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Simultaneous quantification of phytohormones in fermentation extracts of Botryodiplodia theobromae by liquid chromatography–electrospray tandem mass spectrometry

Abstract

Fermentation broth and biomass from three strains of Botryodiplodia theobromae were characterized by high performance liquid chromatography–electrospray tandem mass spectrometry (HPLC–ESI–MS/MS) method, in order to quantify different phytohormones and to identify amino acid conjugates of jasmonic acid (JA) present in fermentation broths. A liquid–liquid extraction with ethyl acetate was used as sample preparation. The separation was carried out on a C18 reversed-phase HPLC column followed by analysis via ESI–MS/MS. The multiple reaction monitoring mode was used for quantitative measurement. For the first time, indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and JA were identified and quantified in the ethyl acetate extracts from the biomass, after the separation of mycelium from supernatant. The fermentation broths showed significantly higher levels of JA in relation to the other phytohormones. This is the first report of the presence of gibberellic acid, abscisic acid, salicylic acid and the cytokinins zeatin, and zeatin riboside in fermentation broths of Botryodiplodia sp. The presence of JA-serine and JA-threonine conjugates in fermentation broth was confirmed using HPLC-ESI tandem mass spectrometry in negative ionization mode, while the occurrence of JA-glycine and JA-isoleucine conjugates was evidenced with the same technique but with positive ionization. The results demonstrated that the used HPLC–ESI–MS/MS method was effective for analysing phytohormones in fermentation samples.

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Acknowledgments

The authors want to thank the Service of Support to the Experimental Sciences (SACE) of the University of Murcia for providing the HPLC–MS facilities and to Dr. Torrellas for providing the microbiological laboratory facilities; also to Agencia Española de Colaboración Internacional para el Desarrollo (AECID, Spain) for providing a MAEC-AECID Grant to G.C., allowing in this way to make a contribution to the research project AGL2008-02472/AGR Granted by MICINN/FEDER (Spain). The authors are very grateful to Dr. O. Miersch for his critical reading of manuscript. Also, we are grateful to Drs. F. Eng and T. Díaz de Villegas for their helpful assistance in preparing the manuscript.

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Correspondence to Grolamys Castillo.

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Castillo, G., Torrecillas, A., Nogueiras, C. et al. Simultaneous quantification of phytohormones in fermentation extracts of Botryodiplodia theobromae by liquid chromatography–electrospray tandem mass spectrometry. World J Microbiol Biotechnol 30, 1937–1946 (2014). https://doi.org/10.1007/s11274-014-1612-5

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Keywords

  • Jasmonic acid
  • Botryodiplodia theobromae
  • HPLC–ESI–MS/MS
  • Phytohormones
  • Fermentation broth
  • Biomass