Antifungal potential of bacterial rhizosphere isolates associated with three ethno-medicinal plants (poppy, chamomile, and nettle)

  • Marija Mojicevic
  • Paul M. D’Agostino
  • Jasmina Nikodinovic-Runic
  • Branka Vasiljevic
  • Tobias A.M. Gulder
  • Sandra VojnovicEmail author
Original Article


The objective of the present study was to isolate Actinobacteria, preferably Streptomyces spp. from the rhizosphere soils of three ethno-medicinal plants collected in Serbia (Papaver rhoeas, Matricaria chamomilla, and Urtica dioica) and to screen their antifungal activity against Candida spp. Overall, 103 sporulating isolates were collected from rhizosphere soil samples and determined as Streptomyces spp. Two different media and two extraction procedures were used to facilitate identification of antifungals. Overall, 412 crude cell extracts were tested against Candida albicans using disk diffusion assays, with 42% (43/103) of the strains showing the ability to produce antifungal agents. Also, extracts inhibited growth of important human pathogens: Candida krusei, Candida parapsilosis, and Candida glabrata. Based on the established degree and range of antifungal activity, nine isolates, confirmed as streptomycetes by 16S rRNA sequencing, were selected for further testing. Their ability to inhibit Candida growth in liquid culture, to inhibit biofilm formation, and to disperse pre-formed biofilms was assessed with active concentrations from 8 to 250 μg/mL. High-performance liquid chromatographic profiles of extracts derived from selected strains were recorded, revealing moderate metabolic diversity. Our results proved that rhizosphere soil of ethno-medicinal plants is a prolific source of streptomycetes, producers of potentially new antifungal compounds.


Antifungal activity Soil isolates Ethno-medicinal plants Streptomyces Screening 



PM D’Agostino would like to thank the TUM University Foundation Fellowship for funding. TAM Gulder thanks the DFG for further funding (GU 1233/1-1 and CIPSM).

Funding information

This work was supported by the Ministry of Education, Science and Technological Development of Serbia (Grant No 173048) and the DAAD (Deutscher Akademischer Austauschdienst, Bilateral Project with Republic of Serbia to J Nikodinović-Runic and TAM Gulder – 2016/2017).

Compliance with ethical standards

This manuscript does not contain human studies or experiments using animals.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10123_2019_54_MOESM1_ESM.docx (33 kb)
ESM 1 (DOCX 33 kb)


  1. Alimuddin A, Widada J, Asmara W, Mustofa M (2011) Antifungal production of a strain of Actinomycetes spp isolated from the rhizosphere of cajuput plant: selection and detection of exhibiting activity against tested fungi. I J Biotech 16:1–10. Google Scholar
  2. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefGoogle Scholar
  3. Bode HB, Bethe B, Hofs R, Zeeck A (2002) Big effects from small changes: possible ways to explore nature’s chemical diversity. Chembiochem 3:619–627.<619::AID-CBIC619>3.0.CO;2-9 CrossRefGoogle Scholar
  4. Capoor MR, Nair D, Deb M, Verma PK, Srivastava L, Aggarwal P (2005) Emergence of non-albicans Candida species and antifungal resistance in a tertiary care hospital. Jpn J Infect Dis 58:344–348Google Scholar
  5. Castillo U, Harper J, Strobel G, Sears J, Alesi K, Ford E, Lin J, Hunter M, Maranta M, Ge H, Yaver D, Jensen J, Porter H, Robison R, Millar D, Hess W, Condron M, Teplow D (2003) Kakadumycins, novel antibiotics from Streptomyces sp NRRL 30566, an endophyte of Grevillea pteridifolia. FEMS Microbiol Lett 29:183–190. CrossRefGoogle Scholar
  6. Castillo U, Strobel G, Ford E, Hess W, Porter H, Jensen J, Albert H, Robison R, Condron M, Teplow D, Stevens D, Yaver D (2002) Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562, endophytic on Kennedia nigriscans. Microbiology 148:2675–2685. CrossRefGoogle Scholar
  7. Challis GL, Hopwood DA (2003) Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc Natl Acad Sci U S A 100(Suppl 2):14555–14561. CrossRefGoogle Scholar
  8. Clinical and Laboratory Standards Institute (2008) Reference method for broth dilution antifungal susceptibility testing of yeasts—third edition: approved standard M27-A3. CLSI W, PA, USAGoogle Scholar
  9. Clinical and Laboratory Standards Institute. (2012) Reference method for broth dilution antifungal susceptibility testing of yeasts: Fourth informational supplement M27-S4. CLSI W, PA, USA.Google Scholar
  10. Cordova-Davalos LE, Escobedo-Chavezávez KG, Evangelista-Martínez Z (2017) Inhibition of Candida albicans cell growth and biofilm formation by a bioactive extract produced by soil Streptomyces strain GCAL-25. Arch Biol Sci 70:387–396. CrossRefGoogle Scholar
  11. Crusemann M, O’Neill EC, Larson CB, Melnik AV, Floros DJ, da Silva RR, Jensen PR, Dorrestein PC, Moore BS (2017) Prioritizing natural product diversity in a collection of 146 bacterial strains based on growth and extraction protocols. J Nat Prod 80:588–597.
  12. Djokic L, Narancic T, Nikodinovic-Runic J, Savic M, Vasiljevic B (2011) Isolation and characterization of four novel Gram-positive bacteria associated with the rhizosphere of two endemorelict plants capable of degrading a broad range of aromatic substrates. Appl Microbiol Biotechnol 91:1227–1238. CrossRefGoogle Scholar
  13. Fguira L, Bejar S, Mellouli L (2012) Isolation and screening of Streptomyces from soil of Tunisian oases ecosystem for nonpolyenic antifungal metabolites. Afr J Biotechnol 11:7512–7519. Google Scholar
  14. Genilloud O (2017) Actinomycetes: still a source of novel antibiotics. Nat Prod Rep 34:1203–1232. CrossRefGoogle Scholar
  15. Golinska P, Wypij M, Agarkar G, Rathod D, Dahm H, Rai M (2015) Endophytic actinobacteria of medicinal plants: diversity and bioactivity. Antonie van Leeuwenhoek 108:267–289. CrossRefGoogle Scholar
  16. Hamid ME, Assiry MM, Joseph MR, Haimour WO, Abdelrahim IM, Al-Abed F, Fadul AN, Al-Hakami AM (2014) Candida and other yeasts of clinical importance in Aseer region, southern Saudi Arabia. Presentation of isolates from the routine laboratory setting. Saudi Med J 35:1210–1214Google Scholar
  17. Hartmann A, Rothballer M, Schmid M (2008) Lorenz Hiltner, a pioneer in rhizosphere microbial ecology and soil bacteriology research. Plant Soil 312:7–14. CrossRefGoogle Scholar
  18. Ikeda H, Ishikawa J, Hanamoto A, Shinose M, Kikuchi H, Shiba T, Sakaki Y, Hattori M, Ōmura S (2003) Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotech 21:526–531. CrossRefGoogle Scholar
  19. Ilic-Tomic T, Genčić MS, Živković MZ, Vasiljevic B, Djokic L, Nikodinovic-Runic J, Radulovic NS (2015) Structural diversity and possible functional roles of free fatty acids of the novel soil isolate Streptomyces sp. NP10. Appl Microbiol Biotechnol 99:4815–4833CrossRefGoogle Scholar
  20. Ilic S, Konstantinovic S, Todorovic Z (2005) UV/VIS analysis and antimicrobial activity of Streptomyces isolates. F U Med Biol 12:44–46Google Scholar
  21. Jog R, Pandya M, Nareshkumar G, Rajkumar S (2014) Mechanism of phosphate solubilization and antifungal activity of Streptomyces spp. isolated from wheat roots and rhizosphere and their application in improving plant growth. Microbiology 160:778–788. CrossRefGoogle Scholar
  22. Khamna S, Yokota A, Lumyong S (2009a) Actinomycetes isolated from medicinal plant rhizosphere soils: diversity and screening of antifungal compounds, indole-3-acetic acid and siderophore production. World J Microbiol Biotechnol 25:649–655. CrossRefGoogle Scholar
  23. Khamna S, Yokota A, Peberdy J, Lumyong S (2009b) Antifungal activity of Streptomyces spp. isolated from rhizosphere of Thai medicinal plants. Int J Integr Biol 6:143–147 Google Scholar
  24. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces Genetics. John Innes Foundation, Norwich, UK.Google Scholar
  25. Kostiala AAI, Kostiala I (1984) Broth dilution and disc diffusion methods in the susceptibility testing of pathogenic Candida albicans against four antimycotics. Mycopathologia 87(1–2):121–127. CrossRefGoogle Scholar
  26. Köberl M, Schmidt R, Ramadan E, Bauer R, Berg G (2013) The microbiome of medicinal plants: diversity and importance for plant growth, quality and health. Front Microbiol 4:400. CrossRefGoogle Scholar
  27. Kumar D, Bhattacharyya S, Gupta P, Banerjee G, Singh M (2015) Comparative analysis of disc diffusion and E-test with broth micro-dilution for susceptibility testing of clinical Candida isolates against amphotericin B, fluconazole, voriconazole and caspofungin. J Clin Diagn Res 9(11):DC01–DC04Google Scholar
  28. Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. CrossRefGoogle Scholar
  29. Larkin M, Blackshields G, Brown N, Chenna R, McGettigan P, McWilliam H, Valentin F, Wallace I, Wilm A, Lopez R, Thompson J, Gibson T, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948. CrossRefGoogle Scholar
  30. Maleki H, Dehnad A, Hanifian S, Khani S (2013) Isolation and molecula identification of Streptomyces spp. with antibacterial activity from Northwest of Iran. Bioimpacts 3:129–134. Google Scholar
  31. Mellouli L, Ben Ameur-Mehdi R, Sioud S, Salem M, Bejar S (2003) Isolation, purification and partial characterization of antibacterial activities produced by a newly isolated Streptomyces sp. US24 strain. Res Microbiol 154:345–352. CrossRefGoogle Scholar
  32. Nikodinovic J, Barrow KD, Chuck JA (2003) High yield preparation of genomic DNA from Streptomyces. BioTechniques 35:932–936CrossRefGoogle Scholar
  33. Pierce CG, Uppuluri P, Tristan AR, Wormley FL Jr, Mowat E, Ramage G, Lopez-Ribot JL (2008) A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc 3:1494–1500. CrossRefGoogle Scholar
  34. Reysenbach AL, Wickham GS, Pace NR (1994) Phylogenetic analysis of the hyperthermophilic pink filament community in Octopus Spring, Yellowstone National Park. Appl Environ Microbiol 60:2113–2119Google Scholar
  35. Rios JL, Recio MC (2005) Medicinal plants and antimicrobial activity. J Ethnopharmacol 100:80–84. CrossRefGoogle Scholar
  36. Schrey SD, Erkenbrack E, Früh E, Fengler S, Hommel K, Horlacher N, Schulz D, Ecke M, Kulik A, Fiedler H-P, Hampp R, Tarkka MT (2012) Production of fungal and bacterial growth modulating secondary metabolites is widespread among mycorrhiza-associated streptomycetes. BMC Microbiol 12:164–164. CrossRefGoogle Scholar
  37. Sheir DH, Hafez MA (2017) Antibiofilm activity of Streptomyces toxytricini Fz94 against Candida albicans ATCC 10231. Microbial Biosystems 2:26–39. CrossRefGoogle Scholar
  38. Spasic J, Mandic M, Radivojevic J, Jeremic S, Vasiljevic B, Nikodinovic-Runic J, Djokic L (2018) Biocatalytic potential of Streptomyces spp. isolates from rhizosphere of plants and mycorrhizosphere of fungi. Biotechnol Appl Biochem.
  39. Stamenov D, Djuric S, Hajnal Jafari T, Ćirić V, Manojlovic M (2018) Microbiological activity in the soil of various agricultural crops in organic production. Contemp Agr 67:34–39. CrossRefGoogle Scholar
  40. Stankovic N, Senerovic L, Bojic-Trbojevic Z, Vuckovic I, Vicovac L, Vasiljevic B, Nikodinovic-Runic J (2013) Didehydroroflamycoin pentaene macrolide family from Streptomyces durmitorensis MS405T: production optimization and antimicrobial activity. J Appl Microbiol 115:1297–1306. CrossRefGoogle Scholar
  41. Thakur D, Yadav A, Gogoi BK, Bora TC (2007) Isolation and screening of Streptomyces in soil of protected forest areas from the states of Assam and Tripura, India, for antimicrobial metabolites. J Myc Méd 17:242–249. CrossRefGoogle Scholar
  42. Vartak A, Mutalik V, Parab RR, Shanbhag P, Bhave S, Mishra PD, Mahajan GB (2014) Isolation of a new broad spectrum antifungal polyene from Streptomyces sp. MTCC 5680. Lett Appl Microbiol 58:591–596. CrossRefGoogle Scholar
  43. Vicente MF, Basilio A, Cabello A, Peláez F (2003) Microbial natural products as a source of antifungals. Clin Microbiol Infect 9:15–32. CrossRefGoogle Scholar
  44. Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer K-H, Whitman W (2009) Bergey’s Manual of Systematic Bacteriology. Volume 3: The Firmicutes. Second edition. Springer-Verlag New York, New York, USA.Google Scholar
  45. Waksman SA, Henrici AT (1943) The nomenclature and classification of the Actinomycetes. J Bacteriol 46:337–341Google Scholar
  46. Williams ST, Mayfield CI (1971) Studies on the ecology of actinomycetes in soil III. The behaviour of neutrophilic streptomycetes in acid soil. Soil Biol Biochem 3:197–208. CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Institute of Molecular Genetics and Genetic EngineeringUniversity of BelgradeBelgradeSerbia
  2. 2.Department of Biotechnology and Pharmaceutical Engineering, Faculty of TechnologyUniversity of Novi SadNovi SadSerbia
  3. 3.Biosystems Chemistry, Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM)Technical University of MunichMunichGermany

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