Plant and Soil

, Volume 237, Issue 1, pp 47–54 | Cite as

Isolation, partial characterization, and the effect of plant growth-promoting bacteria (PGPB) on micro-propagated sugarcane in vitro

  • M. Sajjad Mirza
  • Waseem Ahmad
  • Farooq Latif
  • Jacqueline Haurat
  • Rene Bally
  • Philippe Normand
  • Kauser A. Malik
Article

Abstract

We report the isolation of nitrogen fixing, phytohormone producing bacteria from sugarcane and their beneficial effects on the growth of micropropagated sugarcane plantlets. Detection of the nitrogen fixing bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) method indicated the presence of up to 106 bacteria per gram dry weight of stem and 107 bacteria per gram dry weight of root of field-grown sugarcane. Two nitrogen fixing bacterial isolates were obtained from stem (SC11, SC20) and two from the roots (SR12, SR13) of field-grown plants. These isolates were identified as Enterobacter sp. strains on the basis of their morphological characteristics and biochemical tests. The isolate SC20 was further characterized by 16S rRNA sequence analysis, which showed high sequence similarity to the sequence of Enterobacter cloacae and Klebsiella oxytoca. All the isolates produced the phytohormone indoleacetic acid (IAA) in pure culture and this IAA production was enhanced in growth medium containing tryptophan. The bacterial isolates were used to inoculate micro-propagated sugarcane in vitro where maximum increase in the root and shoot weight over control was observed in the plantlets inoculated with strain SC20. By using the15N isotope dilution technique, maximum nitrogen fixation contribution (28% of total plant nitrogen) was detected in plantlets inoculated with isolate SC20.

Enterobacter nitrogen fixation phytohormones 16S rRNA 

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References

  1. Alexander M 1965 Most probable number method for microbial population. In Methods of Soil Analysis. Part 2. Eds. C A Black, D D Evans, L E Ensuinger, J K White and F F Clarke. pp 1467–1472. Am. Soc. Agronomy, Madison, W.I.Google Scholar
  2. Arshad M and Frankenberger Jr. W T 1998 Plant growth-regulating substances in the rhizosphere: Microbial production and functions. Adv. Agron. 62, 45–151.Google Scholar
  3. Bashan Y and Holguin G 1997 Azospirillum-plant relationships: environmental and physiological advances (1990-1996). Can. J. Microbiol. 43, 103–121.Google Scholar
  4. Bashan Y and Holguin G 1998 Proposal for the division of plant growth-promoting rhizobacteria into two classifications: Biocontrol-PGPB (plant growth-promoting bacteria) and PGPB. Soil Biol. Biochem. 30, 1225–1228.Google Scholar
  5. Barraquio W L, Segubre E M, Gonzalez M S, Verma S C, James E K, Ladha J K and Tripathi A K 2000 Diazotrophic enterobacteria: What is their role in the rhizosphere of rice? In The Quest for Nitrogen Fixation in Rice. Eds.J K Ladha and P M Reddy. pp 93–118. IRRI, Philippines.Google Scholar
  6. Bilal R and Malik K A 1987 Isolation and identification of a N2-fixing zoogloea-forming bacterium from kallar grass histoplane. J. Appl.Bacteriol. 62, 289–294.Google Scholar
  7. Boddey R M, Oliveira O C de, Urquiaga S, Reis V M, Olivares F L, Baldani V L D and Dobereiner J 1995a Biological nitrogen fixation associated with sugarcane and rice: Contributions and prospects for improvement. Plant Soil 174, 195–209.Google Scholar
  8. Boddey R M, Reis V M, Urquiaga S, daSilva L G, dosReis F B, Baldani J I and Dobereiner J 1995b N2 fixation in sugar cane: the role of Acetobacter diazotrophicus. In Nitrogen Fixation: Fundamental and Applications. Eds. I A Tikhonovich, N A Rovorov and W E Newton. pp 641–646. Kluwer Academic Publishers, Dordrecht, The Netherlands.Google Scholar
  9. Boddey R M, Urquiaga S, Reis V M and Dobereiner J 1991 Biological nitrogen fixation associated with sugarcane. Plant Soil 137, 111–117.Google Scholar
  10. Caballero-Mellado J and Martinez-Romero E 1994 Limited genetic diversity in the endophytic sugarcane bacterium Acetobacter diazotrophicus. Appl. Envir. Microbiol. 60, 1532–1537.Google Scholar
  11. Cavalcante V A and Dobereiner J 1988 A new acid-tolerant nitrogen fixing bacterium associated with sugarcane. Plant Soil 108, 23–31.Google Scholar
  12. Cochran W G 1950 Estimation of bacterial densities by means of “Most Probable Number”. Biometrics 6, 105–115.Google Scholar
  13. Dobereiner J 1959 Influence da cane-de acucarna populaco de Beijerinckia do solo. Rev. Bras.Biol. 19,251.Google Scholar
  14. Dobereiner J 1961 Nitrogen fixing bacteria of the genus Beijerinckia Drex. in the rhizosphere of sugarcane. Plant Soil 15, 211–216.Google Scholar
  15. Dobereiner J, Day J M and Dart P J 1972 Nitrogenase activity in the rhizophere of sugarcane and some other tropical grasses. Plant Soil 37, 191–196.Google Scholar
  16. Dobereiner J, Baldani V L D and Reis V M 1995a Endophytic occurrence of diazotrophic bacteria in non-leguminous crops. In Azospirillum VI and Related Micro-organisms. Eds. I Fendrik, M del Gallo, J Vanderleyden and M de Zamaroczy. pp 3–14. Springer-Verlag, Berlin.Google Scholar
  17. Dobereiner J, Urquiaga S and Boddey R M 1995b Alternatives for nitrogen nutrition of crops in tropical agriculture. Fertil. Res. 42, 339–346.Google Scholar
  18. Dong Z, Heydrich M, Bernard K and McCully M E 1995 Further evidence that the N2-fixing endophytic bacterium from the intercellular spaces of sugarcane stem is Acetobacter diazotrophicus. Appl. Environ. Microbiol. 61, 1843–1846.Google Scholar
  19. Fried M and Middleboe V 1977. Measurement of amount of nitrogen fixed by a legume crop. Plant Soil 41, 713–715.Google Scholar
  20. Fuentes-Ramirez L E, Jimenez-Salgado T, Abarca-Ocampo I R and Caballero-Mellado J 1993 Acetobacter diazotrophicus, an indoleacetic acid producing bacterium isolated from sugarcane cultivars of Mexico. Plant Soil 154, 145–150.Google Scholar
  21. Gillis M, Dobereiner J, Pot B, Goor M, Falsen E, Hoste B, Reinhold B and Kersters K 1991 Taxonomic relationship between (Pseudomonas) rubrisubalbicans, some clinical isolates (EF group 1) Herbaspirillum seropedicae, and (Aquaspirillum) autotrophicum. In Nitrogen Fixation. Eds. M Pollineli, R Materassi and M Vicenzini. pp 292–294. Kluwer Academic Press, Dordrecht, The Netherlands.Google Scholar
  22. Gillis M, Kersters K, Hoste B, Janssens D, Kroppenstedt R M, Stephan M P, Teixeira K R S, Dobereiner J and de Ley J 1989 Acetobacter diazotrophicus sp. nov., a nitrogen fixing acetic acid bacterium associated with sugarcane. Int. J. Syst. Bacterial. 39, 361–364.Google Scholar
  23. Gonzalez-Lopez J, Salmeron V, Martinez-Toledo M V, Sallesteros F and Ramos-Cormenzana A 1986 Production of auxin, gibberellins and cytokinins by Azotobacter vineladii ATCC 12837 in chemically defined media and dialysed soil media. Soil Biol. Biochem. 18, 119–120.Google Scholar
  24. Grimont F and Grimont P A D 1992 The genus Enterobacter. In The Prokaryotes. Vol: III. Eds. A Balows, H G Truper, M Dworkin, W Harder and K H Schleifer. pp 2797–2815. Springer-Verlag, New York.Google Scholar
  25. Grimont F, Grimont P A D and Richard C 1992 The genus Klebsiella. In The Prokaryotes. Vol: III.. Eds. A Balows, H G Truper, M Dworkin, W Harder and K H Schleifer. pp 2775–2795. Springer-Verlag, New York.Google Scholar
  26. Haahtela K, Konkoo R, Laakso T, Williams P H and Korhonem T K 1990 Root associated Enterobacter and Klebsiella in Poa pratensis: Characterization of an iron scavenging system and a substance stimulating root hair production. Mol. Plant-Microbe Interact. 3, 358–365.Google Scholar
  27. Harari A, Kigel J and Okon Y 1988 Involvement of IAA in the interaction between Azospirillum brasilense and Panicum miliaceum root. Plant Soil 110, 275–282.Google Scholar
  28. Hegazi N A, Eid N, Farq R S and Monib M 1979 Asymbiotic nitrogen fixation in the rhizosphere of sugarcane planted under semi-arid conditions of Egypt. Rev. Ecol. Biol. Soil 16, 23–27.Google Scholar
  29. Hoagland D R and Arnon D T 1950 California Agriculture Experimental station Circular 347. University of California, Berkeley, CA, USA.Google Scholar
  30. Holt J G, Kreig N R, Sneath P H A, Staley J T and Williams S T (eds) 1994 Bergey's Manual of Determinative Bacteriology. Williams and Wilkins, Baltimore, USA. pp 178–229.Google Scholar
  31. Hugh R and Leifson E 1953 The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various Gram-negative bacteria. J. Bacteriol. 66, 24–26.Google Scholar
  32. James E K and Olivares F L 1997 Infection and colonization of sugarcane and other graminaceous plants by endophytic bacteria. Cri. Rev. Plant Sci. 17, 77–119.Google Scholar
  33. James E K, Gyaneshwar P, Barraqui W L, Mathan N and Ladha J K 2000 Endophytic diazotrophs associated with rice. In The Quest for Nitrogen Fixation in Rice. Eds. J K Ladha and P M Reddy. pp 119–140. IRRI, Philippines.Google Scholar
  34. Lima E, Boddey R M and Dobereiner J 1987 Quantification of biological nitrogen fixation associated with sugarcane using 15N aided nitrogen balance. Soil Boil. Biochem. 19, 165–170.Google Scholar
  35. MacFaddin J F 1980 Biochemical Tests for Identification ofMedical Bacteria. pp 51–54. Williams and Wilkins, Baltimore.Google Scholar
  36. Malik K A, Bilal R, Mehnaz S, Rasul G, Mirza M S and Ali S 1997 Association of nitrogen fixing and plant growth promoting rhizobacteria (PGPR) with kallar grass and rice. Plant Soil 194, 37–44.Google Scholar
  37. Murashige T and Skoog F A 1962 Revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 473–497.Google Scholar
  38. Normand P 1995 Utilisation des séquences 16S pour le positionnement phylétique d'un organisme inconnu. Oceanis 21, 31–56.Google Scholar
  39. Okon Y and Labandera-Gonzalez C A 1994 Agronomic applications of Azospirillum. In Improving Plant Productivity with Rhizosphere Bacteria. Eds. M H Ryder, P M Stephens and G D Bowen. pp 274–278. Commonwealth Scientific and Industrial Research Organization, Adelaide, Australia.Google Scholar
  40. Patten C L and Glick B R 1996 Bacterial biosynthesis of indole-3-acetic acid. Can. J. Microbiol. 42, 207–220.Google Scholar
  41. Rasul G, Mirza M S, Latif F and Malik K A 1998 Identification of plant growth hormones produced by bacterial isolates from rice, wheat and kallar grass. In Nitrogen Fixation with Non-legumes. Eds. K A Malik, M S Mirza and J K Ladha. pp 25–37. Kluwer Academic Publishers, Dordrecht, The Netherlands.Google Scholar
  42. Reinhold-Hurek B, Hurek T, Gillis M, Hoste B, Vancannyt M, Kersters K and De Ley J 1993 Azoarcus gen. Nov., nitrogen-fixing proteobacteria associated with roots of kallar grass (Leptochloa fusca L. Kunth) and description of two species, Azoarcus indigens sp. nov., and Azoarcus communis sp. nov. Int. J. Syst. Bacteriol. 43, 574–584.Google Scholar
  43. Rennie R J 1981 A single medium for the isolation of acetylenereducing (dinitrogen-fixing) bacteria from soils. Can. J. Microbiol. 27, 8–14.Google Scholar
  44. Rennie R J, DeFreitas J R, Ruschel A P and Vose P B 1983 Isolation and identification of N2-fixing bacteria associated with sugarcane (Saccharum). Can. J. Microbiol. 28, 462–467.Google Scholar
  45. Ruschel A P 1981 Associative N2-fixation by sugarcane. In Associative N2-Fixation. Eds. P B Vose and A P Ruschel. pp 82-90. CRC Press Inc. Florida.Google Scholar
  46. Sevilla M, de Oliveira A, Baldani I and Kennedy C 1998 Contribution of the bacterial endophyte Acetobacter diazotrophicus to sugarcane nutrition: A preliminary study. Symbiosis 25, 181–191.Google Scholar
  47. Strzelczyk E and Pokojska-Burdziej A 1984 Production of auxins and gibberellin-like substance by mycorrhizal fungi, bacteria and actinomycetes isolated from soil and the mycorrhizosphere of pine (Pinus silvestris L.). Plant Soil 81, 185–194.Google Scholar
  48. Tien T M, Gaskins M H and Hubbel D H 1979 Plant growth substances produced by Azospirillum brasilense and their effect on growth of pearl millet (Pennisetum americanum L.). Appl. Environ. Microbiol. 37, 1016–1024.Google Scholar
  49. Urquiaga S, Cruz K H S and Boddey R M 1992 Contribution of nitrogen fixation to sugarcane: Nitrogen-15 and nitrogen balance estimates. Soil Sci. Soc. Am. J. 56, 105–114.Google Scholar
  50. Zafar Y, Wahid A, Rasul E and Malik K A 1986 Root associated nitrogen fixation by sugarcane (Saccharum officinarum L. var. Col-54) in Pakistan. Pak. J. Bot. 18, 221–228.Google Scholar
  51. Zelena E, Kutacek M and Cermak V 1988 Fate of root applied indolylacetic acid and its influence on the growth of plants. In Physiology and Biochemistry of Auxins in Plants. Eds. M Kutacek, R S Bandurski and J Krekule. pp 371–376. SPB Academic Publishing, The Hague. Section editor: T.C. Paulitz Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • M. Sajjad Mirza
    • 1
  • Waseem Ahmad
    • 1
  • Farooq Latif
    • 1
  • Jacqueline Haurat
    • 2
  • Rene Bally
    • 2
  • Philippe Normand
    • 2
  • Kauser A. Malik
    • 2
  1. 1.Biofertilizer Division, National Institute for Biotechnology and Genetic Engineering (NIBGE)FaisalabadPakistan
  2. 2.Laboratoire d'Ecologie Microbienne du Sol, UMR CNRS 5557, UCB Lyon1Villeurbanne CedexFrance

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