Heterotrophic and endophytic diazotrophs were isolated from rhizosphere soil, rhizoplane, roots and stems of different rice varieties. A total of thirteen isolates obtained were subjected to acetylene reduction assay (ARA) and eight isolates recorded significant amount of nitrogenase activity in a rangeof 31.65 to 91.95 nmoles of ethylene mg-1 cells h-1 . Targeted PCR fingerprinting using nif H primers generated specific DNA band of approximately 750 bp, confirming the presence of nif genes in theseisolates. Two isolates of heterotrophic diazotrophs, HDM 7 and HDT 1, generated multiple bands ranging from 500 – 1000 bp

Kumar, K

Sugitha, TCK

English

AJOL - African Journals Online

African Journal of Biotechnology Vol. 8 (22), pp. 6249-6252, 16 November, 2009     Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2009 Academic Journals     Full Length Research Paper  Identification of nif genes of heterotrophic and endophytic diazotrophs associated with rice (Oryza sativa L.,) by targeted DNA finger printing  T. C. Kumari Sugitha and K. Kumar*  Department of Agricultural Microbiology, Tamil Nadu Agricultural University, 641 003 Coimbatore, India.  Accepted 30 May, 2008  Heterotrophic and endophytic diazotrophs were isolated from rhizosphere soil, rhizoplane, roots and stems of different rice varieties. A total of thirteen isolates obtained were subjected to acetylene reduction assay (ARA) and eight isolates recorded significant amount of nitrogenase activity in a range of 31.65 to 91.95 nmoles of ethylene mg-1 cells h-1. Targeted PCR fingerprinting using nif H primers generated specific DNA band of approximately 750 bp, confirming the presence of nif genes in these isolates. Two isolates of heterotrophic diazotrophs, HDM 7 and HDT 1, generated multiple bands ranging from 500 – 1000 bp.  Key words: Acetylene reduction assay, endophytes, heterotrophs, nif gene, targeted PCR fingerprinting.   INTRODUCTION   Symbiotic N2 fixation by legumes is generally expected to be the dominant source of biological of N input in the earth. In recent years, N balance and 15N techniques have provided convincing evidence that non-legumes such as wetland rice can, under certain conditions, derive a considerable amount of N from associated N2 fixing bacteria in the plant rhizosphere (Chalk, 1991; Mark and Craswell, 1992). In non-legume agro systems, rice fields are considered to be ideal niches for biological nitrogen fixation because of their characteristic ecological conditions. It is well known that a remarkable diversity of N2 fixing bacteria are naturally associated with field-grown rice (Balandreau, 1986). The free living heterotro-phic N2 fixers are a potentially important source of N2 fixation in rice fields (Boddey et al., 1995, Mahadevappa and Shenoy, 2000).  The recent isolation and study of endophytic nitrogen fixing bacteria from several grasses (Baldani et al., 1997) represent an exciting phase in the field of biological nitrogen fixation. The search for natural association and endophytic interaction of diazotrophs with rice is consi-dered very promising especially in primitive rice varieties not bred to efficiently respond to N fertilizer (Barraquio et     *Corresponding author. E-mail: azollakumar@rediffmail.com. Tel: +91422-5511294. al., 1997; Stoltzfus et al., 1997). Endophytic diazotrophs usually live with in the root apoplast that is the inter-cellular spaces and or the xylem vessels, and may enter the plants via the root or epidermal cracks at lateral root junctions and might interact more closely with the host with less competition for carbon sources and a more protected environment for N2 fixation (Reinhold and Hurek, 1998).  Nitrogenase, the enzyme highly essential for reducing nitrogen to ammonia, is composed of Fe (dinitrogenase) and Mo-Fe protein (dinitrogenase reductase) is encoded by nif gene. A substantial molecular diversity of N2 fixing bacteria has been detected in field grown rice based on retrieval of nif H or nif D gene fragments from root DNA (da Rocha et al., 1986). Since the nif H gene only occurs in nitrogen fixing microorganisms, it has been used to monitor the presence of these diazotrophs, for example, in pure cultures (Frank et al., 1998), in soil (Widmer et al., 1999) and plants. In view of the above, this study was aimed to identify the diazotrophic nature of bacteria isolated from rice rhizosphere soil of different locations and popular rice cultivars of Tamil Nadu, India, using targeted PCR finger printing with nif H primer.   MATERIALS AND METHODS   Isolation of heterotrophic bacteria from the rhizosphere   Heterotrophic diazotrophs were isolated from rhizosphere soil  sam-  6250         Afr. J. Biotechnol.    Table 1. Nitrogenase activity of the heterotrophic and endophytic diazotrophic isolates.  S/N Isolate Nitrogenase activity  (n moles of ethylene mg-1 of cells h-1) 1 HDC 4 0.00 2 HDPY 1 61.8 3 HDPY 2 0.00 4 HDMY 2 0.00 5 HDT 1 53.4 6 HDC 8 91.8 7 HDM 7 53.1 8 EDA 1 91.95 9 EDC 5 68.05 10 EDA 2 31.65 11 EDM 2 53.75 12 EDC 3 0.00 13 EDC 6 0.00  HD = Heterotrophic diazotrophs; ED = endophytic diazotrophs. The isolates are subjected to acetylene reduction assay. Acetylene was converted to ethylene by the organisms due to the presence of nitrogenase enzyme and the amount of ethylene was quantified in a gas chromatography.    ples collected from different rice growing regions in Tamil Nadu State, India using the medium for total diazotrophs as standardized by Watanabe and Barrquio (1979) consisting of (per liter) glucose (0.5 g), malic acid (0.5 g), yeast (0.1 g), K2HPO4 (0.5 g), FeSO4 (0.04 g), MgSO4 (0.02 g), NaMoO4 (trace), CaCl2 (0.2 g), boric acid (0.15 g), ZnSO4 (0.07 g), CuSO4 (trace), MnCl2 (trace), agar (20 g) and pH 7.0.   Isolation of heterotrophic diazotrophs from rhizoplane   To isolate the diazotrophs from rhizoplane rice seedlings were up-rooted carefully from the field and the shoot portion was cut with sterile scissors. The excised root system along with the soil parti-cles were washed carefully in a beaker containing water without disturbing the root system. One gram of roots washed free of soil was transferred to 10 ml of distilled water, serially diluted upto 10-6 dilutions and plated in Watanabe and Barraquio (1979) medium for isolation of total heterotrophic diazotrophs.    Isolation of endophytic bacteria   Plant samples of five different rice varieties (ADT 38, ADT 39, White ponni, ASD 16 and TPS 1) were collected and washed with tap water to remove all soil particles and the stems, roots and leaves were cut with sterile scissors. The roots and leaves were surface sterilized with 70% ethanol for 5 min and then treated with 0.1% HgCl2 for 30 s. The stems were further cut into small bits, surface sterilized by dipping in 70% ethanol and then treated with 0.1% trypic soy agar (TSA plates). They were then homogenised, under sterile conditions with pestle and mortar in phosphate – buffered saline. The serially diluted homogenate was poured in tubes with N-free medium (Dobereiner and Day, 1976) consisting of (per liter) malic acid (5 g), K2HPO4 (0.5 g), MgSo4, 7 H2O (0.2 g), NaCl (0.1 g), CaCl2 (0.2 g) and 0.5% bromothymol blue in 0.2 N KOH (2 ml), 1.64% Fe-EDTA solution (4 ml) and agar (2 g). The final pH was adjusted to 7.0 by 0.1 N KOH.      Purification and maintenance of cultures   Single, well separated and morphologically, different colonies grow-ing on the plate were picked and purified by streak plate method. The purified colonies were transferred into agar slants and mass multiplied in the respective medium. Thirteen isolates were obtained and were used for further studies. The slant cultures were stored at 4°C. The isolates were designated according to the loca-tion from which it was isolated. The first two letters of the diazotro-phic isolate names denote heterotrophic (HD) and endophytic (ED) and the remaining letters represent the location from which it was isolated with the numerals indicating the isolate number from each location.   Diazotrophy of the isolates   Diazotrophy of the isolates were determined on the basis of ARA and nif H gene amplification by PCR.    i) Assay of acetylene reduction activity (ARA)  The ARA was carried out for all the thirteen isolates grown in N free medium (Watanabe and Barraquio, 1979) for total diazotrophs by following the method of Hardy et al. (1968).    ii) Identification of the nif genes of diazotrophs using nif H primer   The total genomic DNA of the diazotroph was isolated using the method of Chen and Kuo (1993). The respective cultures were grown till log phase and bacterial cells were harvested by centri-fugation for 5 min at 6000 rpm at 4°C. To the cell pellet, 200 µl of washing buffer (0.15 M NaCl 7.5 ml, 0.1 M EDTA 10 ml and the final volume made upto 50 ml) was added, thoroughly vortexed and again centrifuged at 6000 rpm at 4°C for 5 min. Ten per cent SDS was added to the pellet and again spun at 8000 rpm for 10 min at 4°C. The top aqueous layer was transferred to new tubes, added with 2.5 volume of cold ethanol and 1/10 volume of 3 M sodium acetate and then the contents were precipitated at-20°C overnight. After centrifugation at 12,000 rpm at 4°C for 5 min, the pellet was washed with 100 µl of 70% ethanol, again spinned at 12000 rpm at 4°C for 5 min and the pellet was dissolved in 50 µl TE buffer (Tris HCL 10 mM 2 ml, EDTA 1 mM 0.4 ml and volume made upto 100 ml) and stored at 4°C. The quality of DNA obtained was checked by loading 5 µl DNA on 0.8% agarose gel, and viewing the lands in UV transilluminator.  3 µl of this DNA was used as the template for amplification of nif H by PCR with primers (5’ GTTTTACGGCAAGGGCGGTAT-CGGCA-3’ and 5’-TCCTCCAGCTCTCCATGGTGATCsG-3’). PCR wa carried out in a PTC-100TM programmable thermocycler with an initial denature-tion for 5 min at 94°C, then 35 cycles of 1 min at 94°C, 1 min of 50°C, 2 min at 72°C and a final extension for 10 min at 72 and 4°C for storage.    RESULTS AND DISCUSSION  Among the 13 isolates subjected to ARA, nitrogenase activity was not detected in 5 isolates. All the other 8 iso-lates recorded significant amount of nitrogenase activity in a range of 31.65 to 91.95 moles of ethylene as depic-ted in the Table 1. A maximum of 91.95 n moles of ethylene mg-1 cells h-1 was exhibited by the isolate EDA 1 followed by  HDC 8  (91.8 n moles  of  ethylene mg-1 cells        Figure 1. Characterization of nif genes of heterotrophic and endophytic diazotrophs using nif H primers.    h-1), HDPY 1, HDT 1, HDM 7, EDC 5, EDA 2 and EDM 2 recorded a maximum of 61.8, 53.4, 53.1, 68.05, 31.65 and 53.75 n moles of ethylene mg-1 of cells h-1 respec-tively. Investigation on the nitrogen fixing capabilities based on acetylene reduction activity of the heterotrophic and endophytic isolates of rice revealed that 57.14 per cent and 66.66 per cent were nitrogen fixers respectively. Some strains isolated in this study showed relatively low ARA. This could have been due to the reason that the O2 level was not controlled during incubation and diazo-trophs have a varying ability to tolerate O2 (Postgate, 1998).   Identification of diazotrophs using nif gene   The DNA extracted from different cultures was resolved in 0.8% agarose gel. The amplified DNA was found to be in the range of 15 to 20 kbp. The nif H  primers  produced informative and reproducible genetic markers in standard Rhizobium culture (BMBS 1) and eight bacterial isolates,  Sugitha and Kumar         6251    confirming their diazotrophic nature. The molecular wei-ght of the replicon was approximately 750 bp for the stan-dard culture, HDPY 1, HDC 8, EDM 2, EDA 2, EDC 5 and EDA 1 (Figure 1). Two isolates of heterotrophic diazo-trophs, HDM 7, and HDT 1 generated multiple DNA bands ranging from 500-1500 bp. The remaining 5 iso-lates did not generate any replicon.  Rapid and unambiguous identification of diazotrophs has greatly benefited from recent advances in DNA fingerprinting based on polymerase chain reaction (PCR). Though short-term kinetic measurements such as Acety-lene Reduction Assay (ARA) and reduction of 15N2 or 13N2 are available, they are not reliable and also more time con-suming (Rennie and Rennie, 1983). In the present investigation, targeted PCR fingerprinting (TPF) using nif primers was used to as a molecular tool to identify the nif H gene of the selected isolates.  Identification of specific replicon with a molecular weight of approximately 750 bp in HDPY 1, HDC 8, EDM 2, EDA 1, EDC 5 and EDA 2 confirmed the presence of nif genes in the isolates. Perrett and Broughton (1998) employed TPF using primers specific for the nif H and rec A genes to discriminate between Rhizobium species NGR 234 and R. fredii USDA 257, the closely related bacteria in which the symbiotic loci are 98% homologous. Incase of HDT 7 and HDT 1, multiple replicons were for-med, this might be due to minute variations in the con-centration and purity of template DNA in these culture as observed by Berg et al. (1994). The diazotrophs has been intensively studied in the last few decades because of their widespread ecological and know-ledge of the physiological characteristics of nif H will be highly useful to study the diversity and role that these diazotrophs play in the environment.   ACKNOWLEDGEMENTS  The authors are grateful to the Indian Council of Agricul-tural Research, New Delhi for the financial support to carry out this study under national Agricultural Techno-logy Project-Team of Excellence on Biofertilizers for rice-based cropping systems.   REFERENCES  Balandreau J (1986). Ecological factors and adaptive processes in N2 fixing bacterial populations of the plant environment. Plant Soil 90: 73-92. Baldani JI, Caruso L, Baldani VLD, Goi S (1997). Recent advances in BNF with non-legume plants. Soil Biol. Biochem. 29: 911-922. Barraquio WL, Sevilla L, Ladha JK (1997). Isolation of diazotrophic bacteria from wetland rice. Plant Soil 194: 15-24. Berg DE, Akopyants NS, Kernulyte D (1994). Finger printing microbial genomes using the RAPD or Rep-PCR method. Methods Mol. Cell. Biol. 5: 13-24. Boddey RM, de Oliveira OC, Urquiaga S (1995). Biological nitrogen fixation associated with sugarcane and rice : contributions  and  pros- pects for improvement. Plant soil 174: 195-209. Chalk PM (1991). The contribution of associative and symbiotic nitrogen 6252         Afr. J. Biotechnol.    fixation to the nitrogen nutrition of non legumes. Plant Soil 132: 29-39. Chen W, Kuo T (1993) A simple and rapid method for preparation of bacterial genomic DNA. Nucleic Acid Res. 21(8): 2260-2262. da Rocha A, Ohki ST, Hiruki C (1986). Detection of mycoplasm like organisms insitu by indirect immunofluorescence microscopy. Phytopathology. 76: 864-868. Dobereiner J, Day M (1976). Associative symbioses in tropical grasses: Characterization of microorganisms and nitrogen – fixing sites. In: Newton WE, Nyman CJ, (eds) Proceedings of the 1st International Symposium on N2 fixation, Vol. 2, Washington State University Press, Pullman, pp. 518-558.  Frank IH, Fegan M, Hayward AC (1998). Nucleotide sequence of the nif H gene coding for nitrogen reduction in the acetic acid bacterium Acetobacter diazotrophicus. Lett. Appl. Microbiol. 26: 12-16. Hardy RWF, Holstein RD, Jackson EK, Burns RC (1968). The acetylene reduction assays of nitrogen fixation : Laboratory and field evaluations. Plant Physiol. 43: 1185-1207. Mahadevappa M, Shenoy VV (2000). Towards nitrogen fixing rice (Oryza sativa). Adv. Agric. Res. India. 13: 131-139. Mark BP, Craswell ET (1992). Biological nitrogen fixation : investments, expectations and actual contributions to agriculture. Plant Soil. 141: 13-39. Perrett X, Broughton Y (1998). Rapid identification of Rhizobium strains by targeted PCR fingerprinting. Plant Soil. 204: 21-34. Postgate J (1998). Nitrogen fixation, 3rd edn. Cambridge University Press, Cambridge, 28-33.                                                 Reinhold B, Hurek T (1998) Life in grases : diazotrophic endophytes. Trends Microbiol. 6: 139-144. Rennie RJ, Rennie DA (1983). Techniques for quantifying nitrogen fixation in association with non legumes under field and greenhouse conditions. Can. J. Microbiol. 29: 1022-1034. Stoltzfus JR, So R, Malavizhi PP, Ladha  JK, de Bruijn FJ (1997). Isola-tion of endophylic bacteria from rice assessment of their potential for supplying rice with biologically fixed nitrogen. Plant Soil, 194: 25-36. Watanabe I, Barraquio NL (1979) Low levels of fixed nitrogen for isolation of free living nitrogen fixing organisms from rice roots. Nature. 272: 565-566. Widmer F, Shaffer BT, Porteous LA, Seidler RJ (1999) Analysis of nif H gene pool complexity in soil and litter at a Douglas fir Forest site in the Oregan Cascade Mountain Range. Appl. Envion. Microbiol. 65: 374-380.  

Identification of nif genes of heterotrophic and endophytic diazotrophs associated with rice (Oryza sativa L.,) by targeted DNA finger printing

https://www.ajol.info/index.php/ajb/article/download/66128/53843

Identification of nif genes of heterotrophic and endophytic diazotrophs associated with rice (Oryza sativa L.,) by targeted DNA finger printing

Abstract

Similar works

Full text

Available Versions

AJOL - African Journals Online