Symbiosis of nodule bacteria with perennial xerophyte leguminous plants of Central Asia

Zair S. Shakirov, Sardor A. Khakimov

.

DOI: 10.4236/as.2010.11004                

Abstract

From nodules of perennial xerophyte desert leguminous plants – Ammodendron conollyi, Astragalus villossimus, Astragalus unifoliolatus – 151 bacterial isolates have been isolated. The study of nodulation showed that AC8-1, AC11, AC21, AC1-1, AC12-1 isolates (from Ammodendron conollyi), AV1, AV8-1, AV9, AV26-1, AV36-1 isolates (from Astragalus villossimus) and AU17-1, AU30-1, AU30-2, AU20-1, AU23 isolates (from Astragalus unifoliolatus) formed an effective nitrogen-fixing symbiosis with the host plants. As a result of 16S rRNA gene study of the salt-resistant nodule bacteria it has been determined that bacteria were related to Rhizobium, Burkholderia and Achromobacter genera. The study of isolates growth has revealed that there were fast-growing and moderately-grow- ing isolates that possessed with doubling-time varying from 20 to 45 min. Their examination for antibiotic-resistance showed that the number of bacterial colonies of selected strains decreased to some extent in the presence of chloramphenicol, but in all strains the resistance to antibiotics was detected. The further investigations of resistance of the formed symbiosis to stresses (drought, salinity) showed that at 6.41% of moisture the maximal height and biomass of inoculated plants of Ammodendron conollyi were 21 cm and 2320 mg, but at 3.8% moisture the height reduced by 4 times (up to 4.5 cm) and the biomass – by 11 times (203 mg). The analogous effect was observed in Astragalus villossimus and Astragalus unifoliolatus symbiosises. The salinity equal to 100-200 mM NaCl did not affect practically on normal growth and development of desert leguminous plants symbiosis, while for Astragalus villossimus such affecting concentration comprised up to 100 mM NaCl. The light microscopy and electron microscopy of Astragalus villossimus nodule sections showed that V1 nodule bacteria strain efficiently colonized the internal space within nodules, where they were transformed into bacteroids. At 100 mM NaCl salinity concentration the colonization of nodule bacteria within nodule plant cells reduced in comparison with control nodules of plants grown in non-salted conditions.

Keywords

Ammodendron conollyi; Astragalus villossimus; Astragalus unifoliolatus; Nodulation; Nitrogen Fixation; Salinity; Bacteroid; Rhizobium; Burkholderia; Achromobacter

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Dart, P.J. (1994) Microbial symbiosises of tree and shrub legumes. In: R. C. Gutteridge and H. M. Shelton, Ed., Forage Tree Legumes in Tropical Agriculture, CAB International, Wallingford. http://www.betuco.be/agroforestry/ Forage%20Tree%20Legumes%20in%20Tropical%20Agriculture%20FAO
[2]	Zahran, H.H. (2001) Rhizobia from wild legumes: Diversity, taxonomy, ecology, nitrogen fixation and biotechnology. Journal of Biotechnology, 91(2), 143-153.
[3]	Korovin, E.P., Ed. (1955) Flora of Uzbekistan. Publish House of Academy Sciences of UzSSR, Tashkent, 638-640.
[4]	Khotyanovich, A.U., Ed. (1991) Methods for cultivation of nitrogen-fixing bacteria, ways of both their obtaining and preparing of preparations on their base (methodical guides). All-Union Institute of Agricultural Microbiology, Leningrad, 33-60.
[5]	Hardy, D.W., Halstein, R., Jakson, E. and Buens, R.S. (1968) C2H2–C2H4 assay to N2 fixation laboratory and field evaluation. Plant Physiology, 43, 9-13.
[6]	Ferris, M.J., Muyzer, G. and Ward, D.M. (1996) Denaturing gradient gel electrophoresis profiles of 16S rRNA- defined populations inhabiting a hot spring microbial mat community. Applied Environmental Microbiology, 62(2), 340-346.
[7]	Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped: BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research, 25(17), 3389-3402.
[8]	Saitou, N. and Nei, M. (1987) The neighbour-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology Evolution, 4(4), 406-425.
[9]	Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24, 1596-1599. http://www.ncbi.nlm.nih.gov/sites/entrez? cmd=retrieve&db=pubmed&list_uids=17488738&dopt=AbstractPlus
[10]	Collavoli, A., Storti, S., Dell’Amico, C. and Iascone, M.R. (2000) Northern blot analysis with digoxigenin in PCR-labeled probes in research samples from myocardial biopsies. Biochemica, 3, 19-20. http://www.roche-applied- science.com/PROD_INF/BIOCHEMI/no3_2000/PDF/p19- 20.pdf
[11]	Eckhardt, T. (1978) A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria. Plasmid, 1(4), 584-588.
[12]	Priefer, U.B. (1984) Characterization of plasmid DNA by agarose gel electrophoresis. In: Pühler, A. and Timmis, K.N., Ed., Advanced Molecular Genetics, Springer-Verlag, Berlin, 26-37.
[13]	Glazer, V.M., Ed. (1972) Big practical course on genetics of microorganisms. Publish House of Moscow State of University, Moscow, 1-25.
[14]	Nechaeva, N.T., Ed., (1985) Improvement of desert ranges in Soviet Central Asia. Harwood Academic Publishers, London, 88.
[15]	Cubo, M.T., Buendia-Claveria, A.M., Beringer, J.E. and Ruiz-Sainz, J.E. (1988) Melanin production by Rhizobium strains. Applied Environmental Microbiology, 54(7), 1812-1817.
[16]	Teketay, D. (1998) Germination of Acacia origena, Acacia pilispirina and Pterolobium stellatum in response to different pre-sowing seed treatments, temperature and light. Journal of Arid Environments, 38, 551-560.
[17]	Abulfatih, H.A. (1995) Seed germination in Acacia species and their relation to altitudinal gradient in south- western Saudi Arabia. Journal of Arid Environments, 31(2), 171-178.
[18]	Yakovleva, Z.M. and Mishustin, E.N., Ed. (1975) Bacteroids of nodule bacteria. Publish House Nauka, Moscow, 171.
[19]	Gordienko, N.Y. and Yakovleva, Z.M. (1979) Topography of infection threads in root nodules of legume plants. Proceedings of the USSR Academy of Sciences, Series Biologic, 3, 466-471.
[20]	Yakovleva, Z.M. (1981) Microstructure of pea nodules upon infectioning by neomycin-resistant nodule bacteria mutant. Mikrobiologiya, 50(3), 528-534.
[21]	Newcomb, W., Syono, K. and Torrey, J.G. (1977) Development of an ineffective pea root nodule: Morphogenesis, fine structure and cytokinin biosynthesis. Canadian Journal of Botany, 55(14), 1891-1907.
[22]	B. Dreyfus, J.L. Garcia, M. Gillis, “Characterization of Azorhizobium caulinodans gen.nov. sp.nov., a stem-nodulating nitrogen-fixing bacterium isolated from Sesbania rostrata”, Internation Journal of Systematic Bacteriology, vol. 38, 1988, pp. 89-98.
[23]	Rana, D. and Krishnan, H.B. (1995) A new root-nodulating symbiont of the tropical legume Sesbania, Rhizobium sp. SIN-1, is closely related to Rhizobium galegae, a speсies that nodulate temperate Legumes. FEMS Microbiology Letters, 134(1), 19-25.
[24]	Wdowiak, S. and Malek, W. (2000) Numerical analysis of Astragalus cicer microsymbionts. Current Microbiology, 41(2), 142-148.
[25]	Chen, W.X., Li, G.S., Qi, Y.L., Wang, E.T., Yuan, H.L. and Li, J.L. (1991) Rhizobium huakuii sp. nov., isolated from the root nodules of Astragalus sinicus. International Journal of Systematic Bacteriology, 41(2), 275-280.
[26]	Zhang, X.X., Turner, S.L., Guo, X.W., Yang, H.J., Debelle, F., Yang, G.P., Denarie, J., Young, J.P. and Li, F.D. (2000) The common nodulation genes of Astragalus sinicus rhizobia are conserved despite chromosomal diversity. Applied Environmental Microbiology, 66(7), 2988- 2995.
[27]	Wang, S. and Chen, W. (1997) A study of taxonomy of Rhizobia isolated from Astragalus sp. Acta Microbiologica Sinica, 37(5), 335-343.
[28]	Gao, J., Terefework, Z., Chen, W. and Lindstrom, K. (2001) Genetic diversity of rhizobia isolated from Astragalus adsurgens growing in different geographical regions of China. Journal of Biotechnology, 91(2-3), 155- 168.
[29]	Haukka, K., Lindstrom, K. and Young, P.W. (1998) Three phylogenetic groups of nodA and nifH genes in Sinorhizobium and Mezorhizobium isolates from Lehuminous trees in Africa and Latin America. Applied Environmental Microbiology, 64, 419-426.