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Research Article

volume 54 issue 6 (december 2020) : 763-768,   Doi: 10.18805/IJARe.A-5232

Un-culturable Microbial Community Analysis of Paddy Ecosystem at Different Depths using Nif H Gene DGGE Analysis Approach

C
C. Daphy Meurial
K
K. Kumar
1Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India.
Cite article:- Meurial Daphy C., Kumar K. (2020). Un-culturable Microbial Community Analysis of Paddy Ecosystem at Different Depths using Nif H Gene DGGE Analysis Approach. Indian Journal of Agricultural Research. 54(6): 763-768. doi: 10.18805/IJARe.A-5232.

ABSTRACT

Background: Biological N2 fixation is gaining importance in rice ecosystem, because of current concern on the environmental and soil health that are caused by the continuous use of nitrogenous fertilizers and the need for improved sustainable rice productivity. BNF is the conversion of atmospheric nitrogen by certain microorganism’s viz., N2 fixing microorganisms or diazotrophs into forms that plants can use. A substantial molecular diversity of N2 fixing bacteria has been detected in the rice field soil based on retrieval of nif H gene fragment from DNA by molecular approach, i.e., DGGE (Denaturant Gradient Gel Electrophoresis). The present investigation aimed to assess the diversity of N2 fixing bacteria by sequencing and phylogenetic analysis of nif H gene of the metagenomic DNA collected from different depth of flooded rice soil.
Methods: In this laboratory investigation, soil samples were collected from the paddy field at different depths viz., rhizosphere region, 5 cm, 10 cm, 15 cm depth using the core sampler during active tillering stage. In the laboratory, the collected soil samples were analyzed by molecular methods involving the use of primer sets based on nif H sequences (nitrogenase reductase). 
Result: Our investigations in the genetic diversity of diazotrophs associated with rice field soil using nif H revealed the existence of seven discrete bands. The sequencing results showed all sequences were classified as uncultured bacterium clone nitrogenase iron protein (nif H) gene, partial cds. The present works based on the applications of molecular methods have facilitated the study of individual ecosystem and their specific activity.

REFERENCES

  1. Barer, M.R. and Harwood, C.R. (1999). Bacterial viability and culturability. Advances in Microbial Physiology. 41: 93-137.
  2. Berg, D.E., Akopyants, N.S., Kersulyte, D. (1994). Fingerprinting microbial genomes using the RAPD or Rep- PCR method. Methods in Molecular and Cellular Biology. 5: 13-24.
  3. Chou, J.H., Jiang, S.R., Cho, J.C., Song, J., Lin, M.C., Chen, W.M. (2008). Azonexus hydrophilus sp. nov., a nif H gene-harbouring bacterium isolated from freshwater. International Journal of Systematic and Evolutionary Microbiology. 58: 946-51.
  4. Da Rocha, A., Okhi, S.T., Hiuki, C. (1986). Detection of mycoplasma like organisms in situ by indirect immune fluorescence microscopy. Phytopathology. 76: 864-868.
  5. Desnoues, N., Lin, M., Guo, X., Ma, L., Carren O-Lopez, R., Elmerich, C. (2003). Nitrogen fixation genetics and regulation in a Pseudomonas stutzeri strain associated with rice. Microbiology. 149: 2251–2262.
  6. Elbeltagy, A. and Ando, Y. (2008). Expression of nitrogenase gene (nif H) in roots and stems of rice, Oryza sativa, by endophytic nitrogen fixing communities. African Journal of Biotechnology. 7: 1950-1957.
  7. Ellis, J. (1994). Climate variability and complex ecosystem dynamics: Implications for pastoral development. In. Scoones J (ed). Living with uncertainty. Intermediate Technology Publications, London.
  8. Engelhard, M., Hurek, T., Reinhold-Hurek, B. (2000). Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races. Environmental Microbiology. 2: 131-141.
  9. Frank, I.H., Fegan, M., Hayward, A.C., Sly, L. (1998). Nucleotide sequence of the nif H gene coding for nitrogen reduction in the acetic acid bacterium Acetobacter diazotrophicus. Letters in Applied Microbiology. 26: 12-16.
  10. Hooper, D.U. and Johnson, L. (1999). Nitrogen limitation in dryland ecosystems: Responses to geographical and temporal variation in precipitation. Biogeochemistry. 46:247-293.
  11. Jenkinson, D.S. (2001). The impact of humans on the nitrogen cycle, with focus on temperate arable agriculture. Plant and Soil. 228:3-15.
  12. Kronzucker, H.J., Glass, A.D.M., Siddiqi, M.Y., Kirk, G.J.D. (2000). Comparative kinetic analysis of ammonium and nitrate acquisition by tropical lowland rice: Implications for rice cultivation and yield potential. New Phytologist. 145:471-476.
  13. Krotzky, A. and Werner, D. (1987). Nitrogen fixation in Pseudomonas stutzeri. Archives of Microbiology. 147: 48-57.
  14. Muyzer, G., De Waal, E.C., Uitterlinden, A.G. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S rRNA. Applied and Environmental Microbiology. 59: 695-670.
  15. Ohkuma, M., Noda, S., Kudo, T. (1999). Phylogenetic diversity of nitrogen fixation genes in the symbiotic microbial community in the gut of diverse termites. Applied and Environmental Microbiology. 65: 4926-4934.
  16. Pinto-Tomás, A.A., Anderson, M.A., Suen, G., Stevenson, D.M., Chu, F.S., Cleland, W.W., Weimer, P.J., Currie, C.R. (2009). Symbiotic nitrogen fixation in the fungus gardens of leaf-cutter ants. Science. 326: 1120-1123.
  17. Perez, P.G., Ye, J., Wang, S., Wang, X., Huang, D. (2014). Analysis of the occurrence and activity of diazotrophic communities in organic and conventional horticultural soils. Applied Soil Ecology. 79: 37-48.
  18. Poly, F., Monrozier, L.J., Bally, R. (2001a). Improvement in the RFLP procedure for studying the diversity of nif H genes in communities of nitrogen fixers in soil. Research in Microbiology. 152: 95-103. 
  19. Poly, F., Ranjard, L., Nazaret, S., Gourbiere, F., Monrozier, L.J. (2001b). Comparison of nif H gene pools in soils and soil micro environments with contrasting properties. Applied and Environmental Microbiolology. 67: 2255-2262.
  20. Ramasamy, K., Kalaichelvan, G., Nagamani, B. (1992). Working with Anaerobes: Methanogens. A Laboratory Manual. 
  21. Rosenblueth, M., Martínez, L., Silva, J., Martínez-Romero, E. (2004). Klebsiella variicola, a novel species with clinical and plant-associated isolates. Systematic and Applied Microbiology. 27:27 -35
  22. Saitou, N. and Nei, M. (1987). The neighbour-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution. 4: 406-425.
  23. Song, T., Martensson, L., Eriksson, T., Zheng, W., Rasmussen, U. (2005) Biodiversity and seasonal variation of the cyanobacterial assemblage in rice paddy field in Fujian, China. FEMS Microbiology Ecology. 54: 131-140.
  24. Shivprasad, S. and Page, W.J. (1989). Catechol formation and melanization by Na+-dependent Azotobacter chroococcum: A protective mechanism for aeroadaptation? Applied and Environmental Microbiology. 55:1811-1817.
  25. Tamura, K., Dudley, J., Nei, M., Kumar, S. (2007). MEGA 4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution. 24: 1596-1599.
  26. Vermeiren, H., Willems, A., Schoofs, G., De Mot, R., Keijers, V., Hai, W.,Vanderleyden, J. (1999). The rice inoculant strain Alcaligenes faecalis A15 is a nitrogen-fixing Pseudomonas stutzeri. Systematic and Applied Microbiology. 22: 215-224.
  27. Wang, M.Y., Siddiqi, M.Y., Ruth, T.J., Glass, A. (1993). Ammonium uptake by rice roots (I.fluxes and subcellular distribution of 13NH4 +). Plant Physiology. 103:1249-1258.
  28. Wartiainen, I., Eriksson, T., Zheng, W., Rasmussen, U. (2008). Variation in the active diazotrophic community in rice paddy-nif H PCR-DGGE analysis of rhizosphere and bulk soil. Applied Soil Ecology. 39:65-75 
  29. Widmer, F., Shaffer, B.T., Porteous, L.A., Seidler, R.J. (1999). Analysis of nif H gene pool complexity in soil and litter at a Douglas fir forest site in the Oregon cascade mountain range. Applied and Environmental Microbiology. 65: 374–380.
  30. Xie, C.H. and Yokota, A. (2006). Sphingomonas azotifigens sp. nov., a nitrogen-fixing bacterium isolated from the roots of Oryza sativa. International Journal of Systematic and Evolutionary Microbiology. 56: 889-893.
  31. Zehr, J.P., Mellon, M.T., Zani, S. (1998). New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nif H) genes. Applied and Environmental Microbiology. 64: 3444-3450.
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Indian Journal of Agricultural Research
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    Research Article

    volume 54 issue 6 (december 2020) : 763-768,   Doi: 10.18805/IJARe.A-5232

    Un-culturable Microbial Community Analysis of Paddy Ecosystem at Different Depths using Nif H Gene DGGE Analysis Approach

    C
    C. Daphy Meurial
    K
    K. Kumar
    1Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India.
    Cite article:- Meurial Daphy C., Kumar K. (2020). Un-culturable Microbial Community Analysis of Paddy Ecosystem at Different Depths using Nif H Gene DGGE Analysis Approach. Indian Journal of Agricultural Research. 54(6): 763-768. doi: 10.18805/IJARe.A-5232.

    ABSTRACT

    Background: Biological N2 fixation is gaining importance in rice ecosystem, because of current concern on the environmental and soil health that are caused by the continuous use of nitrogenous fertilizers and the need for improved sustainable rice productivity. BNF is the conversion of atmospheric nitrogen by certain microorganism’s viz., N2 fixing microorganisms or diazotrophs into forms that plants can use. A substantial molecular diversity of N2 fixing bacteria has been detected in the rice field soil based on retrieval of nif H gene fragment from DNA by molecular approach, i.e., DGGE (Denaturant Gradient Gel Electrophoresis). The present investigation aimed to assess the diversity of N2 fixing bacteria by sequencing and phylogenetic analysis of nif H gene of the metagenomic DNA collected from different depth of flooded rice soil.
    Methods: In this laboratory investigation, soil samples were collected from the paddy field at different depths viz., rhizosphere region, 5 cm, 10 cm, 15 cm depth using the core sampler during active tillering stage. In the laboratory, the collected soil samples were analyzed by molecular methods involving the use of primer sets based on nif H sequences (nitrogenase reductase). 
    Result: Our investigations in the genetic diversity of diazotrophs associated with rice field soil using nif H revealed the existence of seven discrete bands. The sequencing results showed all sequences were classified as uncultured bacterium clone nitrogenase iron protein (nif H) gene, partial cds. The present works based on the applications of molecular methods have facilitated the study of individual ecosystem and their specific activity.

    REFERENCES

    1. Barer, M.R. and Harwood, C.R. (1999). Bacterial viability and culturability. Advances in Microbial Physiology. 41: 93-137.
    2. Berg, D.E., Akopyants, N.S., Kersulyte, D. (1994). Fingerprinting microbial genomes using the RAPD or Rep- PCR method. Methods in Molecular and Cellular Biology. 5: 13-24.
    3. Chou, J.H., Jiang, S.R., Cho, J.C., Song, J., Lin, M.C., Chen, W.M. (2008). Azonexus hydrophilus sp. nov., a nif H gene-harbouring bacterium isolated from freshwater. International Journal of Systematic and Evolutionary Microbiology. 58: 946-51.
    4. Da Rocha, A., Okhi, S.T., Hiuki, C. (1986). Detection of mycoplasma like organisms in situ by indirect immune fluorescence microscopy. Phytopathology. 76: 864-868.
    5. Desnoues, N., Lin, M., Guo, X., Ma, L., Carren O-Lopez, R., Elmerich, C. (2003). Nitrogen fixation genetics and regulation in a Pseudomonas stutzeri strain associated with rice. Microbiology. 149: 2251–2262.
    6. Elbeltagy, A. and Ando, Y. (2008). Expression of nitrogenase gene (nif H) in roots and stems of rice, Oryza sativa, by endophytic nitrogen fixing communities. African Journal of Biotechnology. 7: 1950-1957.
    7. Ellis, J. (1994). Climate variability and complex ecosystem dynamics: Implications for pastoral development. In. Scoones J (ed). Living with uncertainty. Intermediate Technology Publications, London.
    8. Engelhard, M., Hurek, T., Reinhold-Hurek, B. (2000). Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races. Environmental Microbiology. 2: 131-141.
    9. Frank, I.H., Fegan, M., Hayward, A.C., Sly, L. (1998). Nucleotide sequence of the nif H gene coding for nitrogen reduction in the acetic acid bacterium Acetobacter diazotrophicus. Letters in Applied Microbiology. 26: 12-16.
    10. Hooper, D.U. and Johnson, L. (1999). Nitrogen limitation in dryland ecosystems: Responses to geographical and temporal variation in precipitation. Biogeochemistry. 46:247-293.
    11. Jenkinson, D.S. (2001). The impact of humans on the nitrogen cycle, with focus on temperate arable agriculture. Plant and Soil. 228:3-15.
    12. Kronzucker, H.J., Glass, A.D.M., Siddiqi, M.Y., Kirk, G.J.D. (2000). Comparative kinetic analysis of ammonium and nitrate acquisition by tropical lowland rice: Implications for rice cultivation and yield potential. New Phytologist. 145:471-476.
    13. Krotzky, A. and Werner, D. (1987). Nitrogen fixation in Pseudomonas stutzeri. Archives of Microbiology. 147: 48-57.
    14. Muyzer, G., De Waal, E.C., Uitterlinden, A.G. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S rRNA. Applied and Environmental Microbiology. 59: 695-670.
    15. Ohkuma, M., Noda, S., Kudo, T. (1999). Phylogenetic diversity of nitrogen fixation genes in the symbiotic microbial community in the gut of diverse termites. Applied and Environmental Microbiology. 65: 4926-4934.
    16. Pinto-Tomás, A.A., Anderson, M.A., Suen, G., Stevenson, D.M., Chu, F.S., Cleland, W.W., Weimer, P.J., Currie, C.R. (2009). Symbiotic nitrogen fixation in the fungus gardens of leaf-cutter ants. Science. 326: 1120-1123.
    17. Perez, P.G., Ye, J., Wang, S., Wang, X., Huang, D. (2014). Analysis of the occurrence and activity of diazotrophic communities in organic and conventional horticultural soils. Applied Soil Ecology. 79: 37-48.
    18. Poly, F., Monrozier, L.J., Bally, R. (2001a). Improvement in the RFLP procedure for studying the diversity of nif H genes in communities of nitrogen fixers in soil. Research in Microbiology. 152: 95-103. 
    19. Poly, F., Ranjard, L., Nazaret, S., Gourbiere, F., Monrozier, L.J. (2001b). Comparison of nif H gene pools in soils and soil micro environments with contrasting properties. Applied and Environmental Microbiolology. 67: 2255-2262.
    20. Ramasamy, K., Kalaichelvan, G., Nagamani, B. (1992). Working with Anaerobes: Methanogens. A Laboratory Manual. 
    21. Rosenblueth, M., Martínez, L., Silva, J., Martínez-Romero, E. (2004). Klebsiella variicola, a novel species with clinical and plant-associated isolates. Systematic and Applied Microbiology. 27:27 -35
    22. Saitou, N. and Nei, M. (1987). The neighbour-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution. 4: 406-425.
    23. Song, T., Martensson, L., Eriksson, T., Zheng, W., Rasmussen, U. (2005) Biodiversity and seasonal variation of the cyanobacterial assemblage in rice paddy field in Fujian, China. FEMS Microbiology Ecology. 54: 131-140.
    24. Shivprasad, S. and Page, W.J. (1989). Catechol formation and melanization by Na+-dependent Azotobacter chroococcum: A protective mechanism for aeroadaptation? Applied and Environmental Microbiology. 55:1811-1817.
    25. Tamura, K., Dudley, J., Nei, M., Kumar, S. (2007). MEGA 4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution. 24: 1596-1599.
    26. Vermeiren, H., Willems, A., Schoofs, G., De Mot, R., Keijers, V., Hai, W.,Vanderleyden, J. (1999). The rice inoculant strain Alcaligenes faecalis A15 is a nitrogen-fixing Pseudomonas stutzeri. Systematic and Applied Microbiology. 22: 215-224.
    27. Wang, M.Y., Siddiqi, M.Y., Ruth, T.J., Glass, A. (1993). Ammonium uptake by rice roots (I.fluxes and subcellular distribution of 13NH4 +). Plant Physiology. 103:1249-1258.
    28. Wartiainen, I., Eriksson, T., Zheng, W., Rasmussen, U. (2008). Variation in the active diazotrophic community in rice paddy-nif H PCR-DGGE analysis of rhizosphere and bulk soil. Applied Soil Ecology. 39:65-75 
    29. Widmer, F., Shaffer, B.T., Porteous, L.A., Seidler, R.J. (1999). Analysis of nif H gene pool complexity in soil and litter at a Douglas fir forest site in the Oregon cascade mountain range. Applied and Environmental Microbiology. 65: 374–380.
    30. Xie, C.H. and Yokota, A. (2006). Sphingomonas azotifigens sp. nov., a nitrogen-fixing bacterium isolated from the roots of Oryza sativa. International Journal of Systematic and Evolutionary Microbiology. 56: 889-893.
    31. Zehr, J.P., Mellon, M.T., Zani, S. (1998). New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nif H) genes. Applied and Environmental Microbiology. 64: 3444-3450.
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