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Isolation of endophytic diazotrophic bacteria from wetland rice

Chapter
Part of the Developments in Plant and Soil Sciences book series (DPSS, volume 75)

Abstract

Endophytic nitrogen-fixing bacteria are believed to contribute substantial amounts of N to certain gramineous crops. We have been interested to find (a) a diazotroph(s) in rice which can aggressively and stably persist and fix nitrogen in interior tissues and (b) unique rice-diazotrophic endophyte combinations. To achieve these objectives, it has been essential to find an efficient method to surface sterilize rice tissues. The method described here consists of exposing tissues to 1% Chloramine T for 15 min followed by shaking with glass beads. It has proven very efficient since (a) surface bacterial populations on the root and culm were found to be reduced by more than 90%, (b) the number of the internal colonizers was found to be significantly higher than the number of surface bacteria, and (c) colonization of root but not subepidermal tissue by gusA-marked Herbaspirillum seropedicae Z67 bacteria was found to be virtually eliminated. Nitrogen-fixing putative endophytic populations (MPN g dry wt−1) in the root (7.94 × 107) and culm (2.57 × 106) on field-grown IR72 plants grown in the absence of N fertilizer was found to be significantly higher near heading stage. The corresponding total putative endophyte populations in the tissues of 25 highly diverse genotypes of rice and their relatives was found to range from 105−108and 104−109, in the roots and culms, respectively. Generally, the resident bacteria were found to be non-diazotrophic, although in isolated cases diazotrophs were found, for example in the roots and culm of IR72 rice plants, or the culm of Zizaniopsis villanensis plants. The size of populations of diazotrophic bacteria in different rice genotypes was found to be 103−107 for the roots and 104−106 for the culms, respectively. The rice genera-related plants Potamophila pariffora and Rhynchoryza subulata showed the highest levels.

Key words

endophytic diazotrophs rice endophytes surface sterilization 

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References

  1. Bell C R, Dickie G A, Harvey W L G and Chan J W Y F 1995 Endophytic bacteria in grapevine. Can. J. Microbiol. 41, 46–53.Google Scholar
  2. Baldani V L D, Baldani J I, Olivares F and Dobereiner J 1992 Identification and ecology of Herbaspirillum seropedicae and the closely related Pseudomonas rubrisubalbicans. Symbiosis 13, 65–73.Google Scholar
  3. Baldani J I, Baldani V L D, Seldin L and Dobereiner J 1986 Characterization of Herbaspirillum seropedicae gen. nov., sp. nov., a root-associated nitrogen-fixing bacterium. Int. J. Syst. Bacteriol. 36, 86–93.Google Scholar
  4. Boddey R M, de Oliveira O C, Urquiga S, Reis V M, de Olivares F L, Baldani V L D and Dobereiner J 1995 Biological nitrogen fixation associated with sugar cane and rice: contributions and prospects for improvement. Plant Soil 174, 195–209.CrossRefGoogle Scholar
  5. Cameron, H. R. 1970. Pseudomonas content of cherry trees. Phytopathology 60, 1343–1346.Google Scholar
  6. Cavalcante V A and Dobereiner J 1988 A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane. Plant Soil 108, 23–31.CrossRefGoogle Scholar
  7. Diem G, Rougier M, Hamad-Fares I, Balandreau J P and Dommergues Y R. 1978 Colonization of rice roots by diazotroph bacteria. In Environmental Role of Nitrogen-fixing Blue-green Algae and Asymbiotic Bacteria. Ed. U Granhall. Ecol. Bull. ( Stockholm ) 26, 305–311.Google Scholar
  8. Dobereiner J 1980 Forage grasses and grain crops. In Methods for Evaluating Biological Nitrogen Fixation. Ed. F J Bergerson. pp 535–555. John Wiley and Sons, New York.Google Scholar
  9. Fisher P J, Petrini O and Lappinscott H M 1992 The distribution of some fungal and bacterial endophytes in maize (Zea mays L.). New Phytol. 122, 299–305.CrossRefGoogle Scholar
  10. Gagne S, Richard C, Rousseau H and Antoun H 1987 Xylem-residing bacteria in alfalfa roots. Can. J. Microbiol. 33, 996–1000.Google Scholar
  11. Gardner J M, Feldman A W and Zablotowicz R M 1982 Identity and behavior of xylem-residing bacteria in rough lemon roots of Florida citrus trees. Appl. Environ. Microbiol. 43, 1335–1342.Google Scholar
  12. Hurek T, Reinhold-Hurek B, van Montagu M and Kellenberger E 1991 Infection of intact roots of Kallar grass and rice seedlings by Azoarcus. In Developments in Plant and Soil Sciences, Vol. 48. Nitrogen fixation. Eds. M Posinelli, R Materassi and M Vincenzini. pp 235–242. Kluwer Acad. Publ., Dordrecht.Google Scholar
  13. Hurek T, Reinhold-Hurek B, van Montagu M and Kellenberger E 1994 Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses. J. Bacteriol. 176, 1913–1923.PubMedGoogle Scholar
  14. Ito O, Cabrera D A and Watanabe I 1980 Fixation of dinitrogen-15 associated with rice plants. Appl. Environ. Microbiol. 39, 554–558.Google Scholar
  15. Jacobs M J, Bugbee W M and Gabrielson D A 1985 Enumeration, location, and characterization of endophytic bacteria within sugar beet roots. Can. J. Bot. 63, 1262–1265.Google Scholar
  16. James E K, Reis V M, Olivares F L, Baldani J I and Dobereiner J 1994 Infection of sugarcane by the nitrogen-fixing bacterium Acetobacter diazotrophicus. J. Exp. Bot. 45, 757–766.Google Scholar
  17. Ladha J K, Tirol-Padre A, Daroy M G, Punzalan G C, Ventura W and Watanabe I 1986 Plant-associative N2 fixation (C2H2 reduction) by five rice varieties, and relationship with plant growth characters as affected by straw incorporation. Soil Sci. Plant Nutr. 32, 91–106.Google Scholar
  18. Ladha J K, Tirol-Padre A, Punzalan and Watanabe I 1987 Nitrogen-fixing (C2H2-reducing) activity and plant growth characters of 16 wetland rice varieties. Soil Sci. Plant Nutr. 33, 187–200.Google Scholar
  19. Ladha J K and Reddy P M 1995 Extension of nitrogen fixation to rice–necessity and possibilities. Geojournal 35, 363–372.CrossRefGoogle Scholar
  20. Li R P and MacRae I C 1992 Specific identification and enumeration of Acetobacter diazotrophicus in sugarcane. Soil Biol. Biochem. 24, 413–419.Google Scholar
  21. Magalhaes F M M, Patriquin D and Dobereiner J 1979 Infection of field grown maize with Azosprillum spp. Rev. Brasil. Biol. 39, 587–596.Google Scholar
  22. McClung C R, van Berkum P, Davis R E and Sloger C 1983 Enumeration and localization of N2-fixing bacteria associated with roots of Spartina alterniflora Loisel. Appl. Environ. Microbiol. 45, 1914–1920.Google Scholar
  23. Mclnroy J A and Kloepper J W 1995a Population dynamics of endophytic bacteria in field-grown sweet corn and cotton. Can. J. Microbiol. 41, 895–901.CrossRefGoogle Scholar
  24. Mclnroy J A and Kloepper J W 1995b Survey of indigenous bacterial endophytes from cotton and sweet corn. Plant Soil 173, 337–342.CrossRefGoogle Scholar
  25. Olivares F L, Baldani V L D, Reis V M, Baldani J I and Dobereiner J 1996 Occurrence of the endophytic diazotrophs Herbaspirillum spp. in roots, stems, and leaves, predominantly of Gramineae. Biol. Feil. Soils 21, 197–200.Google Scholar
  26. Patriquin D G and Dobereiner J 1978 Light microscopy observation of tetrazolium reducing bacteria in endorhizosphere of maize and other grasses in Brazil. Can. J. Microbiol. 24, 734–742.PubMedCrossRefGoogle Scholar
  27. Philipson M N and Blair I D 1957 Bacteria in clover root tissue. Can. J. Microbiol. 3, 125–129.CrossRefGoogle Scholar
  28. Reddy P M, Ladha J K, So R, Hemandez R, Dazzo F B, Angeles O R, Ramos M C and de Bruijn F J 1997 Rhizobial communication with rice roots: induction of phenotypic changes, mode of invasion and extent of colonization. Plant Soil 194, 81–98.CrossRefGoogle Scholar
  29. Reinhold H B, Hurek T, Niemann E G and Fendrik I 1986 Close association of Azospirillum and diazotrophic rods with different root zones of Kallar grass. Appl. Environ. Microbiol. 52, 520–526.Google Scholar
  30. Reis V M, Olivares F L and Dobereiner J 1994 Improved methodology for isolation of Acetobacter diazotrophicus and confirmation of its endophytic habitat. World J. Microbiol. Biotechnol. 10, 401–405.Google Scholar
  31. Shishido M, Loeb B M and Chanway C P 1995 External and internal root colonization of lodgepole pine seedlings by two growth-promoting Bacillus strains originated from different root microsites. Can. J. Microbiol. 41, 707–713.Google Scholar
  32. Stoltzfus J R, So R, Malarvizhi P P, Ladha J K and de Bruijn F J 1997 Isolation of endophytic bacteria from rice and assessment of their potential for supplying rice with biologically fixed nitrogen. Plant Soil 194, 25–36.CrossRefGoogle Scholar
  33. Ueda T, Suga Y, Yahiro N and Matsuguchi T 1995a Genetic diversity of N2-fixing bacteria associated with rice roots by molecular evolutionary analysis of a nu i) library. Can. J. Microbiol. 41, 235–240.Google Scholar
  34. Ueda T, Suga Y, Yahiro N and Matsuguchi T 1995b Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences. J. Bacteriol. 177, 1414–1417.PubMedGoogle Scholar
  35. Umali-Garcia M, Hubell D H, Gaskins M H and Dazzo F B 1980 Association of Azospirillum with grass roots. Appl. Environ. Microbiol. 39, 219–226.Google Scholar
  36. Vaughan D A 1994 The Wild Relatives of Rice. IRRI, Manila.Google Scholar
  37. Watanabe I, Barraquio W L, de Guzman M R and Cabrera D A 1979 Nitrogen-fixing (acetylene reduction activity) and population of aerobic heterotrophic nitrogen-fixing bacteria associated with wetland rice. Appl. Environ. Microbiol. 39, 813–819.Google Scholar
  38. Watanabe I, Cabrera D A and Barraquio W L 1981 Contribution of basal portion of shoot to N2 fixation associated with wetland rice. Plant Soil 59, 391–398.CrossRefGoogle Scholar
  39. Wilson K J, Sessitsch A, Corbo J C, Giller K E, Akkermans A D L and Jefferson R A 1995 Q-glucuronidase activity in studies of rhizobia and other Gram-negative bacteria. Microbiology 141, 1691–1705.PubMedCrossRefGoogle Scholar
  40. Wilson K J 1996 GusA as a reporter gene to track microbes. ln Molecular Microbial Ecology Manual. Eds. A D L Akkermans, F J de Bruijn and J D van Elsas. Kluwer Acad. Publishers, Dordrecht.Google Scholar
  41. Woomer P L 1994 Most probable number counts. In Methods of Soil Analysis, part 2. Eds. R W Weaver, S Angle, P Bottomley, D Bezdicek, S Smith, A Tabatabai and A Wollum. pp 59–79. Soil Sci. Soc. Am., Inc., Wisconsin.Google Scholar
  42. You C and Zhou F 1989 Non-nodular endorhizosphere nitrogen fixation in wetland rice. Can. J. Microbiol. 35, 403–408.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 1997

Authors and Affiliations

  1. 1.Soil Microbiology Laboratory, SWSDInternational Rice Research InstituteLos Banos, LagunaPhilippines

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