Abstract

In the present study, an endophytic fungus with a considerable antimicrobial activity was isolated from the medicinal plant Mentha Pulegium L. The morphological and molecular identification revealed that this fungus is a Penicillium griseofulvum. A preliminary screening was done to choose the suitable culture medium for a maximum production of the bioactive compounds using the dual-culture agar diffusion assay. The malt extract agar (MEA) and potato dextrose agar (PDA) media were the suitable, and the MEA was selected for the further study. Extraction was done with three solvents, n-Hexane, dichloromethane and ethyl acetate, and the crude extracts were tested against Gram-positive and negative bacteria. A high activity was found with ethyl acetate and dichloromethane crude extracts against all bacteria with a maximal inhibition zones of 45.5 and 41 mm respectively obtained against Escherichia coli. The minimum inhibitory and bactericidal concentrations (MIC, MBC) of ethyl acetate crude extract were evaluated using the broth micro-dilution method. A MIC of 50 µg / ml on Gram-negative bacteria and of 100 µg / ml on Gram-positive bacteria was found. The MBCS ranged from 50 and 200 µg/ml. The time kill study has revealed a bactericidal activity of the Penicillium griseofulvum crude extract. At 10 h and for all concentrations (MIC, MIC× 2 and MIC× 4), 99.9% killing of the bacterial cells was achieved. These results prove that the extract of Penicillium griseofulvum can be a promising source of important bioactive molecules.   Keys words: Antibacterial activity, endophytic fungi, Mentha pulegium L., Penicillium griseofulvum.

Highlights

  • Best-scoring maximum likelihood tree based on the Tamura–Nei model calculated using MEGA 7.0 based on against four clinical pathogenic bacteria; this permitted a internal transcribed spacer (ITS) sequences showing the relationships of the newly generated sequences (GenBank accession number MH006592) in this study with previously known taxa in the NCBI GenBank

  • 100 μg / ml for the three Gram-positive pathogenic in our study reveal the opposite, and this can be bacteria

  • Killing growth profile of P. griseofulvum ethyl acetate crude extract against Methicilline-resistant S. aureus ATCC 43300 shows a bactericidal effect at 24 h of incubation; 100% killing of the bacterial cells was achieved for minimum inhibitory concentrations (MICs) × 2 and MIC × 4 concentrations (Figure 6)

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Summary

INTRODUCTION

Medicinal plants like all plants interact continously with different microorganisms, like fungi (both pathogenic and beneficial), living on the surface of plants (epiphytes) and their roots (mycorhizes) and even inside (endophytes). Antibacterial activity of the endophytic fungi was performed using the method of agar plug diffusion explained by Powthong et al (2013) and Sahani et al (2017), with some modification. It is native to America, Europe, North Africa and in Asia Minor and Near East (Mahboubi and Haghi, 2008) It is widely used in traditional medicine (food poisoning, bronchitis, diuretic, cut (6 mm diameter) using a sterile cork borer and placed on previously inoculated Muller Hinton Agar (MHA) (Sigma- Aldrich; 7.3±0.2) plates. (2013) have studied the endophytic fungi of this plant, and have determined the cytotoxicity and anticancer Identification activity of the bioactive metabolites of the endophytic fungus, Stemphylium globuliferum. DNA was extracted from culture using the Ultraclean Microbial DNA Isolation Kit (MoBio, Solana Beach, CA, USA) following the manufacturer’s protocols

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