Biosurfactant based formulation of Pseudomonas guariconensis LE3 with multifarious plant growth promoting traits controls charcoal rot disease in Helianthus annus

Abstract

Biosurfactants are environment compatible surface-active biomolecules with multifunctional properties which can be utilized in various industries. In this study a biosurfactant producing novel plant growth promoting isolate Pseudomonas guariconensis LE3 from the rhizosphere of Lycopersicon esculentum is presented as biostimulant and biocontrol agent. Biosurfactant extracted from culture was characterized to be mixture of various mono- and di-rhamnolipids with antagonistic activity against Macrophomina phaseolina, causal agent of charcoal rot in diverse crops. Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR) analysis confirmed the rhamnolipid nature of biosurfactant. PCR analysis established the presence of genes involved in synthesis of antibiotics diacetylphloroglucinol, phenazine 1-carboxylic acid and pyocyanin, and lytic enzymes chitinase and endoglucanase suggesting biocontrol potential of the isolate. Plant growth promoting activities shown by LE3 were phosphate solubilization and production of siderophores, indole acetic acid (IAA), ammonia and 1-aminocyclopropane-1-carboxylate deaminase (ACCD). To assemble all the characteristics of LE3 various bioformuations were developed. Amendment of biosurfactant in bioformulation of LE3 cells improved the shelf life. Biosurfactant amended formulation of LE3 cells was most effective in biocontrol of charcoal rot disease of sunflower and growth promotion in field conditions. The root adhered soil mass of plantlets inoculated with LE3 plus biosurfactant was significantly higher over control. Biosurfactant amended formulation of LE3 cells caused maximum yield enhancement (80.80%) and biocontrol activity (75.45%), indicating that addition of biosurfactant improves the plant-bacterial interaction and soil properties leading to better control of disease and overall improvement of plant health and yield.

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Data availability

16S rRNA gene sequence of isolate LE3 was submitted in NCBI GenBank. Bacterial isolate P. guariconensis LE3 was submitted to IDA approved culture collection center, NAIMCC, Uttar Pradesh, India.

References

  1. Abdel-Mawgoud A, Lépine F, Déziel E (2010) Rhamnolipids: diversity of structures, microbial origins and roles. Appl Microbiol Biotechnol 86:1323–1336

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Adnan M, Alshammari E, Ashraf SA, Patel K, Lad K, Patel M (2018) Physiological and molecular characterization of biosurfactant producing endophytic fungi Xylaria regalis from the cones of Thuja plicata as a potent plant growth promoter with its potential application. BioMed Res Int 2018:1–11

    Google Scholar 

  3. Aeron A, Khare E, Arora NK, Maheshwari DK (2012) Practical use of CMC-amended rhizobial inoculant for Mucuna pruriens cultivation to enhance the growth and protection against Macrophomina phaseolina. J Gen Appl Microbiol 58:121–127

    CAS  PubMed  Google Scholar 

  4. Ali N, Wang F, Xu B, Safdar B, Ullah A, Naveed M, Wang C, Rashid MT (2019) Production and application of biosurfactant produced by Bacillus licheniformis Ali5 in enhanced oil recovery and motor oil removal from contaminated sand. Molecules 24:4448

    CAS  PubMed Central  Google Scholar 

  5. Al-Tahhan RA, Sandrin TR, Bodour AA, Maier RM (2000) Rhamnolipid-induced removal of lipopolysaccharide from Pseudomonas aeruginosa: Effect on cell surface properties and interaction with hydrophobic substrates. Appl Environ Microbiol 66(8):3262–3268

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Altschul SF, Madden TL, Scaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search program. Nucleic Acids Res 25:3389–3402

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Ariech M, Guechi A (2015) Assessment of four different methods for selecting biosurfactant producing extremely halophilic bacteria. Afr J Biotechnol 14(21):1764–1772

    Google Scholar 

  8. Arnow LE (1937) Colorimetric determination of the components of 3,4-dihydroxy-phenylalanine-tyrosine mixtures. J Biol Chem 118:531–537

    CAS  Google Scholar 

  9. Arora NK, Kang SC, Maheshwari DK (2001) Isolation of siderophore producing strains of Rhizobium meliloti and their biocontrol potential against Macrophomina phaseolina that causes charcoal rot of groundnut. Curr Sci 81:673–677

    Google Scholar 

  10. Arora NK, Fatima T, Mishra I, Verma M, Mishra J (2018) Environmental sustainability: challenges and viable solutions. Environ Sustain 1:309–340

    Google Scholar 

  11. Borah SN, Goswami D, Sarma HK, Cameotra SS, Deka S (2016) Rhamnolipid biosurfactant against Fusarium verticillioides to control stalk and ear rot disease of maize. Front Microbiol 7:1505

    PubMed  PubMed Central  Google Scholar 

  12. Botelho GR, Mendonça-Hagler LC (2006) Fluorescent pseudomonads associated with the rhizosphere of crops: an overview. Braz J Microbiol 37(4):401–416

    CAS  Google Scholar 

  13. Bric JM, Bostock RM, Silversone SE (1991) Rapid in situ assay for indole acetic acid production by bacteria immobilization on a nitrocellulose membrane. Appl Environ Microbiol 57:535–538

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Bustamante M, Durán N, Diez MC (2012) Biosurfactants are useful tools for the bioremediation of contaminated soil: a review. J Soil Sci Plant Nutr 12(4):667–687

    Google Scholar 

  15. Cappuccino JG, Sherman N (1992) Microbiology: a laboratory manual. The Benjamin/Cummings Publishing Company Inc., Menlo Park

    Google Scholar 

  16. Castro MJL, Ojeda C, Cirelli AF (2013) Surfactants in agriculture. In: Lichtfouse E et al (eds) Green materials for energy, products and depollution. Springer, Dordrecht, pp 287–334

    Google Scholar 

  17. Chen J, Wu Q, Hua Y, Chen J, Zhang H, Wang H (2017) Potential applications of biosurfactant rhamnolipids in agriculture and biomedicine. Appl Microbiol Biotechnol 101:8309–8319

    CAS  PubMed  Google Scholar 

  18. Christova N, Tuleva B, Nikolova-Damyanova B (2004) Enhanced hydrocarbon biodegradation by a newly isolated Bacillus subtilis strain. Z Naturforsch 59:205–208

    CAS  Google Scholar 

  19. Cooper DG, Goldenberg BG (1987) Surface-active agents from 2 Bacillus species. Appl Environ Microbiol 53:224–229

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Cox C, Graham R (1979) Isolation of an iron-binding compound from Pseudomonas aeruginosa. J Bacteriol 137:357–364

    CAS  PubMed  PubMed Central  Google Scholar 

  21. D’aes J, de Maeyer K, Pauwelyn E, Höfte M (2009) Biosurfactants in plant–Pseudomonas interactions and their importance to biocontrol. Environ Microbiol Rep 2(3):359–372

    PubMed  Google Scholar 

  22. Das P, Yang XP, Ma LZ (2014) Analysis of biosurfactants from industrially viable Pseudomonas strain isolated from crude oil suggests how rhamnolipids congeners affect emulsification property and antimicrobial activity. Front Microbiol 5:696

    PubMed  PubMed Central  Google Scholar 

  23. De La Fuente L, Mavrodi DV, Landa BB, Thomashow LS, Weller DM (2006) phlD-based genetic diversity and detection of genotypes of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens. FEMS Microbiol Ecol 56:64–78

    Google Scholar 

  24. Fechtner J, Koza A, Dello Sterpaio P, Hapca SM, Spiers AJ (2011) Surfactants expressed by soil pseudomonads alter local soil-water distribution suggesting a hydrological role for these compounds. FEMS Microbiol Ecol 78:50–58

    CAS  PubMed  Google Scholar 

  25. Fenibo EO, Ijoma GN, Selvarajan R, Chikere CB (2019) Microbial surfactants: the next generation multifunctional biomolecules for applications in the petroleum industry and its associated environmental remediation. Microorganisms 7(11):581

    CAS  PubMed Central  Google Scholar 

  26. Fernandes PAV, Arruda IR, Santos AFAB, Araujo AA, Maior AAS, Ximenes EA (2007) Antimicrobial activity of surfactants produced by Bacillus subtilis R14 against multidrug-resistant bacteria. Braz J Microbiol 38:704–709

    Google Scholar 

  27. Garrity G (2005) The proteobacteria, part b the gammaproteobacteria. In: Brenner DJ, Krieg NR, Staley JT (eds) Bergey’s manual of systematic bacteriology, vol 2, 2nd edn. Springer, New York, pp 323–379

    Google Scholar 

  28. Ge S, Zhu Z, Peng L, Chen Q, Jiang Y (2018) Soil nutrient status and leaf nutrient diagnosis in the main apple producing regions in China. Hortic Plant J 4(3):89–93

    Google Scholar 

  29. Gupta S, Pandey S (2019) ACC deaminase producing bacteria with multifarious plant growth promoting traits alleviates salinity stress in french bean (Phaseolus vulgaris) plants. Front Microbiol. https://doi.org/10.3389/fmicb.2019.01506

    Article  PubMed  PubMed Central  Google Scholar 

  30. Ijaz S, Sadaqat HA, Khan MN (2012) A review of the impact of charcoal rot (Macrophomina phaseolina) on sunflower. The J Agr Sci 151(02):222–227

    Google Scholar 

  31. Irorere VU, Tripathi L, Marchant R, McClean S, Banat IM (2017) Microbial rhamnolipid production: a critical re-evaluation of published data and suggested future publication criteria. Appl Microbiol Biotechnol 101(10):3941–3951

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Jarvis FG, Johnson MJ (1949) A glycolipid produced by Pseudomonas aeruginosa. J Am Chem Soc 71:4124–4126

    CAS  Google Scholar 

  33. Kaur S, Dhillon GS, Brar SK, Vallad GE, Chand R, Chauhan VB (2012) Emerging phytopathogen Macrophomina phaseolina: biology, economic importance and current diagnostic trends. Crit Rev Microbiol 38(2):136–151

    CAS  PubMed  Google Scholar 

  34. Kavamura VN, Santos SN, Da Silva JL, Parma MM, Ávila LA, Visconti A et al (2013) Screening of brazilian cacti rhizobacteria for plant growth promotion under drought. Microbiol Res 168:183–191

    CAS  PubMed  Google Scholar 

  35. Khan AN, Shair F, Malik K, Hayat Z, Khan MA, Hafeez FY, Hassan MN (2017) Molecular identification and genetic characterization of Macrophomina phaseolina strains causing pathogenicity on sunflower and chickpea. Front Microbiol 8:1309

    PubMed  PubMed Central  Google Scholar 

  36. 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(1):64–68

    CAS  Google Scholar 

  37. Khare E, Arora NK (2015) Effects of soil environment on field efficacy of microbial inoculants. In: Arora NK (ed) Plant microbe symbiosis: applied facets. Springer, Netherland, pp 353–380

    Google Scholar 

  38. Kiefer J, Radzuan M, Winterburn J (2017) Infrared spectroscopy for studying structure and aging effects in rhamnolipid biosurfactants. Appl Sci 7(5):533

    Google Scholar 

  39. Liu S, Lin N, Chen Y, Liang Z, Liao L, Lv M, Chen Y, Tang Y, He F, Chen S, Zhou J, Zhang L (2017) Biocontrol of sugarcane smut disease by interference of fungal sexual mating and hyphal growth using a bacterial isolate. Front Microbiol 8:778

    PubMed  PubMed Central  Google Scholar 

  40. Luzuriaga-Loaiza WP, Schellenberger R, De Gaetano Y, Obounou Akong F, Villaume S, Crouzet J, Haudrechy A, Baillieul F, Clément C, Lins L, Allais F, Ongena M, Bouquillon S, Deleu M, Dorey S (2018) Synthetic rhamnolipid bolaforms trigger an innate immune response in Arabidopsis thaliana. Sci Rep. https://doi.org/10.1038/s41598-018-26838-y

    Article  PubMed  PubMed Central  Google Scholar 

  41. Maidak BL, Cole JR, Lilburn TG, Parker CT, Saxman PR, Stredwick JM, Garrity GM, Li B, Olsen GL, Pramanik S, Schmidt TM, Tiedje JM (2000) The RDP (ribosomal database project) continues. Nucleic Acids Res 28:173–174

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Maidak BL, Olsen GL, Larsen N, Overbeek R, McCaughey MJ, Woese CR (1997) The ribosomal database project. Nucleic Acids Res 24:82–85

    Google Scholar 

  43. Meyer JM, Abdallah MA (1978) The fluorescent pigment of Pseudomonas fluorescens: biosynthesis, purification and physicochemical properties. J Gen Microbiol 107(2):319–328

    CAS  Google Scholar 

  44. Mishra I, Fatima T, Egamberdieva D, Arora NK (2020) Novel bioformulations developed from Pseudomonas putida BSP9 and its biosurfactant for growth promotion of Brassica juncea (L.). Plants 9:1349

    CAS  PubMed Central  Google Scholar 

  45. Monnier N, Furlan A, Buchoux S, Deleu M, Dauchez M, Rippa S, Sarazin C (2019) Exploring the dual interaction of natural rhamnolipids with plant and fungal biomimetic plasma membranes through biophysical studies. Int J Mol Sci 20(5):1009

    CAS  PubMed Central  Google Scholar 

  46. Morales DK, Jacobs NJ, Rajamani S, Krishnamurthy M, Cubillos-Ruiz JR, Hogan DA (2010) Antifungal mechanisms by which a novel Pseudomonas aeruginosa phenazine toxin kills Candida albicans in biofilms. Mol Microbiol 78:1379–1392

    CAS  PubMed  Google Scholar 

  47. Moreno R, Rojo F (2014) Features of pseudomonads growing at low temperatures: another facet of their versatility. Environ Microbiol Rep 6(5):417–426

    CAS  PubMed  Google Scholar 

  48. Moussa TAA, Mohamed MS, Samak N (2014) Production and characterization of di-rhamnolipid produced by Pseudomonas aeruginosa TMN. Braz J Chem Eng 31(4):867–880

    Google Scholar 

  49. Mukherjee S, Das P, Sen R (2009) Rapid quantification of a microbial surfactant by a simple turbidometric method. J Microbiol Methods 76:38–42

    CAS  PubMed  Google Scholar 

  50. Nakkeeran S, Dilantha FWG, Siddiqui WA (2005) Plant growth promoting rhizobacteria formulations and its scope in commercialization for the management of pests and diseases. In: Siddiqui ZA (ed) PGPR: biocontrol and biofertilization. Springer, Dordrecht, pp 257–296

    Google Scholar 

  51. Nandakumar R, Babu S, Viswanathan R, Raguchander T, Samiyappan R (2001) Induction of systemic resistance in rice against sheath blight disease by plant growth promoting rhizobacteria. Soil Biol Biochem 33:603–612

    CAS  Google Scholar 

  52. Nielsen TH, Sørensen D, Tobiasen C, Andersen JB, Christophersen C, Givskov M, Sørensen J (2002) Antibiotic and biosurfactant properties of cyclic lipopeptides produced by fluorescent Pseudomonas spp. from the sugar beet rhizosphere. Appl Environ Microbiol 68(7):3416–3423

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Oluwaseun A, Phazang P, Sarin N (2017) Significance of rhamnolipids as a biological control agent in the management of crops/plant pathogens. Curr Trends Biomed Eng Biosci 10(3):555788

    Google Scholar 

  54. Pamp SJ, Tolker-Nielsen T (2007) Multiple roles of biosurfactants in structural biofilm development by Pseudomonas aeruginosa. J Bacteriol 189(6):2531–2539

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Patel RR, Thakkar VR, Subramanian BR (2015) A Pseudomonas guariconensis strain capable of promoting growth and controlling collar rot disease in Arachis hypogaea L. Plant Soil 390(1–2):369–381

    CAS  Google Scholar 

  56. Patowary K, Patowary R, Kalita MC, Deka S (2017) Characterization of biosurfactant produced during degradation of hydrocarbons using crude oil as sole source of carbon. Front Microbiol 8:279

    PubMed  PubMed Central  Google Scholar 

  57. Penrose DM, Glick BR (2003) Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plant 118:10–15

    CAS  PubMed  Google Scholar 

  58. Pereg L, de-Bashan LE, Bashan Y (2016) Assessment of affinity and specificity of Azospirillum for plants. Plant Soil 399:389–414

    CAS  Google Scholar 

  59. Pérez-Miranda S, Cabirol N, George-Téllez R, Zamudio-Rivera LS, Fernández FJ (2007) O-CAS, a fast and universal method for siderophore detection. J Microbiol Methods 70:127–131

    PubMed  Google Scholar 

  60. Pieterse CMJ, Zamioudis C, Berendsen RL, Weller DM, Van Wees SCM, Bakker PAHM (2014) Induced systemic resistance by beneficial microbes. Annu Rev Phytopathol 52(1):347–375

    CAS  Google Scholar 

  61. Pikovskaya RI (1948) Mobilization of phosphorous in soil in connection with vital activity of some microbial species. Mikrobiologiya 17:363–370

    Google Scholar 

  62. Politz M, Lennen R, Pfleger B (2013) Quantification of bacterial fatty acids by extraction and methylation. Bio-Protocol 3(21):e950. https://doi.org/10.21769/BioProtoc.950

    Article  PubMed  PubMed Central  Google Scholar 

  63. Prabhukarthikeyan SR, Raguchander T (2016) Antifungal metabolites of Pseudomonas fluorescens against Pythium aphanidermatum. J Pure Appl Microbiol 10(1):579–584

    CAS  Google Scholar 

  64. Reddy S, Osborne JW (2020) Biodegradation and biosorption of reactive red 120 dye by immobilized Pseudomonas guariconensis: kinetic and toxicity study. Water Environ Res. https://doi.org/10.1002/wer.1319

    Article  PubMed  Google Scholar 

  65. Rosenberg M, Gutnick DL, Rosenberg E (1980) Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett 9:29–33

    CAS  Google Scholar 

  66. Sachdev DP, Cameotra SS (2013) Biosurfactants in agriculture. Appl Microbiol Biotechnol 97(3):1005–1016

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Saitou N, Nei M (1987) The Neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  PubMed  Google Scholar 

  68. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  69. Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56

    CAS  PubMed  Google Scholar 

  70. Sen S, Borah SN, Bora A, Deka S (2020) Rhamnolipid exhibits anti-biofilm activity against the dermatophytic fungi Trichophyton rubrum and Trichophyton mentagrophytes. Biotechnol Rep 27:e00516

    Google Scholar 

  71. Siegmund I, Wagner F (1991) New method for detecting rhamnolipids excreted by Pseudomonas species during growth on mineral agar. Biotechnol Tech 5(4):265–268

    CAS  Google Scholar 

  72. Singh R, Glick BR, Rathore D (2018) Biosurfactants as a biological tool to increase micronutrient availability in soil: a review. Pedosphere 28(2):170–189

    Google Scholar 

  73. Singh N, Pandey P, Dubey RC, Maheshwari DK (2008) Biological control of root rot fungus Macrophomina phaseolina and growth enhancement of Pinus roxburghii (Sarg) by rhizosphere competent Bacillus subtilis BN1. World J Microbiol Biotechnol 24(9):1669–1679

    Google Scholar 

  74. Stringlis IA, Zhang H, Pieterse CMJ, Bolton MD, de Jonge R (2018) Microbial small molecules—weapons of plant subversion. Nat Prod Rep 35(5):410–433

    CAS  PubMed  Google Scholar 

  75. Tahzibi A, Kamal F, Assadi MM (2004) Improved production of rhamnolipids by a Pseudomonas aeruginosa mutant. Iran Biomed J 8(1):25–31

    CAS  Google Scholar 

  76. Tewari S, Arora NK (2018) Role of salicylic acid from Pseudomonas aeruginosa PF23EPS+ in growth promotion of sunflower in saline soils infested with phytopathogen Macrophomina phaseolina. Environ Sustain 1(1):49–59

    Google Scholar 

  77. Toro M, Ramirez-Bahena M-H, Cuesta MJ, Velazquez E, Peix A (2013) Pseudomonas guariconensis sp. Nov., isolated from rhizospheric soil. Int J Syst Evol Microbiol 63(12):4413–4420

    CAS  PubMed  Google Scholar 

  78. Turkovskaya OV, Dmitrieva TV, Yu Muratova A (2001) A biosurfactant-producing Pseudomonas aeruginosa strain. Appl Biochem Microbiol 37(1):71–75

    CAS  Google Scholar 

  79. Vurukonda SS, Vardharajula S, Shrivastava M, SkZ A (2016) Enhancement of drought stress tolerance in crops by plant growth promoting rhizobacteria. Microbiol Res 184:13–24

    PubMed  Google Scholar 

  80. Wittgens A, Kovacic F, Müller MM, Gerlitzki M, Santiago-Schübel B, Hofmann D, Tiso T, Blank LM, Henkel M, Hausmann R, Syldatk C, Wilhelm S, Rosenau F (2016) Novel insights into biosynthesis and uptake of rhamnolipids and their precursors. Appl Microbiol Biotechnol 101(7):2865–2878

    PubMed  PubMed Central  Google Scholar 

  81. Zhang L, Tian X, Kuang S, Liu G, Zhang C, Sun C (2017) Antagonistic activity and mode of action of phenazine-1-carboxylic acid, produced by marine bacterium Pseudomonas aeruginosa PA31x, against Vibrio anguillarum in vitro and in a Zebrafish in vivo model. Front Microbiol 8:289

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Authors are thankful to Director, Center of Biomedical Magnetic Resonance, Lucknow, India for providing facilities. Authors are grateful to the Vice Chancellors, Chhatrapati Shahu Ji Maharaj University, Kanpur and Babasaheb Bhimrao Ambedkar University, Lucknow, India for the support. NKA is thankful to DST, New Delhi for the Grant (No. SEED/SCSP/2019/61/BBAU/C).

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First author EK designed and performed experiments. Corresponding author NKA involved with conceptualization and designing of study. Both the authors equally contributed to the manuscript writing.

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Correspondence to Naveen Kumar Arora.

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Khare, E., Arora, N.K. Biosurfactant based formulation of Pseudomonas guariconensis LE3 with multifarious plant growth promoting traits controls charcoal rot disease in Helianthus annus. World J Microbiol Biotechnol 37, 55 (2021). https://doi.org/10.1007/s11274-021-03015-4

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Keywords

  • Biosurfactant
  • Biocontrol
  • Bioformulation
  • Pseudomonas
  • Rhamnolipid