GJ - Privredna komora Srbije

GJ - Privredna komora Srbije GJ - Privredna komora Srbije

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najkvalitetnijih sojeva za komercijalnu proizvodnju kao i kod prouĉavanje ekologije i kompetitivnih odnosa koji vladaju u prirodnim sredinama. S obzirom na znaĉaj prouĉavanja prirodne populacije Rhizobium sp. i nedostatak takvih podataka za naša uslove, ovim istraţivanjima zapoĉelo se sa izolacijom i identifikacijom autohtonih sojeva u rekultivisanim deponijama jalovišta i pepelišta u Kostolaĉkom ugljenom basenu. Identifikacija autohtonih sojeva je uvek praćena odredjenim teškoćama Problem je što metode za diferencijaciju ĉesto vrlo srodnih sojeva, moraju istovremeno biti pouzdane, dovoljno osjetljive i brze da se mogu primijeniti na veliki broj sojeva. Danas se primjenjuju raznovrsne metode za identifikaciju sojeva kvrţiĉnih bakterija zasnovane na fenotipskim ili genotipskim karakteristikama. Kovencionalne metode ukljuĉuju razliĉite serološke metode (Keyser et al. 1984, Sawada et al. 1989, Weber et al. 1989, Leung et al. 1994); ispitivanje prirodne otpornosti na antibiotike (Josey et al. 1979, Mueller et al. 1988), prouĉavanje ukupnih ćelijskih proteina primjenom SDS- poliakrilamid gel elektroforeze (Roberts et al. 1980, Sadowski et al. 1987, Moreira et al. 1993), utvrdjivanje osjetljivosti na bakteriofage (Schmidt et al. 1986), produkcije rhizobitoksina (Devine et al. 1988). Pojedine metode su se s vremenom pokazale nedovoljno specifiĉnim i osjetljivim. Zato su razvijene metode koje omogućavaju identifikaciju baziranu na ukupnoj genetskoj konstituciji ćelija pre nego na nekoliko izabranih svojstava. U novije vrijeme, razvijene su razliĉite tehnike koje diferenciraju sojeve na molekularnom nivou i koje se široko primjenjuju za karakterizaciju sojeva i utvrdjivanje genetske raznolikosti i odnosa izmedju razlièitih organizama (Caetano-Anolles et al. 1991, Hartman i Amarger 1991, de Brujin 1992, Minamisawa et al1992). Cilj ovih istraţivanja, osim utvrdjivanja raznovsnosti sojeva Rhizobium sp. bio je i unapredjenje metoda rekultivacije pepelišta i jalovišta. Poznato je da su bakterije sa potencijalom za simbiozu i azotofiksaciju osetljive na ekstremne vrednosti pH podloge, povišene koncentracije soli, prisustvo nekih teških metala ili metaloida, ekstremno viosoke i niske temperature i druge nepovoljne faktore sredine (Rice et al., 1977). To je i razlog da se sojevi ovih bakterija ne nalaze u sveţim deponijama pepela ili se u malim koliĉinama i sa malim potencijalom za simbiozu nalaze u deponijama jalovine. Dugogodišnji napori za rekultivacijom deponija pepelišta i jalovišta praćeni su i humifikacijom (unošenjem plodnog zemljišta sa humiusom iz pozajmišta). Opravdana je pretpostavka da na taj naĉin na deponije stiţu i sojevi ovih bakterija koji se posle selekcije difirencerija na otporne, specifiĉne i originalne. Biohemiske i fizološke analize pokazuju opravdanost ovakve pretpostavke, jer je nadjeno da devet izolata ima toleranciju i pozitivan rast na baznoj podlozi pH 8,5. Većina izolata pokazuje pozitivan rast pri 1% NaCl pa ih smatramo umerenim halofilima, dok izolati RL02 i RL14 pokazuju toleranciju na 2% NaCl, pa ih smatramo pravim halofilima. Opšta preporuka ovih istraţivanja je da se u postupku setve leguminoza na pepelu i jalovini apliciraju bakterijski preparati otpornih sojeva bakterija Rhizobium sp. time bi se znatno povećao uĉinak azotofiksacije i gajenja leguminoza kao zelenišnog djubrenja, uštedelo vreme potrebno za rekultivaciju i smanjio utrošak sredstava za aplikaciju azotnih djubriva. LITERATURA 1. Caetano-Anolles G., Bassam B. J., Gresshoff P.M. (1991) DNA amplification fingerprinting using very short arbitrary oligonucleotide primers. Biotehnology 9: 553-557 2. Chabot, R., Antoun H. and Cescas M.P.1996. Growth promotion of maize and lettuce by phosphatesolubilizing Rhizobium leguminosarum biovar phaseoli, Plant Soil, 184: 311–321. 3. Chromosomal Types within an Indigenous Soil Population of Rhizobium leguminosarum bv. trifolii. Appl. Environ. Microbiol. 60: 416-426 4. de Brujin F.J. (1992). Use of Repetitive (Repetitive Extragenic Palindromic and Enterobacterial Repetitive 5. Devine T.E., Kuykendall L.D., O.Neill J.J. (1988). DNA Homology Group and the Identity of Bradyrhizobial Strains Producing Rhizobitoxine-Induced Foliar Chlorosis on Soybean. Crop Science 28: 938-941 6. ĐorĊević-Miloradović, J. , Miloradović, M., Savić, N. (2011) Mikrobiološka aktivnost deposola jalovine spoljnog odlagališta PK Drmno-Kostolac, RUDARSTVO 2011: 556-563 7. ĐorĊević-Miloradović, J., Miloradović, M. i Savić, N. (2012) Rekultivacija i ozelenjavanje deponija jalovišta i pepelišta u Kostolcu, PD Rekultivacija i ozelenjavanje, Kostolac, 137 p. 8. ĐorĊjević-Miloradović, J. Miloradović, M. i Savić, N. (2010) Karakteristike fitoremedijacije deponija pepela u zavisnosti od melioracije, revegetacije i mikrobne zajednice u rizosferi II Savetovanje: Deponije pepela, šljake i jalovine u termoelktranama i rudnicima, 145-154 9. Dreyfus, B., J. L. Garcia, and M. Gillis. 1988. Characterization of Azorhizobium caulinodans gen. nov., sp. nov., a stemnodulating nitrogen-fixing bacterium isolated from Sesbunia rostrata. Int. J. Syst. Bacteriol. 38:89- 98. 10. Graham, P.H. (2000): Nod ule for ma tion in le gumes. In: J.Edelberg (ed.) En cy clo pe dia of mi cro bi ol ogy., 2ed., vol.3., 407 – 417, Academyc Press. San Diego. 295

11. Hajsig, D., Naglić, T., Madić, J., Gamulin, S. (1992): Opća mikrobiologija i imunologija s osnovama epizootiologije. Školska knjiga, Zagreb 12. Hartmann A., Amarger N. (1991). Genotypic diversity of an indigenous Rhizobium meliloti field population assessed by plasmid profiles, DNA fingerprinting, and insertion sequence typing. Can. J. Microbiol. 37: 600- 608. 13. Hofer, A. W. 1941. A characterization of Bacterium radiobacter.J. Bacteriol. 41:193-224. 14. Holt, J.G., N.R. Kreig, P.H.A. Sneath, J.T. Staley and S.T. Williams. 1994. Bergey‘s Manual of Determinative Bacteriology. 9th ed., Williams and Wilkins, (Eds). Baltimore, USA, pp. 40-169. 15. Josey D.P., Beynon J.L., Johnston A.W.B., Beringer J.E. (1979). Strain Identification in Rhizobium Using 16. Keyser H.H., Weber D.F., Uratsu S.L. (1984). Rhizobium japonicum Serogroup and Hydrogenase Phenotype Distribution in 12 States. Appl. Environ. Microbiol. 47: 613-615 17. Lindstrom, K., and S. Lehtomaki. 1988. Metabolic properties, maximum growth temperature and phage sensitivity of Rhizobium sp. (Galega) compared with other fast growing rhizobia. FEMS Microbiol. Lett. 50:277-287. 18. Martensson, A.M. (1992): Effects of agrochemical and heavy metals on fastgrowing rhizobia and their symbiosis with small seed legumes. Soil Biology and Biochemistry 24: 435-445 19. Milošević, N., Jarak, M. (2005): Znaĉaj azotofiksacije u snabdevanju biljaka azotom. U AZOT agrohemijski, agrotehniĉki, fiziološki i ekološki aspekti (Ur. R. Kastori), Nauĉni institut za ratarstvo i povrtarstvo, Novi Sad, 305–352 20. Moreira F.M.S., Gillis M., Pot B., Kersters K., Franco A.A. (1993). Characterization of Rhizobia Isolated from Different Divergence Groups of Tropical Leguminosae by Comparative Polyacrylamide Gel Electrophoresis of their Total Proteins. System. Appl. Microbiol. 16:135-146. 21. Mueller J.G., Skipper H.D., Shipe E.R., Grimes L.W., Wagner S.C. (1988). Intrinsic antibiotic resistance in Nodulating Bacteria Isolated from Japanese Soil. Soil Sci. Plant Nutr. 35: 281-288 22. Olsen, W.A., Collins, M.M. (1995): Symbiotic effectiveness of R. meliloti at low root temperature. Plant and Soil, 170, .351-358. 23. Paul, E.A., Clark, F.F. (1989): Soil and Biochem is try. Academic Press, Inc. New YorkRice, 24. Rao, N.S.S. 1999. Soil Microbiology. Oxford and IBH Publishing Co. Pvt. Ltd. New Dehli, Calcutta, pp. 181. 25. Roberts G.P., Leps W.T., Silver L.E., Brill W. J. (1980). Use of Two-dimensional Polyacrylamide Gel Electrophoresis to Identify and Classify Rhizobium Strains. Appl. Environ. Microbiol. 39: 414-422. 26. Sadowsky M.J., Tully R.E., Cregan P.B., Keyser H.H., (1987). Genetic Diversity in Bradyrhizobium japonicum Serogroup 123 and Its Relation to Genotype-specific Nodulation of Soybean. Appl. Environ. Microbiol. 53: 2624-2630. 27. Schmidt E.L., Zidwick M.J., Abebe H.M. (1986) Bradyrhizobium japonicum serocluster 123 and diversity among member isolates. Appl. Environ. Microbiol. 51: 1212-1215 28. Shah, N.H., F.Y. Hafeez, S. Asad, A. Hussain and K.A. Malik ( 1995) Isolation and characterization of indigenous Rhizobium leguminosarum bv. viciae nodulating Lens culinaris Medic from four Pakistani soils. 211-220. In: Biotechnology for Sustainable Development. (Eds.): K.A. Malik, A. Nasim and A.M. Khalid), NIBGE, Faisalabad, Pakistan 29. Society for Ecological Restoration (2002) The SER Primer on Ecological Restoration. http://www.ser.org/. 30. Strains Producing Rhizobitoxine-Induced Foliar Chlorosis on Soybean. Crop Science 28: 938-941 31. Vincent, J.M. 1970. A manual for the practical study of root-nodule bacteria. IBP Handbook 15,Bleckwell, Oxford, pp. 164. 32. Weber D.F., Keyser H.H., Uratsu S.L. (1989). Serological distribution of Bradyrhizobium japonicum from U.S. soybean production areas. Agron. J. 81:786-78 296

11. Hajsig, D., Naglić, T., Madić, J., Gamulin, S. (1992): Opća mikrobiologija i imunologija s osnovama<br />

epizootiologije. Školska knjiga, Zagreb<br />

12. Hartmann A., Amarger N. (1991). Genotypic diversity of an indigenous Rhizobium meliloti field population<br />

assessed by plasmid profiles, DNA fingerprinting, and insertion sequence typing. Can. J. Microbiol. 37: 600-<br />

608.<br />

13. Hofer, A. W. 1941. A characterization of Bacterium radiobacter.J. Bacteriol. 41:193-224.<br />

14. Holt, J.G., N.R. Kreig, P.H.A. Sneath, J.T. Staley and S.T. Williams. 1994. Bergey‘s Manual of Determinative<br />

Bacteriology. 9th ed., Williams and Wilkins, (Eds). Baltimore, USA, pp. 40-169.<br />

15. Josey D.P., Beynon J.L., Johnston A.W.B., Beringer J.E. (1979). Strain Identification in Rhizobium Using<br />

16. Keyser H.H., Weber D.F., Uratsu S.L. (1984). Rhizobium japonicum Serogroup and Hydrogenase Phenotype<br />

Distribution in 12 States. Appl. Environ. Microbiol. 47: 613-615<br />

17. Lindstrom, K., and S. Lehtomaki. 1988. Metabolic properties, maximum growth temperature and phage<br />

sensitivity of Rhizobium sp. (Galega) compared with other fast growing rhizobia. FEMS Microbiol. Lett.<br />

50:277-287.<br />

18. Martensson, A.M. (1992): Effects of agrochemical and heavy metals on fastgrowing rhizobia and their<br />

symbiosis with small seed legumes. Soil Biology and Biochemistry 24: 435-445<br />

19. Milošević, N., Jarak, M. (2005): Znaĉaj azotofiksacije u snabdevanju biljaka azotom. U AZOT agrohemijski,<br />

agrotehniĉki, fiziološki i ekološki aspekti (Ur. R. Kastori), Nauĉni institut za ratarstvo i povrtarstvo, Novi Sad,<br />

305–352<br />

20. Moreira F.M.S., Gillis M., Pot B., Kersters K., Franco A.A. (1993). Characterization of Rhizobia Isolated from<br />

Different Divergence Groups of Tropical Leguminosae by Comparative Polyacrylamide Gel Electrophoresis of<br />

their Total Proteins. System. Appl. Microbiol. 16:135-146.<br />

21. Mueller J.G., Skipper H.D., Shipe E.R., Grimes L.W., Wagner S.C. (1988). Intrinsic antibiotic resistance in<br />

Nodulating Bacteria Isolated from Japanese Soil. Soil Sci. Plant Nutr. 35: 281-288<br />

22. Olsen, W.A., Collins, M.M. (1995): Symbiotic effectiveness of R. meliloti at low root temperature. Plant and<br />

Soil, 170, .351-358.<br />

23. Paul, E.A., Clark, F.F. (1989): Soil and Biochem is try. Academic Press, Inc. New YorkRice,<br />

24. Rao, N.S.S. 1999. Soil Microbiology. Oxford and IBH Publishing Co. Pvt. Ltd. New Dehli, Calcutta, pp. 181.<br />

25. Roberts G.P., Leps W.T., Silver L.E., Brill W. J. (1980). Use of Two-dimensional Polyacrylamide Gel<br />

Electrophoresis to Identify and Classify Rhizobium Strains. Appl. Environ. Microbiol. 39: 414-422.<br />

26. Sadowsky M.J., Tully R.E., Cregan P.B., Keyser H.H., (1987). Genetic Diversity in Bradyrhizobium<br />

japonicum Serogroup 123 and Its Relation to Genotype-specific Nodulation of Soybean. Appl. Environ.<br />

Microbiol. 53: 2624-2630.<br />

27. Schmidt E.L., Zidwick M.J., Abebe H.M. (1986) Bradyrhizobium japonicum serocluster 123 and diversity<br />

among member isolates. Appl. Environ. Microbiol. 51: 1212-1215<br />

28. Shah, N.H., F.Y. Hafeez, S. Asad, A. Hussain and K.A. Malik ( 1995) Isolation and characterization of<br />

indigenous Rhizobium leguminosarum bv. viciae nodulating Lens culinaris Medic from four Pakistani soils.<br />

211-220. In: Biotechnology for Sustainable Development. (Eds.): K.A. Malik, A. Nasim and A.M. Khalid),<br />

NIBGE, Faisalabad, Pakistan<br />

29. Society for Ecological Restoration (2002) The SER Primer on Ecological Restoration. http://www.ser.org/.<br />

30. Strains Producing Rhizobitoxine-Induced Foliar Chlorosis on Soybean. Crop Science 28: 938-941<br />

31. Vincent, J.M. 1970. A manual for the practical study of root-nodule bacteria. IBP Handbook 15,Bleckwell,<br />

Oxford, pp. 164.<br />

32. Weber D.F., Keyser H.H., Uratsu S.L. (1989). Serological distribution of Bradyrhizobium japonicum from<br />

U.S. soybean production areas. Agron. J. 81:786-78<br />

296

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