farmer can introduce large number of effective rhizobia with superior quality in producing nodules. Legume inoculants are either liquid or solid substances which contain live rhizobia and are inoculated by merely bringing in contact with the seed or legume root. The success of a technology depends on its techno economic feasibility. Production of inoculants first commence with the selection of individual rhizobial strains, those are effective at biological nitrogen fixation with particular legumes. These strains are grown to high population in liquid broth culture (Yeast Extract Mannitol Agar medium) in flasks and are subsequently transferred to larger fermentation vessel at laboratory level. Like all pure bacterial culture rhizobia must be grown under sterile condition and do require minimum aeration provided by shaking or sterilized air by bubbling and grow superlative at 30 ± 2 0 C. After an incubation period of 4-6 days the broth culture is ready to be mixed into a suitable carrier i.e. charcoal/lignite powder. The farmer coats his seed with inoculants (Biofertilizer) before a planting so that a high number of superior rhizobia are present when the legume root emerges. These inoculants, rhizobia can then quickly infect the root and start the process of nodulation. The best type of seed inoculants is very fine powder lignite or peat, which will adhere to the seed. A sticker is recommended to find the rhizobia to the seed. A sticker increase the amount of inoculants that will adhere to seed Gum Arabic is a common sticker. MATERIAL AND METHOD: The isolates were checked for contamination and particularly to distinguish the Rhizobium from Agrobacterium using the following biochemical tests: (1) Congo red Yeast Extract Mannitol Agar medium Incorporated medium (2.5 ml of 1% aqueous solution of the dye per one litre of YEMA) colonies of rhizobia in congo red are of white, translucent, glistening, elevated in nature and comparatively smaller with entire margin in contrast to the stained Agrobacterium colonies (Hahn 1966). The composition of the medium was (Fred et al. 1931) as Mannitol (10 g), K 2 HPO 4 (0.5 g), MgSO 4.7H 2 O (0.2 g), NaCl (0.1 g), Yeast Extract (1 g), CaCO 3 (1 g), Agar Agar (20 g), and distilled water (1000 ml) and a final ph The medium was sterilized at 15 PSI at C for min in an autoclave. Three plates were streaked for isolation from each nodule. Upon incubation up to 10 days, the colonies of bacteria emerging were picked up and transferred to YEMA slants. (2) Hofer s alkaline medium Agrobacteria can grow at higher ph levels than rhizobia and therefore their growth in YEM broth with elevated ph of 11.0 is considered as a useful means to distinguish between the two allied genera (Hofer 1935). (3) Glucose Peptone Agar medium Rhizobia cannot utilize peptone whereas Agrobacterium can utilize and grow very fast on this medium. The composition of the medium used during the experimentation contained Glucose-10g, Peptone-20g, NaCl-5 g, Agar Agar- 20 g, distilled water-1000 ml and ph is maintained at ph-7.2. In this medium 1 ml of 1.6 % Bromocresol purple per liter was added and autoclaved for three consecutive days. Rhizobia showed poor or no growth after 24 hrs with neutral or alkaline reaction Presence of Agrobacterium was acknowledged on the basis of maximum growth and acid reaction. Colour change to yellow due to production of acid by Agrobacteria and other contaminants of the medium (Kleczkowska and Nutman 1968). (4) Ketolactose test Ketolactose medium was prepared by replacing mannitol with lactose in YEMA medium. Sterilization was achieved by steaming for a period of 30 min for two successive days. After sterilization the medium was allowed to cool at room temperature (30±2 0 C) and stored at the same temperature for 3 to 4 days to check the contamination and confirm its sterility. A loopful of the inoculum from a fully grown culture slant (7 days old culture) was transferred to a petri plate containing the ketolactose agar medium. After incubation for 5 to 7 days at C the plates were flooded with a shallow layer of Benedict s solution. The composition of Benedict s solution consisted of two solutions: Solution A (Sodium citrate-173 g; Anhydrous sodium carbonate-100 g; Distilled water-600 ml) and Solution B (Copper sulphate-17.3 g; Distilled water-100 ml). The solutions A and B were prepared separately and later solution B was mixed with A and then filtered. The resultant solution was clear and having transparent blue colour. After pouring the Benedict s solution into above mentioned plates the excess of solution was drained off and the plates were incubated for one hour at 30±1 0 C without any disturbance. The yellow colouration around bacterial colonies confirms the presence of Agrobacterium (Bernaerts and Delay 1963). (5) Growth on Yeast Extract Mannitol Agar (YEMA) slant The Yeast Extract Mannitol Agar medium was prepared and 3 ml of this medium was dispensed in culture tubes (15 x 150 mm) autoclaved at 15 PSI at C for minutes. Slanted at 45 0 C and kept for 24 hrs to check for contamination. For inoculation 7 days old culture of different strains of Rhizobium were used. After seven days of incubation, the growth characteristics of each strain were recorded and categorized into three groups fast, medium and poor growing strains. (6) Gram staining For gram staining the following reagents were prepared; Crystal violet solution (Crystal violet-10 g, Ethyl alcohol- 100 ml, Ammonium oxalate-4 g, Distilled water-400 ml); Iodine solution (Iodine-1 g, Potassium iodide-2 g, Ethyl alcohol-25 ml, Distilled water-100 ml); Iodinated alcohol (Iodine solution (b)- 5ml, Ethyl alcohol-95 ml) Counterstain (2.5% safranin in ethyl alcohol-10 ml, Distilled water-100 ml). Gram stained smear were prepared with a loop full of a selected bacterium and spread over on a slide in a drop of water and allowed to dry in air (Graham and Parker 1964). The slide is dried in the vicinity of the flame and allowed to cool and then stained with crystal violet solution as follows: for 1 min followed by rinsing 128

3 with water and removal of excess water, the slide is then flooded with iodine solution followed by decolourized with iodinated alcohol for one minute, for 5 min the slide is washed in water, drained and counterstained with safranin. Finally the slide is washed in water, drained and air dried and observed under oil immersion (Vincent 1970). (7) Growth in 2% sodium chloride concentration To check the adaptability of Rhizobium at 2% NaCl concentration slants of Yeast Extract Mannitol Agar (YEMA) medium containing 2% salt were inoculated with each isolate and observation of growth was taken after 5 day of incubation (Graham and Parker 1964). (8) Gum Production Gum production by Rhizobium was studied by the method described by Anderson (1938). Flasks containing 100 ml of the Yeast Extract Mannitol (YEM) broth were sterilized at 15 PSI at C for min and inoculated in triplicate with 1 ml of 5 days old culture broth of each isolate and incubated for 15 days on a rotatary shaker. After incubation, with the help of boiling the volume of culture fluid was reduced to 30 ml. A mixture of ethanol and acetone (3 volumes) was added to the flasks for precipitation of the gum and were left undisturbed overnight to ensure complete precipitation. The contents from the flasks were filtered and the retentate (i.e. gum) on the filter paper was dried in oven for 24 hrs at 78 0 C after complete drying the amount of gum produced by the individual isolate was recorded. (9)Reduction of 2, 3, 5 triphenyl tetrazolium chloride (TTC) The method of Herrigan et al. (1966) was used for this test. TTC is redox indicator and it indicates the capability of the isolates to produce dehydrogenase enzyme, by adding this into culture. Tubes containing 5 ml of YEM broth were inoculated with different isolates and after 7 days of incubation, 1 ml of 2, 3, 5 TTC (1% solution) was added and further incubated at 28 0 C for 30 min. Appearance of pink colour in the incubated tubes, indicated the reduction of TTC. (10) Sugar Fermentation Test Andrade's Fermentation Broths produce acid as a metabolic waste when inoculated with bacteria that is capable of metabolizing the constituent substrate. Acid production causes a decrease in ph which results in a colour shift from pale pink to red in the medium. Change in color is the indicator for fermentation of carbohydrates by the isolates. (11) Starch Hydrolysis Nutrient agar medium containing 0.2% starch powder was used to determine the starch hydrolysis by the isolates. Positive test indicates that isolates have the competence to solubilise starch by producing amylase enzyme. After incubation period, plates were flooded with Gram s iodine and presence or absence of halos around the bacterial colonies was recorded. (12) Liquefaction of gelatin Nutrient medium without agar was supplemented with 12% of gelatin and used to determine the ability of the isolates to liquefy the gelatine. The plates were prepared in triplicate and incubated at 28±2 0 C for 3-4 days and then flooded with 0.2 % mercuric chloride in 20% HCl. The presence of clear halos around the colonies due to liquefaction of gelation indicated that the isolates are capable of liquefying the gelatine. (13) Action on Litmus milk Litmus milk is a milk-based medium which is used to distinguish between different species of bacteria. The lactose (milk sugar), litmus (ph indicator), and casein (milk protein) contained within the medium can all be metabolized by different types of bacteria. Since milk is usually the first substrate used to maintain bacteria, this test allows for accurate depiction of bacterial types. The addition of litmus, other than explaining the ph type, acts as an oxidation-reduction indicator. The test itself tells whether the bacterium can ferment lactose, reduce litmus, form clots, form gas, or start peptonization. Bromocresol purple solution was added to the skimmed milk to get a grey blue colour. The milk was distributed in test tube and sterilized for 30 min for consecutive days. The tubes were then inoculated in duplicate with respective culture and incubated at 28±2 0 C. The appearance and change in colour of the milk was particularly examined after 24 hrs of inoculation. (14) Casein hydrolysis The test is conducted to determine if an organism can produce the exoenzyme casesase. Casease is an exoenzyme that is produced by some bacteria in order to degrade casein. Casein is a large protein that is responsible for the white color of milk. This test is conducted on milk agar which is a complex media containing casien, peptone and beef extract. If an organism can produce casein, then there will be a zone of clearing around the bacterial growth. Skimmed milk agar (containing milk 1000 ml, agar-agar g) was prepared, sterilized and poured on to sterile petriplates. The plates were inoculated in the center using short strokes of the culture loop with and incubated at 37 0 C for 48 hrs. After 48 hrs of incubation, the plates were observed against black background and presence or absence of clear halos around the centre of the plate was recorded. (15) Methyl Red and Voges Proskauer test This test is used to decide the capacity of the bacteria to meatbolize pyruvic acid. A negative result of MR and VP test indicates that isolates are using butylenes glycol pathway to metabolize pyruvic acid to neutral end products. Negative results of VP suggest that isolates are not capable to form acetoin by using glucose. Glucose phosphate broth containing glucose-5 g, peptone-5 g, K 2 HPO 4-5 g, and distilled water-1000 ml was prepared and dispersed (5 ml in each test tube) followed by steam sterilized for conducting Methyl Red and Voges Proskauer test. The tubes were inoculated and incubated for 72 hrs. For methyl red (MR) test 5 drops of the indicator (0.1 g methyl red in 300 ml of ethanol ml distilled water) was added to each tube containing 2 ml of liquid culture. Development of colour indicated positive reaction. For the voges-proskauer test (0.6 ml of 5% Alfa nepthol in absolute alcohol and 0.2 ml of 40 % potassium hydroxide were added to one ml culture. (16) Production of ammonia from peptone Production of ammonia from urea and by deamination of 129

4 peptone by bacteria can be examined by this test. Peptone broth containing peptone-1 g, NaCl-0.5 g, potassium nitrate-0.5 g and distilled water 1000 ml was prepared and 5 ml volume of the broth was dispensed in each test tubes followed by sterilization. After inoculation test tubes were incubated at 37 0 C for 48 hrs. After incubation 1 ml of Nessler s reagent was added to culture tube. The development of orange to brown colour indicates the presence of ammonia. (17) Reduction of nitrate to nitrite The nitrate broth containing peptone-5 g, yeast extract-3 g, potassium nitrate-1 g and distilled water-1000 ml was prepared, distributed (5 ml in each test tube) and sterilized. After inoculation culture tubes were kept for seven days incubation period and then the tubes were tested for the nitrate reduction with the sulphanilic acid napthylamine reagent. (18) Production of hydrogen sulphide Peptone containing 0.01% cysteine was prepared and dispersed in 5 ml lot in each test tube. Dry filter paper strips saturated with lead acetate solution was put in each test tube. The test tube was then sterilized, incubated at 28±2 0 C for 48 to 72 hrs. Blackening of lead acetate paper indicated production of hydrogen sulphite. (19)Triple sugar iron agar test The test was performed to determine the capability of isolates to use various carbohydrate sources e.g. sucrose, glucose, lactose, etc as media for growth. Triple sugar iron test is designed to differentiate among the different group which are capable of fermenting glucose with the production of acid and hydrogen sulphide production. Triple Sugar Iron Agar media consisted of beef extract-3 g/l, yeast extract-3 g/l, peptone-15 g/l, NaCl-5 g/l, lactose 10 g/l, sucrose 10 g/l, dextrose-1 g/l, ferrous sulphate-0.2 g/l, sodium thiosulfate-0.3 g/l, phenol red-0.24 g/l, agar-15 g/l and the final ph is adjusted to ph 7.0 (Kligler, 1918; Hajna, 1945). After inoculation and incubation, color on the butt and the slant was observed. (20) Simmon s Citrate Agar Slant Organisms growing on Simmons Citrate Agar are capable Table 1. Morphological characteristics of root nodules bacteria. Growth on glucose Growth on Hofer s peptone agar medium Alkaline medium of using citrate as the sole carbon source and they can metabolize the ammonium salt in the medium. Use of citrate increases the ph of the medium which causes colour change in the bromothymol blue indicator, turning it blue. This colour change is useful because growth on Simmons Citrate Agar is often limited and would be hard to observe if it were not for the colour change. (21) Hugh and Leifson medium A carbohydrate is added to the culture medium, degradation of the carbohydrate to acid is indicated by the ph indicator bromothymol blue which changes its colour to yellow. The degradation is allowed to take place while the medium is exposed to air (degradation may be oxidative or fermentative) or under exclusion of air (degradation by fermentation only). Composition (g/liter) of Hugh and Leifson medium: Peptone from casein-2.0, yeast extract-1, sodium chloride-5, di-potassium hydrogen phosphate-0.2, bromothymol blue-0.08, agar-agar- 2.5 and carbohydrate- 10g/l. (22) Urease production Urease broth is a differential medium that tests the ability of an organism to produce an exoenzyme, called urease, which ydrolyzes urea to ammonia and carbon dioxide. The broth contains two ph buffers, urea, a very small amount of nutrients for the bacteria, and the ph indicator phenol red. Phenol red turns yellow in an acidic environment and fuchsia in an alkaline environment. If the urea in the broth is degraded and ammonia is produced, an alkaline environment is created, and the media turns pink. RESULTS AND DISCUSSION Nine different isolates of Rhizobium were isolated from Mung bean root nodules, cultivated in different Mung bean agricultural fields of Anand Agricultural University, Gujarat. Identification and cultural characteristics of Rhizobium species isolates was carried out. The nine species were studied for their morphological, cultural, physiological and biochemical characteristics. Morphology of root nodule bacteria The morphology features of root nodules bacteria are presented in Table 1. The results indicate that all the 3-Keto Lactose test Gram staining AAU 1 -ve -ve -ve Gram negative AAU 2 -ve -ve -ve Gram negative AAU 3 -ve -ve -ve Gram negative AAU 4 -ve -ve -ve Gram negative AAU 5 -ve -ve -ve Gram negative AAU 6 -ve -ve -ve Gram negative AAU 7 -ve -ve -ve Gram negative AAU 8 -ve -ve -ve Gram negative AAU 9 -ve -ve -ve Gram negative 130

5 Table 2. Growth on Congo red YEMA at incubation temperature of 28±2 ºC Size of colonies (mm.) on Congo red YEMA at incubation temperature of 28±2 ºC After 24 hrs After 40 hrs After 72 hrs After 96 hrs After 112 hrs After 136 hrs AAU ± ± ± ±0.3 AAU ± ± ± ± ±0.4 AAU ± ± ± ±0.2 AAU ± ± ± ±0.1 AAU ± ± ± ± ±0.2 AAU ± ± ± ± ±0.1 AAU ± ± ± ±0.2 AAU ± ± ± ±0.1 AAU ± ± ± ± ±0.3 Table 3. Biochemical characteristics of different isolates Sr. No. Test AAU1 AAU 2 AAU 3 AAU 4 AAU 5 AAU 6 AAU 7 AAU 8 AAU % Peptone Broth % Peptone Broth Liquefaction of gelatin Urease test Triple sugar iron agar test AC/Al AC/Al AC/Al AC/Al AC/Al AC/Al AC/Al AC/Al AC/Al 6. Action on Litmus milk curdling curdlin g curdling curdling curdling curdling curdling curdling curdling 7. Methyl Red Vogues Proskauer s Citrate utilization Sugar Utilization Glucose ± ± ± ± ± ± ± ± ± Xylose ± ± ± ± ± ± ± ± ± Mannitol ± ± ± ± ± ± ± ± ± 7. Starch Agar Plate Indole Phenylalanine Deamination Nitrate reduction H 2S production Organic Acid Production Casein Hydrolysis Gum Production on YEMA broth (mg/100ml) Growth in 2% sodium Very Very Poor Poor Poor Good Good Good chloride concentration Good Good growth growth growth Good 16. Reduction of 2,3,5 triphenyl Moder Modera Moderate Moderate Poor Poor Moderate Moderate Moderate tetrazolium chloride (TTC) ate te 17. Melanin Production Amylase Production Table 4. Physiological characteristics of different isolates (incubation temp. 28±2ºC) Temperature 28 o C 32 o C 37 o C 40 o C AAU AAU AAU AAU AAU AAU AAU AAU AAU ph 131

6 isolates showed a negative growth on glucose-peptone agar and on Hofer s Alkaline medium. Similarly all the nine isolates did not produce yellow coloration around their colonies indicating that Agrobacterium was absent and all of them were Rhizobium. All the nine isolates were reported to be Gram negative in nature. Cultural characteristics The cells of different isolates were small to medium sized rods and were Gram negative. All the isolates grew well on yeast extract mannitol agar slants and congored yeast extract mannitol agar plates but didn t show chromo genesis. All isolates failed to grow on glucose peptone agar medium and Hofer s alkaline broth (ph 11.0). All isolates showed the negative test of rhizobia for the production of 3-ketolactose. None of the nine isolates grew on congored yeast extract mannitol agar medium on 24 hrs (Table 2). By 48 hrs, 3 isolates developed into puntiform (less than 1 to 2mm dia) colonies size indicating their ability to grow fast. After 72 hrs all isolates showed moderate rate of growth. By 4th day the colonies of most of the fast growing strain showing 3-4 mm. dia and slow growing isolates AAU-7 and AAU-8 showed 2 and 2.4 mm dia respectively by this time and were the slowest growing among the nine isolates. The colonies of most of the fast growing strain took 7 days to attain 8 to11 mm. dia whereas the colonies of slow or moderate growing strains showing 5-6 mm dia in the same incubation period. Colonies of both fast and slow growing isolates were rounded with entire margin and smooth surface and none of them showed chromogenesis. Biochemical and physiological characteristics In the present study, the 9 isolated from Vigna radiata to check their growth in 2% (w/v) NaCl. Out of nine isolates 2 isolates grew best, 4 isolates grew good and 3 isolates grew poorly in 2% NaCl. (Table 3). As suggested by previous research Kucuk and Kivanc (2008), we found that fast growing isolates were generally more tolerant to high NaCl concentrations than slow growing isolates. Furthermore, the majority of our isolates had similar demands for carbohydrates i.e. Glucose, xylose and mannitol as several fast growing rhizobia (Kucuk and Kivanc 2008). Among the isolates we studied all were able to at grow 37 and 40 ºC, whereas 2 isolates (AAU 7 and AAU 9) showed only minimal growth at 40 and 45 ºC (Table 4). All isolates were grown in YEM medium with ph values of 4, 5, 7 and 9, but differences were detected at ph 5 (Table 4). Of all the isolates, 3(AAU2, 6, 9) isolates even grew at a acidic condition as low as ph 5. None of the test isolates gave positive results for casein, hydrolysis, gelatin liquefaction but gave positive tests of methyl red and voges-proskauer test. Milk was curdled by all test isolate and the colour of Bromocresol purple (BCP) was not changed. All test isolates reduced nitrate to nitrite but didn t produced H 2 S from peptone. The biochemical characteristics showed similarity of nine test isolates. AAU1 produced maximum gum and AAU6 lowest gum produced by isolates. The isolates were identical morphologically but exhibited variations in cultural and physiological characters (Wright 1925) studies made at the isolates differed in their morphological, physiological and biochemical characters (Graham and Parker 1964; Nakul 1990; Tiwari 2003). CONCLUSION: Nine different isolates were isolated from Mung bean root nodules, cultivated in different Mung bean agricultural fields in Gujarat. All the test isolates were strictly Gram ve in nature. During the identification a notable feature was recorded with them as they produced more gum and were able to utilize nitrogenous compounds. All the isolates were able to grow on 2% salt concentration. Similar findings were also reported by Keneni et al.(2010); Nakul (1990); Sindhu and Dadarwal (2000) and a final conclusion is that all the isolates were Rhizobium. In future the same isolates may be checked for ARDRA (Amplified rdna (Ribosomal DNA) Restriction Analysis), nif gene, nod gene etc. to access the capacity of the isolates to produce nodules. REFERENCES: 1. Anderson D.A. (1933) The production of gum by certain species of Rhizobium. Iowa. Agr. Expt. Sta. Res. Bull. 158: Bernaerts MJ, Deley JA (1963) Biochemical test for crown gall bacteria. Nature. 197: Fred EB, Baldwin IL, Mccoy E (1931) Root nodule bacteria and leguminous plants Studies no. 52, Science 5. University of Wisconsin Press, Madison, Wisconsin. 4. Graham PH, Parker CA (1964) Diagnostic features in the characterization of the root nodule bacteria of legumes. Plant Soil.20: Hahn NJ (1966) The congored reaction in bacteria and its usefulness in the identification of rhizobia. 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7 Seeds Res.20: Vincent JM (1970) A Manual for the practical study of Root-Nodule Bacteria, Blackwell Scientific, Oxford. I.B.P Handbook, Wright WH (1925) The nodule bacteria of soybean. I bacteriology of strains. Soil Sci. 20:95. Source of support: Nil; Conflict of interest: None declared 133