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A resourceful methodology to profile indolic auxins produced by rhizo-fungi using spectrophotometry and HPTLC

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Abstract

Plant growth-promoting fungi play an important role in development of sustainable agriculture. In the current study, 13 fungal strains were isolated from the rhizosphere of healthy Triticum aestivum (wheat) plant and screened for their indolic auxin production potential. Aspergillus flavus strain PGFW, Aspergillus niger strain BFW and Aspergillus caespitosus strain DGFW were amongst the most efficient indolic auxin-producing strains. Indolic auxins such as indole 3 acetate (IAA), indole 3 butyrate (IBA) and indole 3 propionate (IPA) are produced by fungi. The conventional method to assess the IAA production is through a spectrophotometric assay using Salkowski’s reagent, which quantifies all indolic auxins and not individual auxins. Moreover, it was also observed that the absorption maxima (λmax) of the samples, when compared to that of standard indole-3-acetic acid, showed deviation from the latter, indicative of production of a mixture of indolic derivatives by the fungi. Hence, for further profiling of these indolic compounds, high-performance thin layer chromatography (HPTLC) based protocol was standardized to precisely detect and quantify individual indolic auxins like IAA, IBA and IPA in the range of 100–1000 ng per spot. HPTLC analysis also showed that the fungal strains produce different indolic auxins in media with and without fortification of tryptophan, with the production of indolic auxins being enhanced in presence of tryptophan. Thus, this standardized HPTLC protocol is an efficient and sensitive methodology to separate and quantify the indolic derivatives.

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References

  • Afzal I, Iqrar I, Shinwari ZK, Yasmin A (2017) Plant growth-promoting potential of endophytic bacteria isolated from roots of wild Dodonaea viscosa L. J Plant Growth Regul 81(3):399–408

    Article  CAS  Google Scholar 

  • Ahemad M, Kibret M (2014) Mechanisms and applications of plant growth promoting rhizobacteria: current perspective. J King Saud Univ Sci 26(1):1–20

    Article  Google Scholar 

  • Ahmad F, Ahmad I, Khan MS (2008) Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol Res 163(2):173–181

    Article  CAS  Google Scholar 

  • Angel SMM, Flores MAS, Badillo MGC, Saavedra MTR, Osuna MAI, Flores SC (2011) The plant growth-promoting fungus Aspergillus ustus promotes growth and induces resistance against different lifestyle pathogens in Arabidopsis thaliana. J Microbiol Biotechnol 21(7):686–696

    Article  Google Scholar 

  • Arora NK, Verma M (2017) Modified microplate method for rapid and efficient estimation of siderophore produced by bacteria. 3 Biotech 7(6):381

    Article  Google Scholar 

  • Barazani OZ, Friedman J (2000) Effect of exogenously applied l-tryptophan on allelochemical activity of plant-growth-promoting rhizobacteria (PGPR). ‎J Chem Ecol 26(2):343–349

    Article  CAS  Google Scholar 

  • Beni A, Soki E, Lajtha K, Fekete I (2014) An optimized HPLC method for soil fungal biomass determination and its application to a detritus manipulation study. J Microbiol Methods 103:124–130

    Article  CAS  Google Scholar 

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

    Google Scholar 

  • Carreno-Lopez R, Campos-Reales N, Elmerich C, Baca BE (2000) Physiological evidence for differently regulated tryptophan-dependent pathways for indole-3-acetic acid synthesis in Azospirillum brasilense. Mol Gen Genet 264(4):521–530

    Article  CAS  Google Scholar 

  • Cassán F, Vanderleyden J, Spaepen S (2014) Physiological and agronomical aspects of phytohormone production by model plant growth promoting rhizobacteria (PGPR) belonging to the genus Azospirillum. J Plant Growth Regul 33(2):440–459

    Article  Google Scholar 

  • Chanclud E, Morel JB (2016) Plant hormones: a fungal point of view. Mol Plant Pathol 17(8):1289–1297

    Article  Google Scholar 

  • De Battista JP, Bacon CW, Severson R, Plattner RD, Bouton JH (1990) Indole acetic acid production by the fungal endophyte of tall fescue. Agron J 82(5):878–880

    Article  Google Scholar 

  • De Palma M, D’Agostino N, Proietti S, Bertini L, Lorito M, Ruocco M, Tucci M (2016) Suppression subtractive hybridization analysis provides new insights into the tomato (Solanum lycopersicum L.) response to the plant probiotic microorganism Trichoderma longibrachiatum MK1. J Plant Growth Regul 190:79–94

    Google Scholar 

  • Dhandhukia PC, Thakkar VR (2008) Separation and quantitation of jasmonic acid using HPTLC. J Chromatogr Sci 46(4):320–324

    Article  CAS  Google Scholar 

  • Dhandhukia PC, Thakker JN (2011) Quantitative analysis and validation of method using HPTLC. In: Srivastava M (ed) High-performance thin-layer chromatography (HPTLC). Springer, Berlin, pp 203–221

    Chapter  Google Scholar 

  • Etesami H, Alikhani HA, Hosseini HM (2015) Indole 3 acetic acid (IAA) production trait, a useful screening to select endophytic and rhizosphere competent bacteria for rice growth promoting agents. MethodsX 2:72 78

    Article  Google Scholar 

  • Glickmann E, Dessaux Y (1995) A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Appl Environ Microbiol 61(2):793–796

    CAS  PubMed  PubMed Central  Google Scholar 

  • Goswami D, Vaghela H, Parmar S, Dhandhukia P, Thakker JN (2013) Plant growth promoting potentials of Pseudomonas spp. strain OG isolated from marine water. J Plant Interact 8(4):281–290

    Article  CAS  Google Scholar 

  • Goswami D, Pithwa S, Dhandhukia P, Thakker JN (2014) Delineating Kocuria turfanensis 2M4 as a credible PGPR: a novel IAA-producing bacterium isolated from saline desert. J Plant Interact 9(1):566–576

    Article  CAS  Google Scholar 

  • Goswami D, Thakker JN, Dhandhukia PC (2015) Simultaneous detection and quantification of indole 3 acetic acid (IAA) and indole 3 butyric acid (IBA) produced by rhizofungi from l tryptophan (TRP) using HPTLC. J Microbiol Methods 110:7–14

    Article  CAS  Google Scholar 

  • Goswami D, Thakker JN, Dhandhukia PC (2016) Portraying mechanics of plant growth promoting rhizobacteria (PGPR): a review. Cogent Food Agric 2(1):1127500. https://doi.org/10.1080/23311932.2015.1127500

    Article  CAS  Google Scholar 

  • Gravel V, Antoun H, Tweddell RJ (2007) Growth stimulation and fruit yield improvement of greenhouse tomato plants by inoculation with Pseudomonas putida or Trichoderma atroviride: possible role of indole acetic acid (IAA). Soil Biol Biochem 39(8):1968–1977

    Article  CAS  Google Scholar 

  • Hansda A, Kumar V (2017) Cu-resistant Kocuria sp. CRB15: a potential PGPR isolated from the dry tailing of Rakha copper mine. 3 Biotech 7(2):132

    Article  Google Scholar 

  • Hartmann A, Singh M, Klingmuller W (1983) Isolation and characterization of Azospirillum mutants excreting high amounts of indole acetic acid. Can J Microbiol 29:916–923

    Article  CAS  Google Scholar 

  • Hayat R, Ali S, Amara U, Khalid R, Ahmed I (2010) Soil beneficial bacteria and their role in plant growth promotion: a review. Ann Microbiol 60(4):579–598

    Article  Google Scholar 

  • Idris EE, Iglesias DJ, Talon M, Borriss R (2007) Tryptophan-dependent production of indole-3-acetic acid (IAA) affects level of plant growth promotion by Bacillus amyloliquefaciens. Mol Plant Microbe Interact FZB42(6):619–626 20(

    Article  Google Scholar 

  • Jasim B, Jimtha John C, Shimil V, Jyothis M, Radhakrishnan EK (2014) Studies on the factors modulating indole-3-acetic acid production in endophytic fungal isolates from Piper nigrum and molecular analysis of ipdc gene. J Appl Microbiol 117(3):786–799

    Article  CAS  Google Scholar 

  • Karnwal A (2009) Production of indole acetic acid by fluorescent Pseudomonas in the presence of l tryptophan and rice root exudates. J Plant Pathol Microbiol 91:61–63

    CAS  Google Scholar 

  • Khati P, Bhatt P, Kumar R, Sharma A (2018) Effect of nanozeolite and plant growth promoting rhizobacteria on maize. 3 Biotech 8(3):141

    Article  Google Scholar 

  • Manici LM, Kelderer M, Caputo F, Mazzola M (2015) Auxin-mediated relationships between apple plants and root inhabiting fungi: impact on root pathogens and potentialities of growth-promoting populations. Plant Pathol 64(4):843–851

    Article  CAS  Google Scholar 

  • Maor R, Haskin S, Levi-Kedmi H, Sharon A (2004) In planta production of indole-3-acetic acid by Colletotrichum gloeosporioides f. sp. aeschynomene. Appl Environ Microbiol 70(3):1852–1854

    Article  CAS  Google Scholar 

  • Martínez-Morales LJ, Soto-Urzúa L, Baca BE, Sánchez-Ahédo JA (2003) Indole-3-butyric acid (IBA) production in culture medium by wild strain Azospirillum brasilense. FEMS Microbiol Lett 228(2):167–173

    Article  Google Scholar 

  • Meiners SJ, Phipps KK, Pendergast TH, Canam T, Carson WP (2017) Soil microbial communities alter leaf chemistry and influence allelopathic potential among coexisting plant species. Oecologia 203(4):1155–1165

    Article  Google Scholar 

  • Mohite B (2013) Isolation and characterization of indole acetic acid (IAA) producing bacteria from rhizospheric soil and its effect on plant growth. J Soil Sci Plant Nutr 13(3):638–649

    Google Scholar 

  • Mondal G, Dureja P (2000) Fungal metabolites from Aspergillus niger AN27 related to plant growth promotion. Indian J Exp Biol 38:84–87

    CAS  PubMed  Google Scholar 

  • Patel T, Saraf M (2017) Biosynthesis of phytohormones from novel rhizobacterial isolates and their in vitro plant growth-promoting efficacy. J Plant Interact 12(1):480–487

    Article  CAS  Google Scholar 

  • Pérez Montaño F, Alías Villegas C, Bellogín RA, Del Cerro P, Espuny MR, Jiménez Guerrero I, Cubo T (2014) Plant growth promotion in cereal and leguminous agricultural important plants: from microorganism capacities to crop production. Microbiol Res 169(5):325–336

    Article  Google Scholar 

  • Robinson M, Riov J, Sharon A (1998) Indole 3 acetic acid biosynthesis in Colletotrichum gloeosporioides f. sp. aeschynomene. Appl Environ Microbiol 64(12):5030–5032

    CAS  PubMed  PubMed Central  Google Scholar 

  • Shameer S, Prasad TNVKV (2018) Plant growth promoting rhizobacteria for sustainable agricultural practices with special reference to biotic and abiotic stresses. J Plant Growth Regul 84:1–13

    Article  Google Scholar 

  • Shawky E (2013) Determination of synephrine and octopamine in bitter orange peel by HPTLC with densitometry. J Chromatogr Sci 52(8):899–904

    Article  Google Scholar 

  • Sivasakthi S, Usharani G, Saranraj P (2014) Biocontrol potentiality of plant growth promoting bacteria (PGPR)-Pseudomonas fluorescens and Bacillus subtilis: a review. Afr J Agric Res 9(16):1265–1277

    Google Scholar 

  • Swain MR, Naskar SK, Ray RC (2007) Indole 3 acetic acid production and effect on sprouting of yam (Dioscorea rotundata L.) minisetts by Bacillus subtilis isolated from culturable cowdung microflora. Pol J Microbiol 56(2):103

    CAS  PubMed  Google Scholar 

  • Szkop M, Bielawski W (2013) A simple method for simultaneous RP-HPLC determination of indolic compounds related to fungal biosynthesis of indole 3 acetic acid. Antonie Van Leeuwenhoek J Microbiol 103(3):683 691

    Article  Google Scholar 

  • Szkop M, Sikora P, Orzechowski S (2012) A novel, simple, and sensitive colorimetric method to determine aromatic amino acid aminotransferase activity using the Salkowski reagent. Folia microbiol 57(1):1–4

    Article  CAS  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. ‎Mol Biol Evol 24(8):1596–1599

    Article  CAS  Google Scholar 

  • Vassilev N, Malusa E, Requena AR, Martos V, López A, Maksimovic I, Vassileva M (2017) Potential application of glycerol in the production of plant beneficial microorganisms. J Ind Microbiol Biotechnol 44:735–743

    Article  CAS  Google Scholar 

  • Wani SH, Kumar V, Shriram V, Sah SK (2016) Phytohormones and their metabolic engineering for abiotic stress tolerance in crop plants. Crop J 4(3):162–176

    Article  Google Scholar 

Download references

Acknowledgements

Authors are thankful to the Gujarat State Biotechnology Mission (GSBTM) for providing the funding under FAP 2016 GSBTM/MD/PROJECTS/SSA/5041/2016-17 project and St. Xavier’s College Ahmedabad for providing necessary facilities.

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Authors and Affiliations

  1. Department of Biotechnology and Microbiology, Parul Institute of Applied Science and Research, Parul University, Bopal Guma Road, Ahmedabad, Gujarat, 380058, India

    Dhavalkumar Patel & Dhaval Acharya

  2. Department of Biochemistry and Biotechnology, St. Xavier’s College (Autonomous), Ahmedabad, Gujarat, 380009, India

    Anoshi Patel, Disha Vora, Sudeshna Menon, Sebastian Vadakan & Dweipayan Goswami

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  1. Dhavalkumar Patel
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  2. Anoshi Patel
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Correspondence to Dweipayan Goswami.

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Patel, D., Patel, A., Vora, D. et al. A resourceful methodology to profile indolic auxins produced by rhizo-fungi using spectrophotometry and HPTLC. 3 Biotech 8, 413 (2018). https://doi.org/10.1007/s13205-018-1428-3

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