Sanjeet Kumar
Isolation and Characterization of Symbiotic Bacteria using molecular tools from selected wild legumes of Odisha
The nitrogen-fixing plants (Legumes) are key components of the natural succession. Legumes establish rhizobial and mycorrhizal symbioses and constitute a fundamental way of source for nitrogen input to the ecosystem. Inoculation with indigenous N2-fixing bacteria, not only enhanced the establishment of key plant species, but also increased soil fertility and quality (Requena et al., 2001). This dual symbiosis system enhance soil nitrogen content and organic matters, hydrostable soil aggregates, and nitrogen transfer from N2-fixing to non-fixing species associated within the natural succession. Nitrogen was known to be an essential nutrient for plant growth and development. Intensive farming practices that accomplish high yields need chemical fertilizers, which are not only cost effective, but may also create environmental problems. The extensive use of chemical fertilizers in agriculture is currently under debate due to environmental concern and fear for human health. Consequently, there has recently been a growing level of interest in environmental friendly sustainable agricultural practices and organic farming systems (Rigby et al., 2001; Lee et al., 2007).
Increasing and extending the role of biofertilizers such as Rhizobium would decrease the need for chemical fertilizers and reduce adverse environmental effects. Bacterial symbiosis with legumes species is of special importance, producing 50% of 175 million tones of total biological nitrogen fixation, annually providing nearly half of all nitrogen used in agriculture (Hatice et al., 2008). Therefore urgent need the identification and addition of new symbiotic bacteria from wild legume plants to fight against above problems. Using biotechnological process it can be done easily such as polymerase chain reaction (PCR) can create highly characteristic patterns when distinguished in agarose gels, providing well separation on strain level (Adiguzel, 2006). Recently, wild legumes and their symbionts have drawn the attention of ecologist, because of their tolerance to extreme environmental conditions, such as severe drought, salinity, and elevated temperatures.
Bacteria of several genera have been isolated from legume tissues, including Aerobacter, Aeromonas, Agrobacterium, Bacillus, Chryseomonas, Curtobacterium, Enterobacter, Erwinia, Flavimonas, Pseudomonas and Sphingomonas . (Gagne et al., 1987; Sturz et al., 1997). The legume shows a remarkable diversity because of its long history of cultivation and its selection under various agro-climatic conditions. Odisha is rich in wild legumes such as Canavalia spp, Mucuna spp, Indigofera spp., Glycine spp. Abrus spp., Crotolaria spp., Pueraria spp., etc. Therefore an attempt has been made to isolate the symbiotic bacteria from wild legumes of Odisha.
Objective of the Research
1. Selection of wild legumes available in Odisha.
2. Isolation the symbiotic bacteria from selected wild legumes.
3. Phenotypic Characterization of Isolated symbiotic bacteria found in experimental legumes.
4. Chemotaxonomic characterization (analysis of peptidoglycan diminoacids, quinine system etc.) of isolated bacteria.
5. Characterization using 16S rDNA sequencing and G+C content determination as well as RFLP and RAPD analysis.
6. Symbiotic properties.
Methodology
Isolation of symbiotic bacteria from legumes
↓
Authentication of isolates
↓
Phenotypic characterization
↓
Colony morphology
↓
Bromothymol blue test.
↓
Salt tolerance test
↓
PCR-based 165 rDNA RFLP and partial sequencing
↓
Estimation of DNA base compositions and DNA-DNA hybridization
↓
Symbiotic properties
Symbiotic bacteria will be isolated from fresh surface sterilized nodules by the standard method (Van Berkum et al., 1996). Hence, nodules were immersed in 95% ethanol (v/v) for 10 s and were surface sterilized in HgCl2 for 4 min, and were washed three times in autoclaved distilled water. Effectiveness of sterilization was monitored to eliminate the possibility of isolating surface-attached bacteria. Sterilized nodules were crushed with a sterile glass rod in a sterile test tube, using some drops of NaCl (9‰) to make it slurry (Beck et al., 1993). One loop full of the nodule content suspension was streaked on yeast mannitol agar (YMA) plates (Vincent, 1970) containing 0.0025% (w/v) Congo red. After incubation for 3 to 7 days at 28°C, single colonies were selected and restreaked on YMA for purity (Jordan, 1984). Pure cultures were preserved in 20% glycerol at -80°C until further use (Elbanna et al., 2009; El-Akhal et al., 2009).
Probable Research Output
1. Documentation of diversity and phenotypic characterization of isolated Symbiotic Bacteria found in wild legumes of Odisha.
2. Can addition of new species of a particular genus of Bacteria.
3. Isolated Symbiotic Bacteria can exert several beneficial effects on host plants, such as stimulation of plant growth, nitrogen fixation and induction of resistance to plant pathogens.
4. Establishment of some wild legume plant species for increasing soil fertility and quality.
References
1) Zhi Yuan Tan, En Tao Wang, J Gui Xiang Peng, Ming E. Z. Characterization of bacteria isolated from wild legumes in the North-Western regions of China. International Journal of Systematic Bacteriology (1999), 49, 1457-1 469.
2) Halima Berrada, Imen Nouioui, Mohammed Iraqui Houssaini, Naïma el Ghachtouli, Maher Gtari and Kawtar Fikri Benbrahim. phenotypic and genotypic characterizations of rhizobia isolated from root nodules of multiple legume species native of Fez, Morocco. African journal of Microbiology Research. (2012), 6(25): 5314-5324.
3) Hamdi Hussein Zahran. Rhizobia from wild legumes: diversity, taxonomy, ecology, nitrogen fixation and biotechnology. Journal of Biotechnology. (2001), 91: 143–153.
4) Pham Quang Hung and K. Annapurna. Isolation and Characterization of Endophytic bacteria in soybean (Glycine sp.). Omonrice. (2004), 12: 92-101.
