Broad-Spectrum Antimicrobial Action of Cell-Free Culture Extracts and Volatile Organic Compounds Produced by Endophytic Fungi Curvularia Eragrostidis

Abstract

Endophytes are the mutualistic microorganisms that reside within the host plant and promote plant growth in adverse conditions. Plants and their endophytes are engaged in a symbiotic relationship that enables endophytes to access bioactive genes of the ethnomedicinal plants, and, as a result, endophytes are constantly addressed in the sector of pharmaceuticals and agriculture for their multidomain bio-utility. The gradual increase of antimicrobial resistance can be effectively countered by the endophytic metabolites. In these circumstances, in the present investigation, endophytic Curvularia eragrostidis HelS1 was isolated from an ethnomedicinally valuable plant Helecteris isora from East India's forests. The secondary volatile and non-volatile metabolites are extracted from HelS1 and are found to be effective broad-spectrum antimicrobials. A total of 26 secondary metabolites (9 volatiles and 17 non-volatiles) are extracted from the isolate, which exhibits effective antibacterial [against six Gram-positive and seven Gram-negative pathogens with a minimum inhibitory concentrations (MIC) value ranging from 12.5 to 400 μg ml^-1] and antifungal (against seven fungal plant pathogens) activity. The secondary metabolite production was optimised by one variable at a time technique coupled with the response surface methodology. The results revealed that there was a 34% increase in antibacterial activity in parameters with 6.87 g L^-1 of fructose (as a carbon source), 3.79 g L^-1 of peptone (as a nitrogen source), pH 6.75, and an inoculation period of 191.5 h for fermentation. The volatile metabolite production was also found to be optimum when the medium was supplemented with yeast extract and urea (0.2 g L^-1) along with dextrose (40 g L^-1). Amongst extracted volatile metabolites, 1-H-indene 1 methanol acetate, tetroquinone, N, N-diphenyl-2-nitro-thio benzamide, Trans 1, 2-diethyl-trans-2-decalinol, naphthalene, and azulene are found to be the most effective. Our investigation opens up opportunities in the sector of sustainable agriculture as well as the discovery of novel antimicrobials against dreadful phyto and human pathogens.

Keywords: broad-spectrum antimicrobial; endophyte; fungi; sustainable agriculture; volatile metabolite.

FIGURE 1

(A) A 6-day old culture of Curvularia eragrostidis HelS1 grown on PDA. (B) Sterile mycelial hyphae of the isolate seen under a stereo-microscope. (C) Septate sterile hyphae seen under a light microscope.

FIGURE 2

A phylogenetic tree of the endophytic isolate C. eragrostidis HelS1.

FIGURE 3

The effect of different concentrations (minimum inhibitory concentrations: MIC/2, MIC, MIC*2, MIC*4) of antibacterial compounds of HelS1 on the growth of bacterial pathogens [(A) methicillin-resistant Staphylococcus aureus (MRSA), (B) S. epidermidis, (C) S. dysenteriae, (D) K. pneumonia] is represented here in the form of a time-kill curve. The CFUs (colony-forming units) are counted after 0, 2, 4, 6, 8, 10, 12, and 24 h of treatment. Values on the graphs are the means ± standard error (SE) of the three replicates.

FIGURE 4

Effect of different concentrations of HelS1 ethyl acetate (EA) extract on the essential enzymes of microorganisms (A MRSA, B L. monocytogenes, C V. parahaemolyticus, and D P. aeruginosa) involved in central energy (carbohydrate) metabolism. Values on the graphs are the means ± standard error (SE) of the three replicates. Tukey’s multiple comparison test was performed. The different letters a, b, and c in each case [Control, MIC, minimum bactericidal concentrations (MBC)] represent a significant difference between them (At, p < 0.05).

FIGURE 5

An antifungal gas test performed by a split plate method (A–F). The growth of pathogenic [(A) Geotrichum candidum, (B) Botrytis cinerea, (C) Cercospora beticola, (D) Rhizoctonia solani, (E) Pythium ultimum, (F) Cercospora beticola] fungi has been inhibited by the VOCs of endophytic fungi HelS1 (except in one case-C). (G) Represents how endophytic fungi are grown on GC-glass vials for VOC analysis. (H) GC-chromatogram of the VOCs produced by the endophytic HelS1.

FIGURE 6

(A) Thin-layer chromatographic (TLC) analysis of EA fraction of HelS1. (B) Zones of inhibition produced by bioactive compounds present in fractions (F_A–F_F) obtained from a TLC plate against pathogenic bacteria-MRSA. (C) GC-MS chromatogram of Fraction A showing the peaks of bioactive compounds. (D) GC-MS chromatogram of Fraction B, showing the peaks of bioactive compounds.