World Journal of Microbiology and Biotechnology

, Volume 30, Issue 2, pp 719–725 | Cite as

Molecular characterization and identification of plant growth promoting endophytic bacteria isolated from the root nodules of pea (Pisum sativum L.)

  • Mohsin TariqEmail author
  • Sohail Hameed
  • Tahira Yasmeen
  • Mehwish Zahid
  • Marriam Zafar
Original Paper


Root nodule accommodates various non-nodulating bacteria at varying densities. Present study was planned to identify and characterize the non-nodulating bacteria from the pea plant. Ten fast growing bacteria were isolated from the root nodules of cultivated pea plants. These bacterial isolates were unable to nodulate pea plants in nodulation assay, which indicate the non-rhizobial nature of these bacteria. Bacterial isolates were tested in vitro for plant growth promoting properties including indole acetic acid (IAA) production, nitrogen fixation, phosphate solubilization, root colonization and biofilm formation. Six isolates were able to produce IAA at varying level from 0.86 to 16.16 μg ml−1, with the isolate MSP9 being most efficient. Only two isolates, MSP2 and MSP10, were able to fix nitrogen. All isolates were able to solubilize inorganic phosphorus ranging from 5.57 to 11.73 μg ml−1, except MSP4. Bacterial isolates showed considerably better potential for colonization on pea roots. Isolates MSP9 and MSP10 were most efficient in biofilm formation on polyvinyl chloride, which indicated their potential to withstand various biotic and abiotic stresses, whereas the remaining isolates showed a very poor biofilm formation ability. The most efficient plant growth promoting agents, MSP9 and MSP10, were phylogenetically identified by 16S rRNA gene sequence analysis as Ochrobactrum and Enterobacter, respectively, with 99 % similarity. It is suggested the potential endophytic bacterial strains, Ochrobactrum sp. MSP9 and Enterobacter sp. MSP10, can be used as biofertilizers for various legume and non-legume crops after studying their interaction with the host crop and field evaluation.


Pisum sativum L. Plant growth promoting bacteria 16S rRNA sequencing Nodules 


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410Google Scholar
  2. Angus A, Hirsch AM (2010) Insights into the history of the legume beta-proteobacterial symbiosis. Mol Ecol 19:28–30Google Scholar
  3. Aravind R, Kumar A, Eapen SJ (2012) Pre-plant bacterisation: a strategy for delivery of beneficial endophytic bacteria and production of disease-free plantlets of black pepper (Piper nigrum L.). Arch Phytopathol Plant Prot 45:1115–1126CrossRefGoogle Scholar
  4. Arnon DI, Hoagland DR (1940) Crop production in artificial culture solution sand in soil with special reference to factors influencing yields and absorption of inorganic nutrient. Soil Sci 50:463–483Google Scholar
  5. Bansal RK (2009) Synergistic effect of Rhizobium, PSB and PGPR on nodulation and grain yield of mung bean. J Food Legum 22:37–39Google Scholar
  6. Beijerinck MW, Delden AV (1902) Uber die Assimilation des freien Stickstoffs durch Bakerien. Centralbl Bakt Abt II 9:3–43Google Scholar
  7. Benhizia Y, Benhizia H, Benguedouar A, Muresu R, Giacomini A, Squartini A (2004) Gamma proteobacteria can nodulate legumes of the genus Hedysarum. Syst Appl Microbiol 27:462–468CrossRefGoogle Scholar
  8. Bhattacharyya PN, Jha DK (2012) Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture. World J Microbiol Biotechnol 28:1327–1350CrossRefGoogle Scholar
  9. Birnboim HC, Doly A (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523CrossRefGoogle Scholar
  10. Dong Y, Iniguez LA, Ahmer BMM (2003) Kinetics and strain specificity of rhizosphere and endophytic colonization by enteric bacteria on seedlings of Medicago sativa and Medicago truncatula. Appl Environ Microbiol 69:1783–1790CrossRefGoogle Scholar
  11. Duangpaeng A, Phetcharat P, Chanthapho S, Boonkantong N, Okuda N (2012) The study and development of endophytic bacteria for enhancing organic rice growth. Procedia Eng 32:172–176CrossRefGoogle Scholar
  12. Ekin Z (2010) Performance of phosphate solubilizing bacteria for improving growth and yield of sunflower (Helianthus annuus L.) in the presence of phosphorus fertilizer. Afr J Biotechnol 9:3794–3800Google Scholar
  13. Fabre F, Planchon C (2000) Nitrogen nutrition, yield and protein content in soybean. Plant Sci 152:51–58CrossRefGoogle Scholar
  14. Fedorova E, Redondo FJ, Koshiba T, de Felipe MR, Pueyo JJ, Lucas MM (2005) Aldehyde oxidase (AO) in the root nodules of Lupinus albus and Medicago truncatula: identification of AO in meristematic and infection zones. Mol Plant Microbe Interact 18:405–413CrossRefGoogle Scholar
  15. Fraile B, Paniagua R, Rodriguez MC (1988) Long day photoperiods and temperature of 20 °C induce spermatogenesis in blinded and non-blinded marbled newts during the period of testicular quiescence. Biol Reprod 39:649–655CrossRefGoogle Scholar
  16. Fujishige NA, Kapadia NN, De Hoff PL, Hirsch AM (2006) Investigations of Rhizobium biofilm formation. FEMS Microbiol Ecol 56:195–206CrossRefGoogle Scholar
  17. Garbeva P, Overbeek LS, Vuurde JW (2001) Analysis of endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments. Microb Ecol 41:369–383Google Scholar
  18. Gordon SA, Weber RP (1951) Calorimetric estimation of indoleacetic acid. Plant Physiol 26:192–195CrossRefGoogle Scholar
  19. Gough C, Vasse J, Galera C, Webster G, Cocking E, Denarie J (1997) Interactions between bacterial diazotrophs and non-legume dicots: Arabidopsis thaliana as a model plant. Plant Soil 194:123–130CrossRefGoogle Scholar
  20. Hameed S, Yasmin S, Malik KA, Zafar Y, Hafeez FY (2004) Rhizobium, Bradyrhizobium and Agrobacterium strains isolated from cultivated legumes. Biol Fertil Soils 39:179–185CrossRefGoogle Scholar
  21. Hardy RWF, Holsten RD, Jackson EK, Burns RE (1968) The acetylene-ethylene assay for nitrogen fixation: laboratory and field evaluation. Plant Physiol 43:1185–1207CrossRefGoogle Scholar
  22. Hirsch AM (2010) How rhizobia survive in the absence of a legume host, a stressful world indeed. In: Seckbach J, Grube M (eds) Symbiosis and stress: cellular origin, life in extreme habitats and astrobiology. Springer 17, pp 375–391Google Scholar
  23. Hirsch AM (2010b) Insights into the history of the legume beta-proteobacterial symbiosis. Mol Ecol 19:28–30CrossRefGoogle Scholar
  24. Ibanez F, Angelini J, Taurian T, Tonelli ML, Fabra A (2009) Endophytic occupation of peanut root nodules by opportunistic Gammaproteobacteria. Syst Appl Microbiol 32:49–55CrossRefGoogle Scholar
  25. Kobayashi DY, Palumbo JD (2000) Bacterial endophytes and their effects on plants and uses in agriculture. In: James CW, White JF (eds) Microbial endophytes. Marcel Dekker Inc, New York, pp 199–233Google Scholar
  26. Ma W, Guinel FC, Glick BR (2003) Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants. Appl Environ Microbiol 69:4396–4402CrossRefGoogle Scholar
  27. Marsudi NDS, Glenn AR, Dilworth MJ (1999) Identification and characterization of fast- and slow-growing root nodule bacteria from South-Western Australian soils able to nodulate Acacia saligna. Soil Biol Biochem 31:1229–1238CrossRefGoogle Scholar
  28. Mishra PK, Mishra S, Selvakumar G, Bisht JK, Kundu S, Gupta HS (2009) Coinoculation of Bacillus thuringeinsis-KR1 with Rhizobium leguminosarum enhances plant growth and nodulation of pea (Pisum sativum L.) and lentil (Lens culinaris L.). World J Microbiol Biotechnol 25:753–761CrossRefGoogle Scholar
  29. Nair A, Juwarkar A, Singh S (2007) Production and characterization of siderophores and its application in arsenic removal from contaminated soil. Water Air Soil Pollut 180:199–212CrossRefGoogle Scholar
  30. Okan YS, Albercht L, Burris RH (1977) Methods of growing Spirillum lipoferum and for counting the pure culture in association and with plant. Appl Environ Microbiol 33:85–88Google Scholar
  31. Patel HA, Patel RK, Khristi SM, Parikh K, Rajendran G (2012) Isolation and characterization of bacterial endophytes from Lycopersicon esculentum plant and their plant growth promoting characteristics. Nepal J Biotechnol 2:37–52CrossRefGoogle Scholar
  32. Patten CL, Glick BR (2002) Role of Pseudomonas putida indole acetic acid in development of the host plant root system. Appl Environ Microbiol 68:3795–3801CrossRefGoogle Scholar
  33. Phetcharat P, Duangpaeng A (2012) Screening of endophytic bacteria from organic rice tissue for indole acetic acid production. Procedia Eng 32:177–183CrossRefGoogle Scholar
  34. Pikovskaya R (1948) Mobilization of P in soil in connection with vital activity by some microbial species. Microbiologica 17:362–370Google Scholar
  35. Selvakumar G, Kundu S, Gupta AD, Shouche YS, Gupta HS (2008) Isolation and characterization of nonrhizobial plant growth promoting bacteria from nodules of Kudzu (Pueraria thunbergiana) and their effect on wheat seedling growth. Curr Microbiol 56:134–139CrossRefGoogle Scholar
  36. Seneviratne G, Weerasekara MLMAW, Seneviratne KACN, Zavahir JS, Kecskes ML, Kennedy IR (2011) Importance of biofilm formation in plant growth promoting rhizobacterial action. Microbiol Monogr 18:81–95CrossRefGoogle Scholar
  37. Steel RGD, Torrie JH (1980) Analysis of covariance. In: Principles and procedures of statistics: a biometrical approach. McGraw-Hill, New YorkGoogle Scholar
  38. Sturz AV, Christie BR, Matheson BG, Nowak J (1997) Biodiversity of endophytic bacteria which colonize red clover nodules, roots, stems and foliage and their influence on host growth. Biol Fertil Soils 25:13–19CrossRefGoogle Scholar
  39. Tan Z, Xu X, Wang E, Gao J, Martinez-Romero E, Chen W (1997) Phylogenetic and genetic relationships of Mesorhizobium tianshanense and related rhizobia. Int J Syst Bacteriol 47:874–879CrossRefGoogle Scholar
  40. Tariq M, Hameed S, Yasmeen T, Ali A (2012) Non-rhizobial bacteria for improved nodulation and grain yield of mung bean [Vigna radiata (L.) Wilczek]. Afr J Biotechnol 11:15012–15019Google Scholar
  41. Taurian T, Ibanez F, Angelini J, Tonelli ML, Fabra A (2012) Endophytic bacteria and their role in legumes growth promotion. Bact Agrobiol Plant Probiotics, Springer-Verlag, Berlin, Heidelberg, pp 141–168 Google Scholar
  42. Tien TM, Gaskins MH, Hubbell DH (1979) Plant growth substances produced by Azospirillum brazilense and their effect on the growth of pearl millet. Appl Environ Microbiol 37:1016–1024Google Scholar
  43. Tripathi AK, Verma SC, Chowdhury SP, Lebuhn M, Gattinger A, Schloter M (2006) Ochrobactrum oryzae sp. nov., an endophytic bacterial species isolated from deep-water rice in India. Int J Syst Evol Microbiol 56:1677–1680CrossRefGoogle Scholar
  44. Vincent JM (1970) A manual for the practical study of root nodule bacteria. Blackwell Science Publisher, OxfordGoogle Scholar
  45. Xu Y, Yokota A, Sanada H, Hisamatsu M, Araki M, Cho HJ, Morinaga T, Murooka Y (1994) Enterobacter cloacae A105, isolated from the surface of root nodules of Astragalus sinicus cv. Japan, stimulates nodulation by Rhizobium huakuii bv. Renge. J Ferment Bioeng 77:630–635CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Mohsin Tariq
    • 1
    • 2
    Email author
  • Sohail Hameed
    • 1
  • Tahira Yasmeen
    • 1
    • 2
  • Mehwish Zahid
    • 1
  • Marriam Zafar
    • 1
    • 3
  1. 1.Microbial Physiology LaboratoryNational Institute for Biotechnology and Genetic Engineering (NIBGE)/ PAECIslamabadPakistan
  2. 2.Government College University Faisalabad (GCUF)FaisalabadPakistan
  3. 3.Centre of Agricultural Biochemistry and Biotechnology (CABB)University of Agriculture FaisalabadFaisalabadPakistan

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