International Journal of Advances in Science and Technology, Vol. 3, No.

6, 2011

Isolation and characterization of rhizobium from Trigonella foenumgraecum and Mimosa pudica.
Mani Ramakrishnan1*, Lingaiah Rajanna2, Lijisha Edachira 1, Sunita Kumari1 and Rwivoo Baruah1
1

PG Department of Biotechnology, CMR Institute of Management Studies (Autonomous), C. A. #2, 3rd Cross, 6th A Main, HRBR Layout, 2nd Block, Kalyana Nagar, Bangalore 560 043. 2 Department of Botany, Bangalore University, Jnana Bharathi campus, Bangalore 560 056. *maniramiyer@yahoo.com

Abstract
Rhizobium species have the ability to infect roots of leguminous plants, form nodules and work symbiotically with their host in fixing molecular nitrogen. In this paper, morphological and biochemical characteristization of Rhizobium species isolated from Trigonella foenumgraecum and Mimosa pudica were reported. Bergeys manual was followed for morphological and biochemical studies. Similar characteristics were observed in morphology and staining studies and variations were also observed in growth response to different media in Rhizobium isolated from both the plants. NaCl concentrations favored the growth of Rhizobium isolated from Mimosa pudica at 39C and 40C, while the strain from Trigonella foenumgraecum in 2% NaCl growth was not observed at 39C and 40C as well. Rhizobium isolated from Trigonella foenumgraecum has responded to biotin, thiamine and its combinations. Results indicate that the Rhizobium isolated from Mimosa pudica found to be similar to MSSP strain of Burkholderia species. Based on the present methods of characterization, the classification of Rhizobia, phylogenetic analysis of the family Rhizobiaceae and related genera can be upgraded.

Keywords: Rhiziobium, Burkholderia, Trigonella foenumgraecum, Mimosa pudica.

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Introduction It is a fact that, Man lives in an environment dominated by nitrogen gas, but he is unable to use this element in its molecular form. Gaseous nitrogen must first be fixed that is incorporated into biomolecules which animals and plants can utilize as food. Certain bacteria, Rhizobium species infect roots of leguminous plants and form nodules to fix molecular nitrogen. The genus Rhizobium was first named by Frank in the year 1889 from Latin meaning root living. Beijernick, a Dutch microbiologist isolated the causal organisms, Rhizobia, from the leguminous nodules in 1888. Rhizobium is the most well known species of a group of bacteria that acts as the primary symbiotic fixer of nitrogen. These bacteria can infect the roots of leguminous plants, leading to the formation of lumps or nodules where the nitrogen fixation takes place. Biological nitrogen fixation (BNF) is an important source of nitrogen and the various legume crops and pasture species often fix as much as 200-300 kg of nitrogen per hectare [1]. Effective symbiotic associations supply sufficient amounts of reduced nitrogen to the plant host for development and ineffective associations do not reduce nitrogen [2]. Rhizhobium enzyme system supplies a constant source of reduced nitrogen to the host plant and the plant furnishes nutrients and energy for the activities of the bacterium and by this principle around 90% of legumes can become nodulated. Rhizobium induces the fertility of soil. Because of this property of Rhizobium, farmers applied leguminous plants in their field to increase the soil fertility. The Rhizobium-leguminous plant association offers the greatest promise of all systems to provide nutritious protein food needed in the years ahead. Intensive farming practices for high productivity need chemical fertilizers and are not cost effective. Their extensive use in agriculture may create environmental problems and currently its under debate due to soil fertility concerns and fear for consumers health. Further the organic farming system and interest in environmental friendly sustainable agricultural practices are growing and rhizobia have been widely used in agriculture for enhancing the ability of legumes to fix atmospheric nitrogen [3]. Therefore, the role of biofertilizers such as Rhizobium would decrease the need for chemical fertilizers and reduce adverse affects on soil fertility. Isolation, characterisation, identification, development and implementation of Rhizhobium species from leguminous plants irrespective of edible or non edible; for sustainable agriculture techniques; are of major importance in alleviating environmental pollution and the deterioration of nature [4]. Despite large diversity of legumes (up to 18000 species) and a great number of species produce nodules, around 11200 rhizobia of nodulating species are uncharacterized yet [5]. Taxonomy and phylogeny of Rhizobia are based on isolates from nodules of only 10% of 750 genera. The rhizobia, which are widely used in agricultural systems, are represented by 7 genera containing about 40 species as Alphaproteobacteria: Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium [6] and a species in the genus Methylobacterium [7]. Symbiotic nitrogen fixing species have also been defined among the genera Burkholderia and Cupriavidus within the beta subclass of proteobacteria [8]. Isolation of rhizobia and characterisation from edible and non edible legumes could yield novel strains which may provide comparable symbiotic benefits to their legume

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hosts in terms of nodule nitrogenase activity and plant biomass gain in the biofertiliser application. Therefore, in the present study, Rhizobium was isolated from root nodules of Mimosa pudica and Trigonella foenumgraecum to characterize and identify based on morphological, biochemical studies and nutritional requirements. Implications of the study on Rhizobium-leguminous plant association offers the greatest promise of all systems for providing the nutritious protein food which will be needed in the years ahead.

Materials and Methods Isolation of Rhizobium from root nodules Plants of Mimosa pudica were uprooted from nearby places of the CMR Institute of Management Studies, HRBR lay out 2nd Block, Kalyana Nagar, Bangalore, India and the Trigonella foenumgraecum seedlings were grown in pots. Fresh root nodules were collected, washed in running tap water, surface sterilized with 75% ethanol, 0.1% mercuric chloride, washed thoroughly in sterile distilled water, crushed in 1 ml of sterile distilled water and 0.1 ml was inoculated on YEMA media (Yeast Extract Mannitol Agar, pH 7.0) by spread plate method and incubated at 28o 1 C [9]. Pure culture was isolated and used for further analysis in triplicates. To determine the optimum temperature Rhizobium cultures from Trigonella foenumgraecum and Mimosa pudica were incubated between 2530oC. Characterization Morphological and Biochemical Characterization Morphological studies like colour of the colony, cell shape and arrangement; gram staining, endospore staining, negative staining, capsule staining reactions and bacterial motility test were conducted. Biochemical characterization namely acid production in YEM broth, catalase test, oxidase test, H2S production, gelatin and starch hydrolysis, growth on 0.1, 0.5, 1 and 2% NaCl, at 39C and 40C were carried out with a control [9]. Growth Response in different media The bacterium was tested on Yeast Extract Mannitol Agar (YEMA), Nutrient Agar slants (NA), yeast extract mannitol broth (YEMB) and Nutrient Broth (NB) for their growth response. Vitamin Requirements The defined basal medium used as per the method of [10] was tested for vitamins requirements like thiamine, biotin, thiamine and biotin combination. Results and Discussion In the host plants (Trigonella foenumgraecum and Mimosa pudica) nodules of varying sizes found distributed both on the top and lateral roots (Fig. 1 A, B). The nodules were loosely attached in Mimosa pudica roots (Fig. 1 B). In Trigonella foenumgraecum, nodules are generally large compared to Mimosa pudica and nodules on the lateral roots are loosely attached and the one on the main top root are firmly attached (Fig. 1 B). The shape and size of the nodules varied with the host. In general the shape of the nodules was round to oval but irregular shape was also noticed. The surface of the nodules was rough in Mimosa pudica and their young nodules were pinkish and the matured one turned pink to brown.

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Figure 1 A) Roots nodules of Trigonella foenumgraecum B) Roots nodules of Mimosa pudica, C-D) Microscopic view (100x oil immersion) Rhizobium strains isolated and Gram stained (C - Trigonella foenumgraecumand D - Mimosa pudica), E-F) Microscopic view (100x oil immersion) of Rhizobium capsules (E - Trigonella foenumgraecumand F - Mimosa pudica) Rhizobia isolated from Trigonella foenumgraecum and Mimosa pudica were identified on the basis of morphological, biochemical characteristics and nutritional requirements (Table 1). The rhizobium colonies belong to Trigonella foenumgraecum and Mimosa pudica produced similar colony characteristics such as gummy, translucent, circular and convex with entire or smooth margins. Baljinder Singh (2008) [11] reported that Rhizobium isolated from Trigonella foenumgraecum (fenugreek) were rod shaped, gram negative, acid and mucous producing. The size of the two rhizobial colonies at 4th day was 4 mm as maximum. Manoj et al. (2010) [12] reported slow growing isolates

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BRC-S-1 and 6 show 4 mm dia at 4th day and they further reported that these isolates were the slow Table 1. Characteristics of rhizobium isolated from two plants Parameters Morphological Characteristics Colony diameter (mm) Cell shape Cell arrangement Endospores Capsules Motility Staining Reaction Gram staining Negative staining Growth response to different media YEMA media Colonies Optimum temperature Growth Margins Elevations Density Nutrient Agar Growth Colour Nutrient broth Growth Clouding Sediment Biochemical characteristics Acid production (YEM broth) Oxidase Activity Catalase Activity H2S Production. Vitamin Requirements Biotin Thiamine Biotin and Thiamine Growth in Nacl / temperatures 0.1% 0.5% 1% 2% + Positive, - Negative Trigonella foenumgraecum 2.5 to 4 Rods Clusters Absent Present Motile Gram negative rods Cells with dark background Mimosa pudica 3 to 4 Rods Clusters Absent Present Motile Gram negative rods Cells with dark background

Gummy pinkish to red 30C Abundant Entire Raised Translucent Abundant white Ring, Flaky Heavy Abundant + + + + + 39C + + + 40C + + + +

Gummy pinkish to red 28C Moderate Entire Convex Semi translucent Moderate White Ring, Flaky Heavy Abundant + + + + + + 39C + + + + 40C + + + +

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growing ones in the collection. Based on the findings of Manoj et al. (2010) [12], strains isolated in the present study could be slow growing ones. They expressed that the colonies of most of the fast growing strain took 7 days to attain 8 to10 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. The Gram staining technique showed that both the plants Rhobial strains were Gram-negative and pleomorphic rods; capsules were present and motile. Similarly, negative staining techniques showed rod shaped cells with dark back ground (Table 1). Strains isolated from both the study plants subjected to growth response in different media such as YEMA, NA, YEMB and NB. The optimum temperature recorded for Rhizobium strain isolated from Trigonella foenumgraecum was 30oC and it was as 28oC for Mimosa pudica. In all the media for both the strains growth was recorded and in YEMA media gummy and pinkish to red colonies with entire margins was found. Rhizobium growth was abundant and the colonies were translucent, with raised elevation in the case of Trigonella foenumgraecum whereas in Mimosa pudica growth was moderate, semi translucent colonies with convex elevations. In NA also growth of Trigonella foenumgraecum Rhizobium was abundant and in the Mimosa pudica Rhizhbium it was moderate. In NB all the growth characteristics observed were similar for the strains from both the plants. Similarly, in the present study, biochemical characteristics such as Acid production on YEM broth, Oxidase Activity, Catalase Activity and H2S Production found to be similar for both the Rhizobium (Table 1). Results of the present study coincides with the findings of Bertand et al. (1985) [13]. According to Bertrand et al. (1985) [13] some of the native Rhizobium isolates exhibited anomalous characteristics in pure culture such as lack of acid production, temperature tolerance, or fast growth on YEM growth media, hence it was essential to verify that these strains were indeed Rhizobium. However, in the present investigation. strains isolated from two plants did not exhibit major differences in their biochemical characters. A test was conducted to screen and differentiate the growth requirements and to identify the difference among two strains based on vitamin supplementation requirements such as Thiamine, Thiamine and Biotin and Biotin alone in the growth media. Interestingly, Rhizobium isolated from Trigonella foenumgraecum has not shown any growth in media containing biotin alone while normal growth was observed in Thiamine and Biotin and Thiamine combinations. Results shows that Rhizobium isolated from two plants are of different of strains. Growth of the test organism in different NaCl (0.1 %, 0.5 %, 1 % and 2%) at 39C and 40C were performed and found that Rhizobium strain of Trigonella foenumgraecum has shown growth in 0.1 %, 0.5 % and 1 % NaCl at 39C and 40C but not grown in 2% NaCl at 39C and 40C while Rhizhobium strain isolated from Mimosa pudica has survived in all. Interestingly, Piyush et al (2005) [14] have reported similar observation in their study on strain MMSP of Rhizobium isolated from Mimosa pudica. They have verified the rhizobial colonies for Kochs postulates and identified by rDNA analysis.

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Based on the morphological and biochemical characterisation studies of two Rhizobial strains isolated from Trigonella foenumgraecum Rhizobium and Mimosa pudica we found that they are morphologically similar. In the biochemical characterisation, the vitamin growth requirement test confirmed that Rhizobium isolated from Mimosa pudica has shown growth in media containing biotin alone while the other strain from Trigonella foenumgraecum did not. While, differences in growth response in 2% NaCl concentration at 39C and 40C between two strains were observed. Hence, the strain isolated from Mimosa pudica is tolerant to high salt and temperature conditions as they were grown in dry lands. Based on the present investigation we conclude that Rhizobium isolated from Mimosa pudica found to be close to MSSP strain of Burkholderia species. To conclude, the two different strains isolates may be useful for inducing nodulation in certain non-legumes and also to increase the symbiotic nitrogen fixation in legumes used for agriculture. Further studies are required to screen nitrogen fixing organisms from different ecosystems, with emphasis on their biochemical characteristics and genetic diversity. Emphasis to reveal the complex association between legumes and their plant symbiotic partners focussed on transfer of nitrogen fixation (nif) or nodulation (nod) genes will pave the way for efficient strains in the modern organic agricultural practices.

References
[1] Peoples, M. B., D. F. Herridge, J.K. Ladha, Biological nitrogen fixation: an efficient source of nitrogen for sustainable agricultural production?, Plant and Soil, vol. 174, pp. 3-28, 1995. [2] Craig F. Barrett, Matthew A. Parker. Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. Nodule Bacteria on two Mimosa spp. in Costa Rica. Applied and Environmental microbiology, vol. 72, no. 2, pp. 11981206, 2006. [3] Rigby, D., Caceres, D., Organic farming and the sustainability of agricultural systems.Agricultural Systems, vol. 68, pp. 21-40, 2001. [4] Ogutcu, H., Algur, O. F., Elkoca, E., Kantar, F. The determination of symbiotic effectiveness of Rhizobium strains isolated from wild chickpea collected from high altitudes in Erzurum, Turkish Journal of Agriculture and Forestry, vol. 32, pp. 241-248, 2008. [5] Sessitsch, A., K. J. Wilson, A. D. L. Akkermans, W. M. de Vos, Simultaneous detection of different Rhizobium strains marked with the Escherichia coli gusA gene and the Pyrococcus furiosus celB gene, Applied Environmental Microbiology, vol. 62, pp. 4191-4194, 1996. [6] Wei, G. H., Wang, E. T., Tan, Z. Y., Zhu, M. E., W. X. Chen, Rhizobium indigoferae sp. nov. and Sinorhizobium kummerowiae sp. nov. respectively isolated from Indigofera spp. and Kummerowia stipulacea, International Journal of Systematic and Evolutionary Microbiology, vol. 52, pp. 2231-2239, 2002. [7] Sy, A., Giraud, E., Jourand, P., Garcia, N., Willems, A., De Lajudie, P., Prin, Y., Neyra, M., Gillis, M., Boivin-Masson, C., B. Dreyfus, Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes, The Journal of Bacteriology, vol. 183, pp. 214-220. 2001. [8] Moulin, L., Munive, A., Dreyfus, B., C. Biovin-Masson, Nodulation of legumes by members of the bold beta -subclass of Proteobacteria, Nature, 411, pp. 948-950, 2001. [9] Aneja, K. R., Experiments in microbiology, plant pathology and biotechnology. 4th Edition. New Age International Publishers, India, 2003. [10] Graham, P. H., In symbiotic nitrogen fixation in plants (P. S. Nutman, ed.), pp. 99-112, Cambridge University press, London and New York, 1976.

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[11] Baljinder Singh, Ravneet Kaur, Kashmir Singh, Characterization of Rhizobium strain isolated from the roots of Trigonella foenumgraecum (fenugreek) African Journal of Biotechnology, vol. 7 no. 20, pp. 3671-3676, 2008. [12] Manoj Patidar, Dileep Mandloi, Ashish Agrwala, Anil Gupta, Isolation and identification of root nodule bacteria of soy bean for bio fertilizer production, International journal of pharmaceutical science and biotechnology, vol. 1, no. 1, pp. 48-55, 2010. [13] Bertrand D., Eardly, David B., Ijannaway, Peter J. Bottomley, Characterization of Rhizobia from Ineffective Alfalfa Nodules: Ability to Nodulate Bean Plants Phaseolus vulgaris (L.) Savi, Applied And Environmental Microbiology. vol. 50 no. 6, pp. 1422- 1427, 1985. [14] Piyush Pandey, S. C. Kang, D. K. Maheshwari, Isolation of endophytic plant growth promoting Burkholderia sp. MSSP from root nodules of Mimosa pudica, Current Science, vol. 89, no. 1, pp, 10, 2005.

Authors Profile
Dr. Mani Ramakrishnan received his Ph. D degree in Botany degree from Bangalore University, Bangalore, India in 2011. This author received Summer Research Fellow- Teachers (2011) from Indian Academy of Sciences, Bangalore. He is having 13 years of experience in teaching and research in the field of Biotechnology. He published 5 research paper in National and International Peer Review Journals and presented 14 research papers in National and International conferences. Dr. Lingaiah Rajanna received his Ph.D degree from Mysore University, Mysore, India in the year 2000. This author is Excellent Researcher Award from Bangaore University. He is JRF and SRF awardee from CSIR. A life member of Indian Science congress, Society of Cytologists and Geneticists and Indian Remote Sensing Society. He is also the member of Delhi Botanical University Association, Swamy botanical Club. He is currently working as a Professor in Department of Botany, Bangalore University. He is having overall teaching experience of 15 years including professional colleges. He published 23 research papers in National and International Peer Review Journals and presented 50 research papers in National and International conferences. Mrs. Lijisha Edachira received her Master degree in Biotechnology from PG Department of Biotechnology, CMR Institute of Management Studies (Autonomous), affiliated to Bangalore University, Bangalore, India in the year 2011. Ms. Sunita Kumari received her Master degree in Biotechnology from PG Department of Biotechnology, CMR Institute of Management Studies (Autonomous), affiliated to Bangalore University, Bangalore, India in 2011.

Mr. Rwivoo Baruah received his Master degree in Biotechnology from PG Department of Biotechnology, CMR Institute of Management Studies (Autonomous), affiliated to Bangalore University, Bangalore, India in the year 2011. Currently he is working as JRF at Indian Institute of Technology, Guwahati, India

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