Biological and Environmental Sciences Vol. 6 Number I
102-108, 2010
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Introduction
Nitrogen is generally considered one of the major limiting nutrients in plant growth and the main reserve of nitrogen in the biosphere being molecular nitrogen from the atmosphere. Nitrogen fixing bacteria are able to fix atmospheric nitrogen under different conditions independently, in loose association with other organisms, or in strict symbiosis with them, such as in the Rhizobium- legume-plant symbiosis which is being considered the most efficient type of association between diazotrophic microorganisms and plants.
A wide diversity of nitrogen fixing bacterial species belonging to the most phyla of the bacteria domain have the capacity to colonize the rhizosphere and to interact with plants. Either mutualistic or pathogenic associations can be established between plants and plants-interacting microorganisms. Specifically, nitrogen fixing bacterial symbionts of legume plants collectively termed Rhizobia, and phytopathogenic bacteria
Molecular Characterization of Genetic Diversity of N
2Fixing Microbes of Some Wild Legume Plants of Manipur - A Review
Rajkumar Suraj Singh, P.B. Mazumdar1 and G.D Sharma2
1Department Biotechnology, Assam University, Silchar
2Department of life science, Assam University, Silchar
*Corresponding Author: Email: [email protected]
Abstract
Non-symbiotic diazotrophic systems as well as symbiotic systems for biological nitrogen fixation (BNF) in agriculture are most promising. Accordingly, the present study was conducted for the, Isolation, Identification and Characterization of nitrogen fixing bacterial strains from some wild legume plants. Nitrogen fixation is the reduction of N2 (atmospheric nitrogen) to NH3 (ammonia) which is made possible by the enzyme nitrogenase. It is found only in prokaryotes. The nitrogen microorganisms are classified into the symbiotic and the non-symbiotic(free living) forms. Diverse nitrogen fixing microorganisms both symbiotic (eg.
Rhizobium and Bradyrhizobium) and non-symbiotic (eg.Azotobacter, Acetobacter etc.) grow in the root nodules and the rhizosphere of many wild legumes. Knowledge of their diversity and ecological importance remains incomplete. Isolation of the nitrogen fixers from different sources and molecular characterization could give a bigger insight about their diversity and importance.
Keywords : Diazotrophs, Nitrogen, Fixation , Rhizosphere, Legumes.
have adopted similar strategies and genetic traits to colonize, invade and establish a chronic infection in the plant host, (Soto et al., 2006). Other than leguminous plants, several varieties of economically important plants are capable of developing associations with diazotrophic microorganisms (Dobereiner, 1997).
For years, a limited number of bacterial species were believed to be nitrogen fixers (Postgate, 1981), but in the last 30 years, nitrogen fixation has been found to be a property with species in most of the phyla of Bacteria and also in Methanogenic Archae, (Young, 1992). Symbiotic nitrogen fixing property within nodules of vascular plants is found in two phylogenetically unrelated major groups of bacteria: rhizobia (Alpha- proteo-bacteria), associating essentially with leguminous plants belonging to one superfamily of angiosperms (Fabaceae), and Frankia (in Actinobacteria) with a broader spectrum of plants
from eight families (Huss-Danell, 1997 ; Vessey et al., 2004). Besides this, cyanobacteria is another important group of nitrogen fixing bacteria found in association with a large variety of higher and lower variety of plants, fungi and algae (Meeks and Elhai, 2002).
Taxonomic identification of several unknown nitrogen-fixing organisms can be accomplished through sequencing of the nifH gene, which is also useful to analyze their genetic potential for the nitrogen fixation (Zehr et al., 1995). NifH genes can be employed as markers for the detection and study of the genetic diversity of diazotrophic organisms in microbial communities, like those in rice roots (Ueda et al., 1995) or forest soil (Widmer et al., 1999).
In the present study, an attempt will be made to isolate several bacterial strains from different wild plant sources and characterized by using phenotypic and genotypic methods in order to assess their taxonomic and genetic diversity and investigate their ability to fix atmospheric nitrogen and the occurrence of nifH-like genes.
Methodologies
Ozawa et al., (2003) worked on the isolation and characterization of Diazotrophic bacteria from the surface-sterilized roots of some legumes.
Nitrogen-free, malate containing, semi-solid media (JNFb) was used to isolate the bacterial strains by inoculating 105-fold dilutions of the homogenate of surface sterilized roots of the legume plants.
The nitrogen fixing activity of the isolates in the semi-solid media was assessed by Acetylene Reduction Acitivity (ARA) method. 16S rDNA analysis was done for molecular characterization of the isolates and their phenotypic characters were examined by using the methods described by Smibert and Krieg, (1994).
Park et al., (2005) worked for isolation and characterization of diazotrophic growth promoting bacteria from rhizosphere of agricultural crops of Korea. Burk’s Nitrogen-free semi-solid and solid medium was used throughout the study and the pH of the medium was adjusted to 7±0.1.
Physiological and biochemical characters of the
bacterial isolates were examined according to methods described in Bergey’s Manual of Systematic Bacteriology. Gram reaction was performed as per standard procedures and tests like plate assays, Fatty acid analysis were also done. After the tests nitrogen fixing activity of the isolates was determined by the acetylene reduction assay (Hardy et al., 1968). 16S rDNA gene amplification and sequencing was done for differentiation of the isolates and production of IAA by the cultures were also estimated.
Zhang et al., (1991) did their work on the diversity of Rhizobium bacteria isolated from the root nodules of Leguminous Trees. In their work 60 rhizobial strains isolated from the root nodules of Acacia senegal and Prosopis chilensis in the Sudan were compared with 37 rhizobia isolated from woody legumes in other regions and with 25 representatives of recognized Rhizobium species by performing a numerical analysis of 115 phenotypic characteristics. Cultures were grown to log phase in YEM agar or broth before inoculation. After this strains were streaked onto YEM agar plates and several tests like hydrolysis of urea, precipitation of calcium glycerophosphate, reduction of nitrate, production of melanin, utilization of carbon sources were performed as well as resistance to intrinsic heavy metals and antibiotics and tolerance of NaCl were also determined.
Suliasih and Widawati, (2005) undertook a study to investigate the occurrence of phosphate solubilizing bacteria (PSB) and nitrogen-fixing bacteria (NFB) from soil samples of Wamena Biological Garden (WbiG). Eleven soil samples were collected randomly to estimate microbial population which used plate count method (Ravina et al., 1992; Thompson, 1989). During their work Yeast extract mannitol agar (YEMA) was used for growing Rhizobium and Mannitol Ashby agar medium for isolating Azotobacter and Okon medium for Azospirillum. The number of bacterial colony was estimated after 7 days of incubation at room temperature and then were identified following the methods of Bergey’s Manual of Systematic Bacteriology (Kreig and Holt, 1984).
Mirza et al., (2001) reported about the isolation of nitrogen fixing, phytohormone producing bacteria from sugarcane and their beneficial effects on the growth of micropropagated sugarcane plantlets. For isolation serial dilutions of the bacterial growth in the semi solid medium in ARA positive vials were spread on LB agar plates and incubated at 30°C for 24–48 h. Colonies appearing on plates were picked and streaked on fresh LB agar plates. All the different types of colonies were again inoculated in N-free semisolid media and were assayed for confirmation of acetylene reduction activity. Physiological and biochemical tests were performed using the QTS- 20 miniaturized identification system (DESTO Laboratories, Karachi, Pakistan). Oxidation- fermentation test (Hugh and Leifson, 1953) and catalase test (MacFaddin, 1980) were also performed for the identification of the isolates.
Finally PCR-amplification and 16S rRNA sequence analysis was carried out for differentiating the isolates.
Reinhardt et al., (2008) have isolated several new bacterial strains from cassava, guinea grass, maize, sugarcane and tomato using the selective media NFb described by Hartmann et al., (2006).
It was found that the isolated strains from the above plants reduced acetylene on the chromatography analysis and hence indicating their N2 fixing ability. Further their work has been analyzed by performing Dot blot Hybridization.
Prabudoss and Stella, (2009) isolated Gluconacetobacter diazotrophicus strains from samples of sugar rich crops like sugar cane (root, stem, bud, leaves), sweet potato, pine apple and wild cane by following the methodology of Cavalcante and Dobereiner, (1988). The nitrogen fixing efficiency of all the isolates were found to be efficient with the reference strain indicating the superiority of the local isolates.
Khan et al., (2008) reported about the isolation and identification of nitrogen fixing microorganisms during the seedling (30 days after seed sowing) stage of rice (BR 10) rhizosphere soil grown in Non-Calcareous Grey Flood Plain soil of Bangladesh. Four individual strains were
microbiologically identified based on the selection criteria and it was found out that their biochemical tests were strictly similar to Enterobacter spp., for strain-1, Klebsiella spp. for strain-2, Bacillus spp. for strain-3 and Azospirillum spp. for strain- 4. They were anaerobic in nature.
Holguin et al., (1992) worked on the isolation of 2 new diazotrophic bacteria, Listonella anguillarum and Vibrio campbellii, and one non- nitrogen- fixing bacterium, Staphylococcus sp., from the rhizosphere of mangrove trees. The cellular morphology of the pure isolates was determined with light microscopy (Zeiss). Species identification was done by FAME analysis through gas chromatography of cell fatty acid methyl esters that have a chain length between 9 and 18 carbons long. During their work it was found that Staphylococcus sp., indicates interaction with the above two diazotrophic bacteria and results in the increased or decreased in the nitrogen fixing capacity of the two diazotrophs.
Results
A total of 251 bacteria were isolated from the surface sterilized roots of 11 legumes except Lathyrus odoratus (Ozawa et al., 2003). Thirty- one isolates of these bacteria showed ARA of 2.6 to 450 nmol h-1 culture-1 when grown in the JNFb media. 16S rRNA gene sequence analysis and physiological characteristics of the 31 isolates showed that the isolates were Agrobacterium radiobacter, A. tumefaciens, Azospirillum lipoferum, Bradyrhizobium elkanii, Burkholderia cepacia, Frateuria aurantia, Klebsiella oxytoca, K. pneumonia, Rhizobium gallicum, R. sp., Starkeya novella and Xantobacter flavus.
Park et al., (2005) isolated a total of 27 free- living nitrogen fixing bacterial isolates were isolated from the rhizosphere of agricultural crops of Korea. Out of these only 5 isolates with nitrogenase activity above 150 nmol h mg protein were identified based on phenotypic and 16S rDNA sequences analysis. The strains were identified as Stenotrophomonas maltophilia, Bacillus fusiformis and Pseudomonas fluorescens, respectively. All the isolates
produced indole-3-acetic acid (IAA), in the presence of tryptophan, ranging from 100.4 ìg ml”1 to 255 ìg ml”1. The isolate of Bacillus fusiformis exhibiting highest nitrogenase activity (3677.81 nmol h mg protein) and IAA production (255 ìg ml”1) has a promising potential for developing as a plant growth promoting rhizobacteria.
Zhang et al., (1991), found that 19 clusters were formed below the boundary level of 0.725 average distance, which was the level that separated the reference Rhizobium and Bradyrhizobium species. The work also indicated that tree rhizobia are very diverse with respect to their cross- nodulation patterns, as well as their physiological and biochemical properties, since 12 of the clusters formed consisted of tree rhizobia alone. Two distinctive features of tree rhizobia isolated in the Sudan were their high maximum growth temperature and their high salt tolerance.
Suliasih and Widawati, (2005) showed that the microbial population ranged from 5.0x103-1.5x107 cells of bacteria/gram of soil for NFB respectively.
There were 17 isolates which have been identified till genus and species. The isolated microorganism were identified for NFB i.e. Azotobacter sp., A.
chroococcum, A. paspalii, Rhizobium sp., and Azospirillum sp.
Mirza et al., (2001), using ARA based MPN method indicated the presence of up to 106 bacteria per gram dry weight of stem and 107 bacteria per gram dry weight of root of field-grown sugarcane. Two nitrogen fixing bacterial isolates were obtained from stem (SC11, SC20) and two from the roots (SR12, SR13) of field-grown plants.
These isolates were identified as Enterobacter sp. strains on the basis of their morphological characteristics and biochemical tests. Further characterization of SC20 by 16S rRNA sequence analysis, showed high sequence similarity to the sequence of Enterobacter cloacae and Klebsiella oxytoca. All the isolates produced the phytohormone indoleacetic acid (IAA) in pure culture and this IAA production was enhanced in growth medium containing tryptophan. By using the15N isotope dilution technique, maximum
nitrogen fixation contribution (28% of total plant nitrogen) was detected in plantlets inoculated with isolate SC20.
Reinhardt et al., (2008) isolated and identified 14 strains of nitrogen fixing bacteria from different agricultural plant species after performing the culture characteristics tests and molecular characterization. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. Out of 14 strains 13 were assigned to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Remaining 1 strain was grouped with Acidovorax avenae, but with low similarity and phenotypic results are inconclusive.
It is possible that this strain can be reassigned to another species or genera if further studied.
Prabudoss and Stella, (2009) isolated Gluconacetobacter diazotrophicus from sugar rich crops like sugarcane (root, stem bud and leaves), sweet potato, pine apple and wild cane.
Nitrogen fixing efficiency of the isolates was evaluated by using acetylene reduction activity following the standard procedure. It was found out that the Nitrogen fixing ability of the isolated strains were comparatively high from that of the reference strains, indicating the superiority of the isolates.
Khan et al.,(2008) isolated 34.6% of the total isolates as nitrogen fixing microorganisms during the seedling (30 days) stage of rice (BR 10) rhizosphere soil. Based on the selection criteria, the four individual strains were microbiologically identified. After performing the biochemical tests it was found out that the isolated strains were strictly similar to Enterobacter spp., for strain-1, Klebsiella spp. for strain-2, Bacillus spp. for strain-3 and Azospirillum spp. for strain-4. They were anaerobic in nature.
After the work of Holguin et al.,(1992), it was assumed that the two diazotrophs isolated from the rhizosphere of mangrove trees probably belonged to the genus Vibrio according to their shape, dimensions and motility, in addition to their being Gram-negative, oxidase-positive and
capable of fermenting glucose. During their work it was found that Staphylococcus sp.,indicates interaction with the above two diazotrophic bacteria and results in the increased or decreased in the nitrogen fixing capacity of the two diazotrophs.
Detail study on molecular characterization of the free-living and symbiotic diazotrophs collected from various wild legumes of Manipur is lacking.
The following works in conjugation with molecular tools will help us to access new diazotroph both free-living and symbiotic collected from various wild legume plants of Manipur.
Culture characteristics
Gram-staining characteristics and cell morphologies could be determined by standard methods. Motility can be observed in wet mount using phase contrast microscope. Colony morphology and Transmission Electon Microscopy of flagella can also be done. Preliminary physiological characterization such as catalase test, starch hydrolysis test can also be carried out.
Biochemical Tests
Strains can be characterized by biochemical tests like Acetylene reduction test, N-Labeling and depletion studies, Gram staining, cells for fatty acid analysis, Hydrogen production test, catalase test, H2S production etc. according to the need of the reseach. Additional phenotypical tests could also be performed as described by Holt et al.,(1994), Hartmann and Baldani(2006), and Schmid et al.,(2006).
DNA extraction
DNA could be extracted from the culture which should then be use for various molecular analysis.
The final procedures for DNA extraction and purification can be done according to Bando et al.,(1998).
PCR Techonologies
For molecular characterization of the bacterial strains various types of PCR technologies can be utilized. Suitable technique should be used out of the several PCR techniques such as RAPD
analysis, RFLP analysis, ARDRA analysis or 16S rDNA sequencing.
RAPD Analysis
RAPD analysis are useful for differentiating bacteria at the strain level. The occurrence of strains with identical RAPD patterns suggests that they share a common clonal origin.
RFLP Analysis
Amplified nifH sequences could be digested overnight with suitable restriction-enzymes and should be electrophoresed on a polyacrylamide gel. Then the restriction pattern could be studied for the complexity of the natural population of nifH bearing bacteria.
ARDRA Analysis
ARDRA was proved to be useful for the classification of bacterial strains at different taxonomic levels, depending on selection of conserved or variable regions in the ribosomal genes for analysis.
Detection of nifH genes
For detection of nifH genes, Dot-blot hybridization assays could be carried out using standard protocols. An alternative means of identifying nitrogen fixers is the nif gene cloning and sequencing and of DNA amplification by Polymerase chain reaction (PCR).
Conclusion
Following the above mentioned protocol and methodologies of isolation and characterization can help in finding novel strains of nitrogen fixing microbes. So far isolation of nitrogen fixing microbes from the wild legumes of Manipur and their molecular characterization has not been carried out. So the current work could lead to the discovery of few new and unassigned species of nitrogen fixers from some of the wild legume plants of Manipur. Furthermore, using the new molecular tools and techniques for molecular characterization the ecological importance and the diversity of the nitrogen fixing microbes found associated with the wild legumes of Manipur could be ascertained and described.
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