, Volume 56, Issue 2, pp 77–86 | Cite as

ACC deaminase containing diazotrophic endophytic bacteria ameliorate salt stress in Catharanthus roseus through reduced ethylene levels and induction of antioxidative defense systems

  • Bala Karthikeyan
  • Manoharan Melvin JoeEmail author
  • Md. Rashedul Islam
  • Tongmin SaEmail author


Among a total of 27 cultivable salt tolerant endophytic bacteria isolated from Catharanthus roseus grown in highly salt affected coastal region of cuddalore district, Tamilnadu, India four isolates were found to be positive for nitrogenase activity. The isolates were evaluated for their stress tolerance efficiency and screened for different PGP traits. Based on the above studied parameters, and ability to produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase (4.24 μmol α-ketobutyrate mg_1 protein h_1) the salt tolerant diazotrophic isolate AUM54 was selected for further investigation and identified as Achromobacter xylosoxidans by 16S rRNA gene sequencing. The ability of this isolate to ameliorate salt stress in C. roseus was evaluated under gnotobiotic and pot culture conditions. At 150 mM NaCl level A. xylosoxidans AUM54 treated plants recorded ethylene level of 394.1 p mol ethylene g−1 FW h−1 compared to the ethylene level of 516.0 p mol ethylene g−1 FW h−1 recorded in the un inoculated control. A. xylosoxidans AUM54 inoculated plants recorded the maximum germination percentage of 98.3, vigor index of 2231.4, plant height of 120.4 cm, root dry weight of 53.24 g Plant_1 and ajmalicine content of 1.60 mg g−1, compared to the germination percentage of 91.6%, vigour index of 1511.5, plant height of 105.8, root weight of 47.2 g Plant−1, and ajmalicine content of 1.23 mg g−1 in uninoculated plants grown without NaCl treatment. This isolate also decreased plant ethylene levels by 11–23% and increased the antioxidative enzyme content of inoculated C. roseus plants to the tune of 19–32% for ascorbate peroxidase (APX) activity, 20–30% for superoxide dismutase (SOD) activity and 4–16% for catalase (CAT) under normal and salt affected conditions.


Achromobacter xylosoxidans NaCl stress ACC deaminase Ethylene Ascorbate peroxidase Catalase Superoxide dismutase 



This study was carried out with the support of “Mid-career Researcher Program through NRF grant funded by the MEST (No. 2010-0000418)”. Authors thank the unknown reviewers for the constructive comments in improving the manuscript. M. R. Islam is supported by the research grant of Inha University, Republic of Korea and B. Karthikeyan thanks Annamalai University.


  1. Abdul-Baki AA, Anderson JD (1973) Vigour determination in soybean and multiple criteria. Crop Sci 13:630–633CrossRefGoogle Scholar
  2. Asada K, Takahashi M (1987) Production and scavenging of active oxygen in chloroplasts. In: Kyle DJ, Osmond CB, Arntzen CJ (eds) Photoinhibition. Elsevier, Amsterdam, pp 227–287Google Scholar
  3. Asada K (1992) Ascorbate peroxidase a hydrogen scavenging enzyme in plants. Physiol Plant 85:235–241CrossRefGoogle Scholar
  4. Baldani VLD, Baldani JI, Döbereiner J (1986) Effect of inoculation of Azospirillum spp on the nitrogen assimilation of field grown wheat. Plant Soil 95:109–121CrossRefGoogle Scholar
  5. Beauchamp C, Fridovich I (1971) Superoxide dismutase: improve assays and an essay applicable to acrylamide gels. Ann Biochem 44:276–287CrossRefGoogle Scholar
  6. Blaha CAG, Schrank IS (2003) An Azospirillum brasilense tn5 mutant with modified stress response and impaired in flocculation. Antonie Van Leeuwenhoek 83(1):35–43Google Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Ann Biochem 72:248–253CrossRefGoogle Scholar
  8. Cassan F, Maiale S, Masciarelli O, Vidal A, Luna V, Ruiz O (2009) Cadaverine production by Azospirillum brasiliense and its possible role in plant growth promotion and osmotic stress mitigation. Eur J Soil Biol 45:12–19CrossRefGoogle Scholar
  9. Chandlee JM, Scandalios JG (1984) Analysis of variants affecting the catalase development program in maize scutellum. Theor Appl Genet 69:71–77CrossRefGoogle Scholar
  10. Cheng Z, Park E, Glick BR (2007) 1-Aminocyclopropane-1-carboxylate (ACC) deaminase from Pseudomonas putida UW4 facilitates the growth of canola in the presence of salt. Can J Microbiol 53:912–918PubMedCrossRefGoogle Scholar
  11. Clark WA (1976) A simplified Leifson flagella stain. J Clinical Microbiol 3:632–634Google Scholar
  12. Cohen A, Bottini A, Piccolo P (2008) Azospirillum brasilense sp. 245 produces ABA in chemically defined culture medium and increases ABA content in Arabidopsis plants. Plant Growth Regul 54:97–103CrossRefGoogle Scholar
  13. Doebereiner J (1995) Isolation and identification of aerobic nitrogen fixing bacteria. In: Alef K, Nannipieri P (eds) Methods in applied soil microbiology and biochemistry. Academic, London, pp 134–141Google Scholar
  14. Elkahoui S, Hernández AJ, Abdelly C, Ghrir R, Limam FB (2005) Effects of salt on lipid peroxidation and antioxidant enzyme activities of Catharanthus roseus suspension cells. Plant Sci 168(3):607–613CrossRefGoogle Scholar
  15. Forchetti G, Masciarelli O, Alemano S, Alvarez D, Abdala G (2007) Endophytic bacteria in sunflower (Helianthus annuus L.): isolation, characterization, and production of jasmonates and abscisic acid in culture medium. Appl Microbiol Biotechnol 76:1145–1152PubMedCrossRefGoogle Scholar
  16. Forchetti G, Masciarelli O, Izaguirre JM, Alemano S, Alvarez D, Abdala G (2010) Endophytic bacteria improve seedling growth of sunflower under water stress, produce salicylic acid, and inhibit growth of pathogenic fungi. Curr Microbiol 61:485–493PubMedCrossRefGoogle Scholar
  17. Gamalero E, Berta G, Massa N, Glick BR, Lingua G (2010) Interactions between Pseudomonas putida UW4 and Gigaspora rosea BEG9 and their consequences on the growth of cucumber under salt stress conditions. J Appl Microbiol 108:236–245PubMedCrossRefGoogle Scholar
  18. Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930Google Scholar
  19. Glick BR, Penrose DM, Li JP (1998) A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria. J Theor Biol 190(1):63–68PubMedCrossRefGoogle Scholar
  20. Glick BR, Patten CL, Holguin G, Penrose DM (1999) Biochemical and genetic mechanisms used by plant growth promoting bacteria. Imperial College Press, LondonCrossRefGoogle Scholar
  21. Goldstein AH (1986) Bacterial solubilization of mineral phosphates: historical perspectives and feature prospects. Am J Alt Agric 1:57–65Google Scholar
  22. Gopal H (2004). Development of microbial consortium for improvement for growth, yield and alkaloid content of Ashwagandha. Ph.D. Thesis, Tamilnadu Agricultural University, CoimbatoreGoogle Scholar
  23. Gorden SA, Paleg LG (1957) Quantitative measurement of Indole acetic acid. Physiol Plant 10:347–348Google Scholar
  24. Gyaneshwar P, James EK, Mathan N, Teddy PM, Reinhold-Hurek B, Ladha JK (2001) Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens. J Bacteriol 183:2634–2645PubMedCrossRefGoogle Scholar
  25. Hu YC, Schmidhalter UM (2005) Drought and salinity: a comparison of their effects on the mineral nutrition of plants. J Plant Nut Soil Sci 168:541–549CrossRefGoogle Scholar
  26. Isenberg HD (1995) Essential Procedures for Clinical Microbiology. Am Soc Microbiol. Washington, D.C. p. 87Google Scholar
  27. Ivanova EG, Doronina NV, Trotsenko YA (2000) Aerobic Methylobacteria are capable of synthesizing auxins. Microbiol 70:392–397CrossRefGoogle Scholar
  28. Jaleel CA, Manivannan P, Kishorekumar A, Sankar B, Gopi R, Somasundaram R, Panneerselvam R (2007) Alterations in osmoregulation, antioxidant enzymes and indole alkaloid levels in Catharanthus roseus exposed to water deficit conditions. Colloid Surf B: Bioin 59:150–157CrossRefGoogle Scholar
  29. Jha PN, Kumar A (2007) Endophytic colonization of Typha australis by a plant growth-promoting bacterium Klebsiella oxytoca strain GR-3. J Appl Microbiol 103:1311–1320PubMedCrossRefGoogle Scholar
  30. Jha P, Kumar A (2009) Characterization of novel plant growth promoting endophytic bacterium achromobacter xylosoxidans from wheat plant. Microb Ecol 58:179–188Google Scholar
  31. Kadouri D, Jurkevitch E, Okon Y (2003) Involvement of the reserve material poly-β-hydroxybutyrate in Azospirillum brasilense stress endurance and root colonization. Appl Environ Microbiol 44:3244–3250CrossRefGoogle Scholar
  32. Karthikeyan B, Jaleel CA, Gopi R, Deiveekasundaram M (2007) Alterations in seedling vigour and antioxidant enzyme activities in Catharanthus roseus under seed priming with native diazotrophs. J Zhejiang Univ Sci B 8(7):453–457PubMedCrossRefGoogle Scholar
  33. Karthikeyan B, Jaleel CA, Gopi R, Lakshmanan GMA, Deiveekasundaram M (2008) Studies on the rhizosphere microbial diversity of some commercially important medicinal plants. Colloid Surf B: Bioint 62:143–145CrossRefGoogle Scholar
  34. Kloepper J, Schroth M (1978) Plant growth-promoting rhizobacteria in radish. In: Proceedings of the 4th International Conference on Plant Pathogenic Bacteria, vol II. INRA, Angers, pp 879–882Google Scholar
  35. Lee YP, Kim SH, Lee HS, Kwak SS, Kwon SY (2007) Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts. Plant Cell Rep 26:591–598Google Scholar
  36. Ma Y, Rajkumar M, Fritas H (2008) Inoculation of plant growth promoting bacterium Achromobacter xylosoxidans strain Ax10 for the improvement of copper phytoextraction by Brassica juncea. J Environ Manage 90(2):831–837PubMedCrossRefGoogle Scholar
  37. Maurhofer M, Reimmann C, Sacherer SP, Heeb S, Haas D, Defago G (1998) Salicylic acid biosynthetic genes expressed in Pseudomonas fluorescens strain P3 improve the induction of systemic resistance in tobacco against tobacco necrosis virus. Phytopathol 88:678–684CrossRefGoogle Scholar
  38. Mayak S, Tirosh T, Glick BR (2004) Plant growth- promoting bacteria confer resistance in tomato plants to salt stress. Plant Physiol Biochem 42:565–572PubMedCrossRefGoogle Scholar
  39. Mc Dade JJ, Weaver RH (1959) Rapid methods for the detection of gelatin hydrolysis. J Bacteriol 77(1):60–64Google Scholar
  40. Nadeem SM, Zahair ZA, Naveed M, Arshad M (2007) Preliminary investigations on inducing salt tolerance in maize through inoculation with rhizobacteria containing ACC deaminase activity. Can J Microbiol 53:1141–1149PubMedCrossRefGoogle Scholar
  41. Pandey P, Kang SC, Maheswari DK (2005) Isolation of endophytic plant growth promoting Burkholderia sp from root nodules of Mimosa pudica. Curr Sci 89(1):177–180Google Scholar
  42. Pellegrin V, Juretschko S, Wagner M, Cottenceau G (1999) Morphological and biochemical properties of a Sphaerotilus sp. Isolated from paper mill slimes. Appl Environ Microbiol 65(1):156–162PubMedGoogle Scholar
  43. Penrose DM, Glick BR (2003) Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiologica Plantarum 118:10–15CrossRefGoogle Scholar
  44. Prochazkova D, Sairam RK, Srivastava GC, Singh DV (2001) Oxidative stress and antioxidant activity as the basis of senescence in maize leaves. Plant Sci 161:765–771CrossRefGoogle Scholar
  45. Raja P, Sanville BC, Buchmann RC, Bisaro DM (2008) Viral genome methylation as an epigenetic defense against Gemini viruses. J Virol 82:8997–9007PubMedCrossRefGoogle Scholar
  46. Rajamani K (2004) Production technology for major medicinal plant. Indian Medicinal and Aromatic Plants Today, pp. 18–26Google Scholar
  47. Reeves M, Neilands PL, Ballows A (1983) Absence of siderophore activity in Leginella sp. grown in iron deficient media. J Bacteriol 154:324–329PubMedGoogle Scholar
  48. Reinhold-Hurek B, Hurek T (1998) Life in grasses: diazotrophic endophytes. Trends Microbiol 6:139–144PubMedCrossRefGoogle Scholar
  49. Roth M, Jensen PK (1967) Determination of catalase by means of the Clark oxygen electrode. Biochim Biophys Acta 139:171–173Google Scholar
  50. Saravanakumar D, Samiyappan R (2007) ACC deaminase from Pseudomonas fluorescens mediated saline resistance in groundnut (Arachis hypogea) plants. J Appl Microbiol Biotechnol 102(5):1283–1292Google Scholar
  51. Sheng XF, Xia JJ, Jiang CY, He LY, Qian M (2008) Characterization of heavy metal-resistant endophytic bacteria from rape (Brassica napus) roots and their potential in promoting the growth and lead accumulation of rape. Environ Sci Pollut Res 156:1164–1170Google Scholar
  52. Siddikee MA, Chauhan PS, Anandham R, Han GH, Sa T (2010) Isolation, characterization, and use for plant growth promotion under salt stress, of ACC deaminase-producing halotolerant bacteria derived from coastal soil. J Microbiol Biotechnol 20:1577–1584PubMedCrossRefGoogle Scholar
  53. Siddikee MA, Glick BR, Chauhan PS, Yim WJ, Sa T (2011) Enhancement of growth and salt tolerance of red pepper seedlings (Capsicum annuum L.) by regulating stress ethylene synthesis with halotolerant bacteria containing ACC deaminase activity. Plant Physiol Biochem 49:427–434PubMedCrossRefGoogle Scholar
  54. Simova-Stoilova L, Demirevska K, Petrova T, Tsenov N, Feller U (2009) Antioxidative protection and proteolytic activity in tolerant and sensitive wheat (Triticum aestivum L.) varieties subjected to long term field drought. Plant Growth Regul 58:107–117Google Scholar
  55. Tilak K, Ranganayaki N, Pal K, Saxena DRA, Nautiyal SC, Mittal S, Tripathi A, Johri B (2005) Diversity of plant growth and soil health-supporting bacteria. Curr Sci 89(1):136–150Google Scholar
  56. Tittsler RP, Sandholzer LA (1936) The use of semi-solid agar for the detection of bacterial motility. J Bacteriol 31:575–580PubMedGoogle Scholar
  57. Yordy DM, Rudoff KL (1981) Dissimilatory nitrate reduction to ammonia. In: Delwiche CC (ed) Denitrification, nitrification and atmospheric nitrous oxide. Wiley, New York, pp 171–190Google Scholar
  58. Yue HT, Mo WP, Li C, Zheng YY, Li H (2007) The salt stress relief and growth promotion effect of Rs-5 on cotton. Plant Soil 297:139–145CrossRefGoogle Scholar
  59. Zahir AZ, Ghani U, Naveed M, Nadeem SM, Asghar HM (2009) Comparative effectiveness of Pseudomonas and Serratia sp. Containing ACC- deaminase for improving growth and yield of wheat (Triticum aestivum L.) under salt stressed conditions. Arch Microbiol 191:415–424PubMedCrossRefGoogle Scholar
  60. Zhang YF, He LY, Chen ZJ, Zhang WH, Wang Q, Qian M, Sheng X (2011a) Characterization of lead-resistant and ACC deaminaseproducing endophytic bacteria and their potential in promoting lead accumulation of rape. J Hazard Mat 186:1720–1725Google Scholar
  61. Zhang YF, He LY, Chen ZJ, Wang QY, Qian M, Sheng XF (2011b) Characterization of ACC deaminase-producing endophytic bacteria isolated from copper-tolerantplants and their potential in promoting the growth and copper accumulation of Brassica napus. Chemosphere 83:57–62PubMedCrossRefGoogle Scholar
  62. Zhao J, Zhu W, Hu Q (2000) Enhanced ajmalicine production in Catharanthus roseus cell cultures by combined elicitor treatment from Shake-flask to airlift bioreactor. Biotechnol Lett 22:509–514CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  1. 1.Department of Agricultural MicrobiologyAnnamalai UniversityAnnamalainagarIndia
  2. 2.Department of Agricultural ChemistryChungbuk National UniversityCheongjuRepublic of Korea
  3. 3.Department of Biological SciencesInha UniversityIncheonRepublic of Korea

Personalised recommendations