Metabolic profiling of Fusarium oxysporum f. sp. conglutinans race 2 in dual cultures with biocontrol agents Bacillus amyloliquefaciens, Pseudomonas aeruginosa, and Trichoderma harzianum

Abstract

There are increasing efforts to identify biocontrol-active microbial metabolites in order to improve strategies for biocontrol of phytopathogens. In this work, Fusarium oxysporum f. sp. conglutinans was confronted with three different biocontrol agents: Trichoderma harzianum, Bacillus amyloliquefaciens, and Pseudomonas aeruginosa in dual culture bioassays. Metabolites produced during the microbial interactions were screened by a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). T. harzianum exhibited the strongest inhibition of growth of F. oxysporum resulting in overlay of the pathogen colony with its mycelium. Recorded metabolite profiles suggested a direct attack of F. oxysporum mycelium by T. harzianum and B. amyloliquefaciens by means of membrane-attacking peptaibols and a set of antimicrobial lipopeptides and siderophores, respectively. The direct mode of the biocontrol activity of T. harzianum and B. amyloliquefaciens corresponded to their ability to suppress F. oxysporum production of mycotoxin beauvericin suggesting that this ability is not specific only for Trichoderma species. In the case of P. aeruginosa, siderophores pyoverdine E/D and two rhamnolipids were produced as major bacterial metabolites; the rhamnolipid production was blocked by F. oxysporum. The results showed that this type of biocontrol activity was the least effective against F. oxysporum. The effective application of MALDI-MS profiling to the screening of nonvolatile microbial metabolites produced during the interaction of the phytopathogen and the biocontrol microorganisms was demonstrated.

This is a preview of subscription content, log in to check access.

Fig. 1

References

  1. Angel LPL, Yusof MT, Ismail IS, Ping BTY, Azni INAM, Kamarudin NHJ, Sundram S (2016) An in vitro study of the antifungal activity of Trichoderma virens 7b and a profile of its non-polar antifungal components released against Ganoderma boninense. J Microbiol 54:732–744. https://doi.org/10.1007/s12275-016-6304-4

    CAS  Article  PubMed  Google Scholar 

  2. Aoki T, O’Donnell K, Geiser DM (2014) Systematics of key phytopathogenic Fusarium species: current status and future challenges. J Gen Plant Pathol 80:189–201. https://doi.org/10.1007/s10327-014-0509-3

    CAS  Article  Google Scholar 

  3. Ben Ayed H, Hmidet N, Bechet M, Jacques P, Nasri M (2017) Identification and natural functions of cyclic lipopeptides from Bacillus amyloliquefaciens An6. Eng Life Sci 17:536–544. https://doi.org/10.1002/elsc.201600050

    CAS  Article  Google Scholar 

  4. Benitez T, Rincon AM, Limon MC, Codon AC (2004) Biocontrol mechanisms of Trichoderma strains. Int Microbiol 7:249–260 www.im.microbios.org

    CAS  PubMed  Google Scholar 

  5. Blaszszyk L, Basinska-Barczak A, Cwiek-Kupczynska H, Gromadzka K, Popiel D, Stepien L (2017) Suppressive effect of Trichoderma spp. on toxigenic Fusarium species. Pol J Microbiol 66:85–100

    Article  Google Scholar 

  6. Caldeira AT, Santos Arteiro JM, Coelho AV, Roseiro JC (2011) Combined use of LC-ESI-MS and antifungal tests for rapid identification of bioactive lipopeptides produced by Bacillus amyloliquefaciens CCMI1051. Process Biochem 46:1738–1746. https://doi.org/10.1016/j.procbio.2011.05.016

    CAS  Article  Google Scholar 

  7. Cezard C, Farvacques N, Sonnet P (2015) Chemistry and biology of pyoverdines, Pseudomonas primary siderophores. Curr Med Chem 22:165–186

    CAS  Article  Google Scholar 

  8. DaSilva Araujo FD, Araujo WL, Eberlin MN (2017) Potential of Burkholderia seminalis TC3.4.2R3 as biocontrol agent against Fusarium oxysporum evaluated by mass spectrometry imaging. J Am Soc Mass Spectrom 28:901–907. https://doi.org/10.1007/s13361-017-1610-6

    CAS  Article  Google Scholar 

  9. Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J, Foster GD (2012) The top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13:414–430. https://doi.org/10.1111/J.1364-3703.2011.00783.X

    Article  PubMed  PubMed Central  Google Scholar 

  10. Dhokane D, Karre S, Kushsalppa AC, McCartney C (2016) Integrated metabolo-transcriptomics reveals Fusarium head blight candidate resistance genes in wheat QTL-Fhb2. PLoS One 11(5):e0155851. https://doi.org/10.1371/Journal.pone.0155851

    Article  PubMed  PubMed Central  Google Scholar 

  11. Gu Q, Yang Y, Yuan QM, Shi GM, Wu LM, Lou ZY, Huo R, Wu HJ, Borriss R, Gao XW (2017) Bacillomycin D produced by Bacillus amyloliquefaciens is involved in the antagonistic interaction with the plant-pathogenic fungus Fusarium graminearum. Appl Environ Microbiol 83:UNSP e01075–UNSP e01017. https://doi.org/10.1128/AEM.01075-17

    Article  Google Scholar 

  12. Gunnaiah R, Kushalappa AC (2014) Metabolomics deciphers the host resistance mechanisms in wheat cultivar Sumai-3 against trichothecene producing and non-producing isolates of Fusarium graminearum. Plant Physiol Biochem 83:40–50. https://doi.org/10.1016/j.plaphy.2014.07.002

    CAS  Article  PubMed  Google Scholar 

  13. Haba E, Pinazo A, Jauregui O, Espuny MJ, Infante MR, Manresa A (2003) Physicochemical characterization and antimicrobial properties of rhamnolipids produced by Pseudomonas aeruginosa 47T2 NCBIM 40044. Biotechnol Bioeng 81:316–322. https://doi.org/10.1002/bit.10474

    CAS  Article  PubMed  Google Scholar 

  14. Hof C, Eisfeld K, Welzel K, Antelo L, Foster AJ, Anke H (2007) Ferricrocin synthesis in Magnaportae grisaea and its role in pathogenicity in rice. Mol Plant Pathol 8:163–172. https://doi.org/10.1111/J.1364-2007.00380.X

    CAS  Article  PubMed  Google Scholar 

  15. Kloepper JW, Leong J, Teintze T, Schroth MN (1980) Pseudomonas siderophores: a mechanism explaining disease-suppressive soils. Curr Microbiol 4:317–320. https://doi.org/10.1007/BF02602840

    CAS  Article  Google Scholar 

  16. Lebeau J, Petit T, Clerc P, Dufosse L, Caro Y (2018) Isolation of two novel purple naphthoquinone pigments concomitant with the bioactive red bikaverin and derivatives thereof produced by Fusarium oxysporum. Biotechnol Prog. https://doi.org/10.1002/btpr.2738

    Article  Google Scholar 

  17. Lehner SM, Atanasova L, Neumann NKN, Krska R, Lemmens M, Druzhinina IS, Schuhmacher R (2013) Isotope-assisted screening for iron-containing metabolites reveals a high degree of diversity among known and unknown siderophores produced by Trichoderma spp. Appl Environ Microbiol 79:18–31. https://doi.org/10.1128/AEM.02339-12

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Li B, Li Q, Xu Z, Zhang N, Shen Q, Zhang R (2014) Responses of beneficial Bacillus amyloliquefaciens SQR9 to different soilborne fungal pathogens through the alteration of antifungal compounds production. Front Microbiol 5:636. https://doi.org/10.3389/fmicb.2011.00636

    Article  PubMed  PubMed Central  Google Scholar 

  19. Li E, Wang G, Yang Y, Xiao J, Mao Z, Xie B (2015a) Microscopic nalysis of the compatible and incompatible interactions between Fusarium oxysporum f. sp. conglutinans and cabbage. Eur J Plant Pathol 141:597–609. https://doi.org/10.1007/s10658-014-0567-6

    Article  Google Scholar 

  20. Li E, Ling J, Wang G, Xiao J, Yang Y, Mao Z, Wang X, Xie B (2015b) Comparative proteomics analyses of two races of Fusarium oxysporum f. sp. conglutinans that differ in pathogenicity. Sci Rep-UK 5:13663. https://doi.org/10.1038/srep13663

    Article  Google Scholar 

  21. Li WB, Li CQ, Sun JB, Peng M (2017) Metabolomic, biochemical and gene expression analyses reveal the underlying responses of resistant and susceptible banana species during early infections with Fusarium oxysporum f. sp. cubense. Plant Dis 101:534–543. https://doi.org/10.1094/PDIS-09-16-1245-RE

    CAS  Article  PubMed  Google Scholar 

  22. Ligon JM, Hill SD, Hammer PE, Torkewitz NR, Hofmann D, Kempf HJ, vanPee KH (2000) Natural products with antifungal activity from Pseudomonas biocontrol bacteria. Pest Manag Sci 56:688–695. https://doi.org/10.1002/1526-4998(200008)56:8<688::AID-PS186>3.0.CO;2-V

    CAS  Article  Google Scholar 

  23. Limon MC, Rodríguez- Ortiz R, Avalos J (2010) Bikaverin production and applications. Appl Microbiol Biotechnol 87:21–29. https://doi.org/10.1007/s00253-010-2551-1

    CAS  Article  PubMed  Google Scholar 

  24. Liu S-Y, Liao C-K, Lo C-T, Yang H-H, Lin K-C, Peng K-C (2016) Chrysophenol is involved in the biofertilization and biocontrol activities of Trichoderma. Physiol Mol Plant Pathol 96:1–7. https://doi.org/10.1016/j.pmpp.2016.06.003

    CAS  Article  Google Scholar 

  25. Liu X, Ling J, Xiao Z, Xie B, Fang Z, Yang L, Zhang Y, Lv H, Yang Y (2017) Characterization of emerging populations of Fusarium oxysporum f. sp. conglutinans causing cabbage wilt in China. J Phytopathol 165:813–821. https://doi.org/10.1111/jph.12621

    CAS  Article  Google Scholar 

  26. Liuzzi VC, Mirabelli V, Cimmarusti MT, Haidukowski M, Leslie JF, Logrieco AF, Caliandro R, Fanelli F, Mule G (2017) Enniantin and beauvericin biosynthesis in Fusarium species: production profiles and structural determinant prediction. Toxins 9:45. https://doi.org/10.3390/toxins9020045

    CAS  Article  PubMed Central  Google Scholar 

  27. Lopez-Berges MS, Capilla J, Turra D, Schafferer L, Matthijs S, Joechl C, Cornelis P, Guarro J, Haas H, DiPietro A (2012) HapX-mediated iron homeostasis is essential for rhizosphere competence and virulence of the soilborne pathogen Fusarium oxysporum. Plant Cell 24:3805–3822. https://doi.org/10.1105/tpc.112.098624

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Malmierca MG, Izquirdo-Bueno I, McCormick SP, Cardoza RE, Alexander NJ, Barua J, Lindo L, Casquero PA, Collado IG, Monte E, Gutierrez S (2016) Trichothecenes and aspinolides produced by Trichoderma arundinaceum regulate expression of Botrytis cinerea genes involved in virulence and growth. Environ Microbiol 18:3991–4004. https://doi.org/10.1111/1462-2920.13410

    CAS  Article  PubMed  Google Scholar 

  29. Martin-Rodriguez AJ, Reyes F, Martin J, Perez-Yepez J, Leon-Barrios M, Couttolenc A, Espinoza C, Trigos A, Martin VS, Norte M, Fernandez JJ (2014) Inhibition of bacterial quorum sensing by extracts from aquatic fungi: first report from marine endophytes. Mar Drugs 12:5503–5526. https://doi.org/10.3390/md12115503

    Article  PubMed  PubMed Central  Google Scholar 

  30. Meca G, Sospedra I, Soriano JM, Ritieni A, Moretti A, Manes J (2010) Antibacterial effect of the bioactive compound beauvericin produced by Fusarium proliferatum on solid medium of wheat. Toxicon 6:349–354. https://doi.org/10.1016/j.toxicon.2010.03.022

    CAS  Article  Google Scholar 

  31. Mukherjee PK, Wiest A, Ruiz N, Keightley A, Moran-Diez ME, McCluskey K, Pouchus YF, Kenerley CM (2011) Two classes of new peptaibols are synthesized by a single non-ribosomal peptide synthetase of Trichoderma virens. J Biol Chem 286:4544–4554. https://doi.org/10.1074/jbc.M110.159723

    CAS  Article  PubMed  Google Scholar 

  32. Mutawila C, Vinal F, Halleen F, Lorito M, Mostert L (2016) Isolation, production and in vitro effects of the major secondary metabolite produced by Trichoderma species used for the control of grapevine trunk diseases. Plant Pathol 65:104–113. https://doi.org/10.1111/ppa.12385

    CAS  Article  Google Scholar 

  33. Novák J, Lemr K, Schug KA, Havlíček V (2015) CycloBranch: De novo sequencing of nonribosomal peptides from accurate product ion mass spectra. J Am Soc Mass Spectrom 26:1780–1786. https://doi.org/10.1007/s13361-015-1211-1

    CAS  Article  PubMed  Google Scholar 

  34. Novák J, Sokolová L, Lemr K, Pluháček T, Palyzová A, Havlíček V (2017) Batch-processing of imaging or liquid-chromatography mass spectrometry datasets and de novo sequencing of polyketide siderophores. Biochim Biophys Acta 1865:768–775. https://doi.org/10.1016/j.bbapap.2016.12.003

    CAS  Article  Google Scholar 

  35. Pascale A, Vinale F, Manganiello G, Nigro M, Lanzuise S, Ruocco M, Marra R, Lombardi N, Woo SL, Lorito M (2017) Trichoderma and its secondary metabolites improve yield and quality of grapes. Crop Prot 92:176–181. https://doi.org/10.1016/j.cropro.2016.11.010

    CAS  Article  Google Scholar 

  36. Pathma J, Rahul GR, Kennedy RK, Subashri R, Sakthival N (2011) Secondary metabolite production by bacterial antagonists. J Biol Control 25:165–181

    Google Scholar 

  37. Paulitz TC, Loper JE (1991) Lack of a role for fluorescent siderophore production in the biological control of Pythium damping-off of cucumber by a strain of Pseudomonas putida. Phytopathology 81:930–935. https://doi.org/10.1094/Phyto-81-930

    Article  Google Scholar 

  38. Quecine MC, Kidarsa TA, Goebel NC, Shaffer BT, Henkels MD, Zabriskie TM, Loper JE (2016) An interspecies signaling system mediated by fusaric acid has parallel effects on antifungal metabolite production by Pseudomonas protegens strain Pf-5 and antibiosis of Fusarium spp. Appl Environ Microbiol 82:1372–1382. https://doi.org/10.1128/AEM.02574-15

    CAS  Article  PubMed Central  Google Scholar 

  39. Rajesh PS, Rai VR (2016) Inhibition of QS-regulated virulence factors in Pseudomonas aeruginosa PAO1 and Pectobacterium carotovorum by AHL-lactonase of endophytic bacterium Bacillus cereus VT96. Biocatal Agr Biotechnol 7:154–163. https://doi.org/10.1016/j.bcab.2016.06.003

    Article  Google Scholar 

  40. Reddy KS, Khan MY, Archana K, Reddy MG, Hameeda B (2016) Utilization of mango kernel oil for the rhamnolipid production by Pseudomonas aeruginosa DR1 towards its application as biocontrol agent. Bioresour Technol 221:291–299. https://doi.org/10.1016/j.biortech.2016.09.041

    CAS  Article  Google Scholar 

  41. Romero D, deVicente A, Rakotoaly RH, Dufour SE, Veening JW, Arrebola E, Cazorla FM, Kuipers OP, Paquot M, Perez-Garcia A (2007) The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podospaera fusca. Mol Plant-Microbe Interact 20:430–440. https://doi.org/10.1094/MPMI-20-4-0430

    CAS  Article  PubMed  Google Scholar 

  42. Ruiz JA, Bernar EM, Jung K (2015) Production of siderophores increases resistance to fusaric acid in Pseudomonas protegens Pf-5. PLoS One 10:e0117040. https://doi.org/10.1371/journal.pone.0117040

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Schmidt R, Etalo DW, deJager V, Gerards S, Zweers H, deBoer W, Garbeva P (2016) Microbial small talk: volatiles in fungal-bacterial interactions. Front Microbiol 6:1495. https://doi.org/10.3389/fmicb2015.01495

    Article  PubMed  PubMed Central  Google Scholar 

  44. Sharma V, Salwan R, Sharma PN, Kanwar SS (2017) Elucidation of biocontrol mechanisms of Trichoderma harzianum against different plant fungal pathogens: universal yet host specific response. Int J Biol Macromol 95:72–79. https://doi.org/10.1016/j.ijbiomac.2016.11.042

    CAS  Article  PubMed  Google Scholar 

  45. Shi M, Chen L, Wang X-W, Zhang T, Zhao P-B, Song X-Y, Sun C-Y, Chen X-L, Zhou B-C, Zhang Y-Z (2012) Antimicrobial peptaibols from Trichoderma pseudokoningii induce programmed cell death in plant fungal pathogens. Microbiology 158:166–175. https://doi.org/10.1099/mic.0.052670-0

    CAS  Article  PubMed  Google Scholar 

  46. Singh VK, Singh HB, Upadhyay RS (2017) Role of fusaric acid in the development of Fusarium wilt symptoms in tomato: physiological, biochemical and proteomic perspectives. Plant Physiol Biochem 118:320–332. https://doi.org/10.1016/j.plaphy.2017.06.028

    CAS  Article  PubMed  Google Scholar 

  47. Son SW, Kim HY, Choi GJ, Lim HK, Jang KS, Lee SO, Lee S, Sung ND, Kim J-C (2008) Bikaverin and fusaric acid from Fusarium oxysporum show antioomycete activity against Phytophthora infestans. J Appl Microbiol 104:692–698. https://doi.org/10.1111/j.1365-2672.2007.03581.x

    CAS  Article  PubMed  Google Scholar 

  48. Torres MJ, Brandan CP, Petroselli G, Erra-Balsells R, Audisio MC (2016) Antagonistic effects of Bacillus subtilis subsp. subtilis and B. amyloliquefaciens against Macrophomina phaseolina: SEM study of fungal changes and UV-MALDI-TOF MS analysis of their bioactive compounds. Microbiol Res 182:31–39. https://doi.org/10.1016/j.micres.2015.03.005

    CAS  Article  PubMed  Google Scholar 

  49. Tung TT, Jakobsen TH, Dao TT, Fuglsang AT, Givskov M, Christensen SB, Nielsen J (2017) Fusaric acid and analogues as Gram-negative bacterial quorum sensing inhibitors. Eur J Med Chem 126:1011–1020. https://doi.org/10.1016/j.ejmech.2016.11.044

    CAS  Article  PubMed  Google Scholar 

  50. Wallner A, Blatzer M, Schrettl M, Sarg B, Lindner H, Haas H (2009) Ferricrocin, a siderophore involved in intra- and transcellular iron distribution in Aspergillus fumigatus. Appl Environ Microbiol 75:4194–4196. https://doi.org/10.1128/AEM.00479-09

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Wang Q, Xu L (2012) Beauvericin, a bioactive compound produced by fungi: a short review. Molecules 17:2367–2377. https://doi.org/10.3390/molecules17032367

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. Zhang F, Chen C, Zhang F, Gao L, Liu J, Chen L, Fan X, Liu C, Zhang K, He Y, Chen C, Ji X (2017) Trichoderma harzianum containing 1-aminocyclopropane-1-carboxylate deaminase and chitinase improved growth and diminished adverse effect caused by Fusarium oxysporum in soybean. J Plant Physiol 210:84–94. https://doi.org/10.1016/j.jplph.2016.10.012

    CAS  Article  PubMed  Google Scholar 

Download references

Funding

The work was supported by QJ1510088 project of Ministry of Agriculture of the Czech Republic and LO1509 and LTC18009 projects of the Ministry of Education, Youth and Sports of the Czech Republic.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Čeněk Novotný.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 707 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Palyzová, A., Svobodová, K., Sokolová, L. et al. Metabolic profiling of Fusarium oxysporum f. sp. conglutinans race 2 in dual cultures with biocontrol agents Bacillus amyloliquefaciens, Pseudomonas aeruginosa, and Trichoderma harzianum. Folia Microbiol 64, 779–787 (2019). https://doi.org/10.1007/s12223-019-00690-7

Download citation