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Potential of Zingiber zerumbet endophytic Fusarium oxysporum as biopriming agents to control Pythium mediated soft-rot and optimization of fermentation conditions for cytotoxic metabolite(s) production

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Abstract

Biosynthetic potential of fungal endophytes from medicinal plants represents an attractive source for discovery of new bioactive metabolites. Present study involved characterizing metabolite(s) of non-pathogenic, endophytic Fusarium oxysporum designated ZzEF8 which was identified previously from Zingiber zerumbet rhizome to display antagonism towards Pythium myriotylum [62.2 ± 2.58% percent of inhibition (PoI)], an oomycetous phytopathogen of significant economic concern. Efficacy of ZzEF8 in suppressing soft rot by P. myriotylum was further evaluated by planting ginger rhizomes primed with ZzEF8 conidial suspension (106 conidia/ ml) followed by transplanting the primed rhizome in sterile soil infested with P. myriotylum (103 zoospores/g soil). Batch fermentation followed by solvent extraction yielded a dark red solid (0.45 mg/ml) which after fractionation by silica column (500 × 30 mm) chromatography (100–200µ) using chloroform: methanol (8:2) solvent yielded three fractions, F1-3. Validation of inhibitory activity of the fractions by disc diffusion assay identified F2 to exhibit absolute P. myriotylum growth inhibition (100%) while F1 and F3 showed 57.62% and 10.73% inhibition respectively. Further purification of F2 by silica column chromatography (300 × 10 mm) yielded three sub-fractions viz., F2a-c. Further LC/Q –TOF MS-analysis of the three sub-fractions identified F2a to contain umbelliferone (tR 6.93 min) as major metabolite. Batch fermentation under submerged conditions was thence optimized for optimal metabolite production. Evaluation of cytotoxicity of F2a and F2b fractions in HeLa cell lines revealed F2a to display cytotoxic effect with IC50 value of 57.9 µg/ml. Anti-Pythium activity of ZzEF8 and anti-cancer activity of ZzEF8 metabolites identified in present experiments signifies its potential for use in pharmaceutical and agricultural industry.

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Abbreviations

LC-QTOF-MS:

Liquid chromatography quadrupole time of flight mass spectrometry

tR :

Retention time

IC50 :

Half maximal inhibitory concentration

PDB:

Potato dextrose broth

PDA:

Potato dextrose agar

DCM:

Dichloromethane

EtoAc:

Ethyl acetate

MtOH:

Methanol

PoI:

Percentage of inhibition

MTT:

3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide

DMEM:

Dulbecco’s Modified Essential Medium

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Acknowledgements

Authors acknowledge the research facilities extended at NITC and CUK for undertaking the present work. The Grant received from UGC (University Grants Commission, Govt. of India) for Junior Research Fellowship is acknowledged by Harsha K (S. No. 2121530466 Ref. no. 20/1/2015(ii)EU-V) and Harshitha K (S. No. 2121530467 Ref no. 20/12/2015(ii)EU-V). KD is thankful for the research fellowship received from Ministry of Human Resource and Development (MHRD), Government of India.

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  1. D. Keerthi

    Present address: Laboratory of Membrane Biophysics (Lab 17), National Centre for Biological Sciences (NCBS-TIFR), GKVK Campus, Bellary Road, Bangalore, 560 065, India

Authors and Affiliations

  1. School of Biotechnology, National Institute of Technology Calicut, Calicut, 673 601, India

    D. Keerthi

  2. Department of Biochemistry and Molecular Biology, Central University of Kerala (CUK), Kasaragod, Kerala, 671 320, India

    K. Harsha, K. Harshitha & Aswati R. Nair

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  1. D. Keerthi
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All authors contributed to the study conception and design of experiments. Material preparation, data collection and analysis were performed by KD, Harsha K and Harshitha K. The manuscript was written by RAN. All authors read and approved the final manuscript.

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Correspondence to Aswati R. Nair.

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Keerthi, D., Harsha, K., Harshitha, K. et al. Potential of Zingiber zerumbet endophytic Fusarium oxysporum as biopriming agents to control Pythium mediated soft-rot and optimization of fermentation conditions for cytotoxic metabolite(s) production. J. Plant Biochem. Biotechnol. 32, 163–173 (2023). https://doi.org/10.1007/s13562-022-00793-2

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