Antonie van Leeuwenhoek

, Volume 112, Issue 12, pp 1827–1839 | Cite as

Isolation, functional characterization and efficacy of biofilm-forming rhizobacteria under abiotic stress conditions

  • Firoz Ahmad Ansari
  • Iqbal AhmadEmail author
Original Paper


Abiotic stresses such as salinity, drought and excessive heat are associated with significant loss of crop productivity globally, and require effective strategies for their reduction or tolerance. Biofilm-forming rhizobacteria, which harbor multifarious plant growth promoting traits and tolerance to abiotic stress, are believed to benefit plant health and production even under environmental stresses. The primary objective of this study was to investigate indigenous biofilm-forming rhizobacteria (Pseudomonas spp., Bacillus sp., Pantoea sp., Brevibacterium sp. and Acinetobacter sp.) for their functional diversity relevant to plant growth promoting activities, biofilm development and tolerance to abiotic stress conditions. The most promising isolates among FAP1, FAP2, FAP3, FAP4, FAP5, FAP10, FAB1, FAB3 and FAA1 were selected. Rhizobacteria exhibited high tolerance to salinity (1.5 M NaCl) and drought stress (up to 55% PEG-6000) conditions in vitro. The isolates demonstrated varying levels of PGP activities (IAA production and phosphate solubilization), biofilm development, and production of alginate and exopolysaccharides in the presence of salinity, drought stress and elevated temperature. Further efficacy of the isolates was demonstrated by inoculating to wheat (Triticum aestivum L.) plants in greenhouse conditions under both normal and drought stress for up to 30 days inoculation. The plant growth potential of the isolates was in the order of FAP3 > FAB3 > FAB1 > FAP10 > FAP5 > FAP4 > FAA1 > FAP2 > FAP1. The present study resulted in successful selection of promising PGPR as identified by 16S rRNA gene sequence analysis. Field study is needed to evaluate their relative performance in both ‘normal’ and stressed environments in order to be exploited for plant stress management.


Abiotic stress Biofilm Isolation PCR Plant growth Rhizosphere 



The authors gratefully acknowledge the University Grant Commission (UGC), New Delhi, for research support as a research fellowship (MAN-SRF). We also acknowledge the support of the University Sophisticated Instrumentation Facility (USIF), Aligarh Muslim University, Aligarh and Macrogen Sequencing Team, Seoul, South Korea, for providing instrumentation facilities. We are highly thankful to Prof. John Pichtel, Ball State University, USA, for critical review and language editing of the manuscript.

Author’s contribution

The authors, F.A. Ansari and I. Ahmad designed the experiment and F.A. Ansari performed the experiments and data analysis. F.A. Ansari wrote the manuscript, and I. Ahmad edited the manuscript critically and very carefully. All authors have read the manuscript and approved the data of the manuscript in its present form.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10482_2019_1306_MOESM1_ESM.tif (841 kb)
Supplementary material 1 (TIFF 840 kb)
10482_2019_1306_MOESM2_ESM.tif (531 kb)
Supplementary material 2 (TIFF 530 kb)
10482_2019_1306_MOESM3_ESM.doc (36 kb)
Supplementary material 3 (DOC 35 kb)
10482_2019_1306_MOESM4_ESM.doc (94 kb)
Supplementary material 4 (DOC 91 kb)
10482_2019_1306_MOESM5_ESM.doc (104 kb)
Supplementary material 5 (DOC 101 kb)
10482_2019_1306_MOESM6_ESM.doc (79 kb)
Supplementary material 6 (DOC 77 kb)


  1. Abiala MA, Odebode AC, Hsu SF, Blackwood CB (2015) Phytobeneficial properties of bacteria isolated from the rhizosphere of maize in southwestern Nigerian soils. Appl Environ Microbiol 81:4736–4743CrossRefGoogle Scholar
  2. Adler J (1966) Chemotaxis in bacteria. Science 153:708–716CrossRefGoogle Scholar
  3. Ahemad M, Kibert M (2014) Mechanisms and application of plant growth promoting rhizobacteria: current perspective. J King Saud Univ-Sci 26(1):1–20CrossRefGoogle Scholar
  4. Ahmad F, Ahmad I, Khan MS (2008) Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol Res 163:173–181. CrossRefPubMedGoogle Scholar
  5. Ansari FA, Ahmad I (2018a) Biofilm development, plant growth promoting traits and rhizosphere colonization by Pseudomonas entomophila FAP1: a promising PGPR. Adv Microbiol 8(03):235CrossRefGoogle Scholar
  6. Ansari FA, Ahmad I (2018b) Plant growth promoting attributes and alleviation of salinity stress to wheat by biofilm forming Brevibacterium sp. FAB3 isolated from rhizospheric soil. Saudi J Biol Sci. CrossRefGoogle Scholar
  7. Ansari FA, Ahmad I (2019) Fluorescent pseudomonas-FAP2 and Bacillus licheniformis interact positively in biofilm mode enhancing plant growth and photosynthetic attributes. Sci Rep 9(1):4547CrossRefGoogle Scholar
  8. Ansari FA, Ahmad I, Pichtel J (2019) Growth stimulation and alleviation of salinity stress to wheat by the biofilm forming Bacillus pumilus strain FAB10. Appl Soil Ecol 143:45–54CrossRefGoogle Scholar
  9. Bechtold U, Field B (2018) Molecular mechanisms controlling plant growth during abiotic stress. J Exp Bot 69(11):2753–2758CrossRefGoogle Scholar
  10. Bharucha U, Patel K, Trivedi UB (2013) Optimization of indole acetic acid production by Pseudomonas putida UB1 and its effect as plant growth-promoting rhizobacteria on mustard (Brassica nigra). Agric Res 2:215–221. CrossRefGoogle Scholar
  11. Bogino P, Oliva M, Sorroche F, Giordano W (2013) The role of bacterial biofilms and surface components in plant-bacterial associations. Int J Mol Sci 14(8):15838–15859CrossRefGoogle Scholar
  12. Cardinale M, Ratering S, Suarez C, Montoya AMZ, Geissler Plaum R, Schnell S (2015) Paradox of plant growth promotion potential of rhizobacteria and their actual promotion effect on growth of barley (Hordeum vulgare L.) under salt stress. Microbiol Res 181:22–32. CrossRefPubMedGoogle Scholar
  13. Cattelan AJ, Hartel PG, Furhmann JJ (1999) Screening for plant growth promoting rhizobacteria to promote early soybean growth. Soil Sci Soc Am J 63:1670–1680CrossRefGoogle Scholar
  14. Chaudhry V, Bhatia A, Bharti SK, Mishra SK, Chauhan PS, Mishra A, Sidhu OP, Nautiyal CS (2015) Metabolite profiling reveals abiotic stress tolerance in Tn5 mutant of Pseudomonas putida. PLoS ONE 10:e0113487. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Chauhan PS, Nautiyal CS (2010) The purB gene controls rhizosphere colonization by Pantoea agglomerans. Lett Appl Microbiol 50:205–210. CrossRefPubMedGoogle Scholar
  16. Davey ME, O’Toole GA (2000) Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 64:847–867CrossRefGoogle Scholar
  17. Fahad S, Bajwa AA, Nazir U, Anjum SA, Farooq A, Zohaib A, Sadia S, Nasim W, Adkins S, Saud S, Ihsan MZ (2017) Crop production under drought and heat stress: plant responses and management options. Front Plant Sci 29(8):1147CrossRefGoogle Scholar
  18. Fiske CH, Subbarow Y (1925) The colorimetric determination of phosphorus. J Biol Chem 66:375–400Google Scholar
  19. Gontia-Mishra I, Sapre S, Sharma A, Tiwari S (2016) Amelioration of drought tolerance in wheat by the interaction of plant growth-promoting rhizobacteria. Plant Biol. 18(6):992–1000CrossRefGoogle Scholar
  20. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98Google Scholar
  21. Islam S, Akanda AM, Prova A, Islam MT, Hossain MM (2016) Isolation and identification of plant growth promoting rhizobacteria from cucumber rhizosphere and their effect on plant growth promotion and disease suppression. Front Microbiol 2(6):1360. CrossRefGoogle Scholar
  22. Kasim WA, Gaafar RM, Abou-Ali RM, Omar MN, Hewait HM (2016) Effect of biofilm forming plant growth promoting rhizobacteria on salinity tolerance in barley. Ann Agric Sci 61(2):217–227CrossRefGoogle Scholar
  23. Khare E, Arora NK (2010) Effect of indole-3-acetic acid (IAA) produced by Pseudomonas aeruginosa in suppression of charcoal rot disease of chickpea. Curr Microbiol 61:64–68. CrossRefPubMedGoogle Scholar
  24. Kloepper JW, Beauchamp CJ (1992) A review of issues related to measuring of plant roots by bacteria. Can J Microbiol 38:1219–1232. CrossRefGoogle Scholar
  25. Laditi MA, Nwoke OC, Jemo M, Abaidoo RC, Ogunjobi AA (2012) Evaluation of microbial inoculants as biofertilizers for the improvement of growth and yield of soybean and maize crops in savanna soils. Afr J Agric Res 7:405–413. CrossRefGoogle Scholar
  26. Martins SJ, Rocha GA, de Melo HC, de Castro Georg R, Ulhôa CJ, de Campos Dianese É, Oshiquiri LH, da Cunha MG, da Rocha MR, de Araújo LG, Vaz KS, Dunlap CA (2018) Plant-associated bacteria mitigate drought stress in soybean. Environ Sci Pollut Res Int. 25(14):13676–13686CrossRefGoogle Scholar
  27. Meena KK, Sorty AM, Bitla UM, Choudhary K, Gupta P, Pareek A, Singh DP, Prabha R, Sahu PK, Gupta VK, Singh HB (2017) Abiotic stress responses and microbe-mediated mitigation in plants: the omics strategies. Front Plant Sci. 9(8):172Google Scholar
  28. Meng X, Yan D, Long X, Wang C, Liu Z, Rengel Z (2014) Colonization by endophytic Ochrobactrum anthropi Mn1 promotes growth of Jerusalem artichoke. Microbiol Biotechnol 7:601–610. CrossRefGoogle Scholar
  29. Meyer JM, Abdallah MA (1978) The florescent pigment of Pseudomonas fluorescens biosynthesis, purification and physical–chemical properties. J Gen Microbiol 107:319–328. CrossRefGoogle Scholar
  30. Mishra S, Mishra A, Chauhan PS, Mishra SK, Kumari M, Niranjan A, Nautiyal CS (2012) Pseudomonas putida NBRIC19 dihydrolipoamide succinyltransferase (SucB) gene controls degradation of toxic allelochemicals produced by Parthenium hysterophorus. J Appl Microbiol 112:793–808. CrossRefPubMedGoogle Scholar
  31. Naseem H, Bano A (2014) Role of plant growth-promoting rhizobacteria and their exopolysaccharide in drought tolerance of maize. J Plant Interact 9(1):689–701CrossRefGoogle Scholar
  32. Nautiyal CS (1997) A method for selection and characterization of rhizosphere-competent bacteria of chickpea. Curr Microbiol 34:12–17. CrossRefPubMedGoogle Scholar
  33. Niu X, Song L, Xiao Y, Ge W (2018) Drought-tolerant plant growth-promoting rhizobacteria associated with foxtail millet in a semi-arid agroecosystem and their potential in alleviating drought stress. Front Microbiol 8:2580CrossRefGoogle Scholar
  34. Oteino N, Lally RD, Kiwanuka S, Lloyd A, Ryan D, Germaine KJ, Dowling DN (2015) Plant growth promotion induced by phosphate solubilizing endophytic Pseudomonas isolates. Front Microbiol 6:745. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Paredes-Páliz K, Rodríguez-Vázquez R, Duarte B, Caviedes MA, Mateos-Naranjo E, Redondo-Gómez S, Caçador MI, Rodríguez-Llorente ID, Pajuelo E (2018) Investigating the mechanisms underlying phytoprotection by plant growth-promoting rhizobacteria in Spartina densiflora under metal stress. Plant Biol. 20(3):497–506CrossRefGoogle Scholar
  36. Penrose DM, Glick BR (2003) Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plant 118:10–15. CrossRefPubMedGoogle Scholar
  37. Podile AR, Vukanti RVNR, Sravani A, Kalam S, Dutta S, Durgeshwar P, Rao VP (2013) Root colonization and quorum sensing are the driving forces of plant growth promoting rhizobacteria (PGPR) for growth promotion. Proc Natl Acad Sci, India, Sect B Biol 80:407–413. CrossRefGoogle Scholar
  38. Qurashi AW, Sabri AN (2012) Bacterial exopolysaccharide and biofilm formation stimulate chickpea growth and soil aggregation under salt stress. Braz J Microbiol 43(3):1183–1191CrossRefGoogle Scholar
  39. Redmile-Gordon MA, Brookes PC, Evershed RP, Goulding KW, Hirsch PR (2014) Measuring the soilmicrobial interface: extraction of extracellular polymeric substances (EPS) from soil biofilms. Soil Biol Biochem 72:163–171CrossRefGoogle Scholar
  40. Smibert RM, Krieg NR (1994) Methods for general and molecular bacteriology. American Society for Microbiology, Washington DCGoogle Scholar
  41. Srivastava S, Yadav A, Seem K, Mishra S, Chaudhary V, Nautiyal CS (2008) Effect of high temperature on Pseudomonas putida NBRI10987 biofilm formation and expression of stress sigma factor RpoS. Curr Microbiol 56:453–457. CrossRefPubMedGoogle Scholar
  42. Srivastava S, Chaudhry V, Mishra A, Chauhan PS, Rehman A, Yadav A, Tuteja N, Nautiyal CS (2012) Gene expression profiling through microarray analysis in Arabidopsis thaliana colonized by Pseudomonas putida MTCC5279, a plant growth promoting rhizobacterium. Plant Signal Behav. 7:235–245. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci USA 101:11030–11035. CrossRefPubMedGoogle Scholar
  44. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Tan IS, Ramamurthi KS (2014) Spore formation in Bacillus subtilis. Environ Microbiol Rep. 6(3):212–225CrossRefGoogle Scholar
  46. Titus S, Gasnkar N, Srivastava KB, Karande AA (1995) Exopolymer production by a fouling marine bacterium Pseudomonas alcaligenes. Indian J Mar Sci 24:45–48Google Scholar
  47. Wang X, Wang Z, Jiang P, He Y, Mu Y, Lv X, Zhuang L (2018) Bacterial diversity and community structure in the rhizosphere of four Ferula species. Sci Rep. 8(1):5345CrossRefGoogle Scholar
  48. World Economic Forum (2011) Realizing a new vision for agriculture: a roadmap for stakeholders. World Economic Forum, DavosGoogle Scholar
  49. Yadav AN, Verma P, Kumar M, Pal KK, Dey R, Gupta A, Padaria JC, Gujar GT, Kumar S, Suman A, Prasanna R, Saxena AK (2015) Diversity and phylogenetic profiling of niche-specific Bacilli from extreme environments of India. Ann Microbiol 65:611–629. CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Biofilm Research Lab, Department of Agricultural Microbiology, Faculty of Agricultural SciencesAligarh Muslim UniversityAligarhIndia

Personalised recommendations