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.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
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.
Abdel-Mawgoud A, Lépine F, Déziel E (2010) Rhamnolipids: diversity of structures, microbial origins and roles. Appl Microbiol Biotechnol 86:1323–1336
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
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
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
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
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
Ariech M, Guechi A (2015) Assessment of four different methods for selecting biosurfactant producing extremely halophilic bacteria. Afr J Biotechnol 14(21):1764–1772
Arnow LE (1937) Colorimetric determination of the components of 3,4-dihydroxy-phenylalanine-tyrosine mixtures. J Biol Chem 118:531–537
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
Arora NK, Fatima T, Mishra I, Verma M, Mishra J (2018) Environmental sustainability: challenges and viable solutions. Environ Sustain 1:309–340
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
Botelho GR, Mendonça-Hagler LC (2006) Fluorescent pseudomonads associated with the rhizosphere of crops: an overview. Braz J Microbiol 37(4):401–416
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
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
Cappuccino JG, Sherman N (1992) Microbiology: a laboratory manual. The Benjamin/Cummings Publishing Company Inc., Menlo Park
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
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
Christova N, Tuleva B, Nikolova-Damyanova B (2004) Enhanced hydrocarbon biodegradation by a newly isolated Bacillus subtilis strain. Z Naturforsch 59:205–208
Cooper DG, Goldenberg BG (1987) Surface-active agents from 2 Bacillus species. Appl Environ Microbiol 53:224–229
Cox C, Graham R (1979) Isolation of an iron-binding compound from Pseudomonas aeruginosa. J Bacteriol 137:357–364
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
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
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
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
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
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
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
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
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
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
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
Jarvis FG, Johnson MJ (1949) A glycolipid produced by Pseudomonas aeruginosa. J Am Chem Soc 71:4124–4126
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
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
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
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
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
Kiefer J, Radzuan M, Winterburn J (2017) Infrared spectroscopy for studying structure and aging effects in rhamnolipid biosurfactants. Appl Sci 7(5):533
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
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
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
Maidak BL, Olsen GL, Larsen N, Overbeek R, McCaughey MJ, Woese CR (1997) The ribosomal database project. Nucleic Acids Res 24:82–85
Meyer JM, Abdallah MA (1978) The fluorescent pigment of Pseudomonas fluorescens: biosynthesis, purification and physicochemical properties. J Gen Microbiol 107(2):319–328
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
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
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
Moreno R, Rojo F (2014) Features of pseudomonads growing at low temperatures: another facet of their versatility. Environ Microbiol Rep 6(5):417–426
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
Mukherjee S, Das P, Sen R (2009) Rapid quantification of a microbial surfactant by a simple turbidometric method. J Microbiol Methods 76:38–42
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
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
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
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
Pamp SJ, Tolker-Nielsen T (2007) Multiple roles of biosurfactants in structural biofilm development by Pseudomonas aeruginosa. J Bacteriol 189(6):2531–2539
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
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
Penrose DM, Glick BR (2003) Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plant 118:10–15
Pereg L, de-Bashan LE, Bashan Y (2016) Assessment of affinity and specificity of Azospirillum for plants. Plant Soil 399:389–414
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
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
Pikovskaya RI (1948) Mobilization of phosphorous in soil in connection with vital activity of some microbial species. Mikrobiologiya 17:363–370
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
Prabhukarthikeyan SR, Raguchander T (2016) Antifungal metabolites of Pseudomonas fluorescens against Pythium aphanidermatum. J Pure Appl Microbiol 10(1):579–584
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
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
Sachdev DP, Cameotra SS (2013) Biosurfactants in agriculture. Appl Microbiol Biotechnol 97(3):1005–1016
Saitou N, Nei M (1987) The Neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Harbor Laboratory Press, Cold Spring Harbor
Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56
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
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
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
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
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
Tahzibi A, Kamal F, Assadi MM (2004) Improved production of rhamnolipids by a Pseudomonas aeruginosa mutant. Iran Biomed J 8(1):25–31
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
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
Turkovskaya OV, Dmitrieva TV, Yu Muratova A (2001) A biosurfactant-producing Pseudomonas aeruginosa strain. Appl Biochem Microbiol 37(1):71–75
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
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
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
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).
Conflict of interest
The authors declare that they have no conflict of interest.
Consent to participate
Both the authors are involved in experimentation and manuscript writing.
Consent for publication
Both the authors give consent for the publication of manuscript in “Biocontrol” and it is not been submitted anywhere else.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Below is the link to the electronic supplementary material.
About this article
Cite this article
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