Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Nov 22;13(12):1116.
doi: 10.3390/antibiotics13121116.

Cell Wall-Mediated Antifungal Activity of the Aqueous Extract of Hedera helix L. Leaves Against Diplodia corticola

Affiliations

Cell Wall-Mediated Antifungal Activity of the Aqueous Extract of Hedera helix L. Leaves Against Diplodia corticola

Christina Crisóstomo et al. Antibiotics (Basel). .

Abstract

Background/Objectives: Cork oak forests have been declining due to fungal pathogens such as Diplodia corticola. However, the preventive fungicides against this fungus have restricted use due to the deleterious effects on human health and the environment, prompting the need for sustainable alternatives. Here, we describe the antifungal activity of an aqueous extract of Hedera helix L. leaves (HAE) against D. corticola and the possible mechanism of action. Results/Methods: The chemical analysis revealed compounds like the saponin hederacoside C, quinic acid, 5-O-caffeoylquinic acid, rutin, and glycoside derivatives of quercetin and kaempferol, all of which have been previously reported to possess antimicrobial activity. Remarkable in vitro antifungal activity was observed, reducing radial mycelial growth by 70% after 3 days of inoculation. Saccharomyces cerevisiae mutants, bck1 and mkk1/mkk2, affected the cell wall integrity signaling pathway were more resistant to HAE than the wild-type strain, suggesting that the extract targets kinases of the signaling pathway, which triggers toxicity. The viability under osmotic stress with 0.75 M NaCl was lower in the presence of HAE, suggesting the deficiency of osmotic protection by the cell wall. Conclusions: These results suggest that ivy extracts can be a source of new natural antifungal agents targeting the cell wall, opening the possibility of preventing fungal infections in cork oaks and improving the cork production sector using safer and more sustainable approaches.

Keywords: Diplodia corticola; Hedera helix water extract; Saccharomyces cerevisiae fungal model; anti-phytopathogen activity; antifungal mechanism of action.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Percentage of growth inhibition of D. corticola by H. helix aqueous extract (HAE). Radial growth of D. corticola mycelium was measured in Petri dishes with PDA medium containing 50, 100, 500, 1000, or 1500 µg/mL of HAE. Representative images of HAE antifungal activity against D. corticola after 6 days of incubation (A). In the negative control (C-), the highest volume of extract used on the assays was replaced by sterilized deionized water (extract solvent). The diameter was measured after 3 (black bars; (B)) and 6 (grey bars; (B)) days of incubation at 25 °C in the dark. Each bar represents the mean ± SD of three independent experiments. A two-way ANOVA was used for the analysis and different letters represent statistical significance. Capital letters were used for comparisons of extract concentrations within the same time of incubation and lowercase letters were used for comparisons of different times of incubation for each concentration tested.
Figure 2
Figure 2
Viability of S. cerevisiae BY4741 (A) and the mutant strains erg2 (B), bck1 (C), and mkk1/mkk2 (D) in the presence of aqueous extract of H. helix (HAE). Cells from exponentially growing cultures were exposed to 10 µg/mL (white squares), 50 µg/mL (black triangles), 75 µg/mL (white inverted triangles), 100 µg/mL (black diamonds), or 250 µg/mL (white circles) of HAE and incubated at 30 °C, 200 rpm. Viability was assessed using CFU after 0, 30, 60, and 90 min of incubation upon spreading 10−4 dilutions on YPDA plates and incubation at 30 °C, 200 rpm. The negative control (black circles) was prepared by replacing the extract by the solvent at the highest volume of extract used in the assays. The data represent the mean ± SD of three independent experiments. A one-way ANOVA and Dunnett’s post hoc test were used for the analysis, and concentrations were compared at each time-point. For clarity of graphical representation, significance is shown only for the 90 min timepoint, where ** means 0.001 < p ≤ 0.01 and **** means p < 0.0001; the absence of significance is not marked with any symbol. Other relevant significances are presented in the text.
Figure 3
Figure 3
Viability of S. cerevisiae BY4741 in the presence of aqueous extract of H. helix (HAE) and osmotic stress. Cells from exponentially growing cultures were exposed to 10 µg/mL (grey bars) or 50 µg/mL (dark grey bars) of HAE at 30 °C, 200 rpm, for 30 min. The viability was assessed using CFU by plating 10−4 dilutions on YPDA plates containing different concentrations of NaCl (0, 0.25, 0.5, or 0.75 M) and further incubation at 30 °C for 48 h. The negative control (black bars) was prepared by replacing the extract with the solvent at the highest volume of extract used in the assays. The data represent the mean ± SD of three independent experiments. A two-way ANOVA and Tukey’s post hoc test were used for the analysis. A letter code was used. Lowercase letters are used for comparisons of extract concentration effects within each salt concentration (p < 0.0001) and capital letters are used for comparisons between salt concentrations for each extract concentration (p < 0.01). Mean values followed by the same letters are not statistically different.
Figure 4
Figure 4
Percentage of growth inhibition of D. corticola by H. helix aqueous extract (HAE) in the absence and presence of osmotic stress. Radial growth of D. corticola mycelium was measured in Petri dishes with PDA medium containing 50, 500, or 1500 µg/mL HAE in the absence or presence of 0.4 M of NaCl. Representative images of HAE antifungal activity against D. corticola after 6 days of incubation (A). In the negative control (C-), the highest volume of extract used on the assays was replaced by sterilized deionized water (extract solvent). The diameter was measured after 6 days of incubation at 25 °C in the dark and the percentage of growth inhibition was calculated, taking C- as a reference (B). Each bar represents the mean ± SD of three independent experiments. A one-way ANOVA and Dunnett’s post hoc test were used for the analysis, where *** means 0.0001 < p ≤ 0.001 and **** means p < 0.0001.

References

    1. Steinberg G., Gurr S.J. Fungi, fungicide discovery and global food security. Fungal Genet. Biol. 2020;144:103476. doi: 10.1016/j.fgb.2020.103476. - DOI - PMC - PubMed
    1. Fang Y., Ramasamy R.P. Current and Prospective Methods for Plant Disease Detection. Biosensors. 2015;5:537–561. doi: 10.3390/bios5030537. - DOI - PMC - PubMed
    1. Paoletti E., Anselmi N., Franceschini A. Pre-Exposure to Ozone Predisposes Oak Leaves to Attacks by Diplodia corticola and Biscogniauxia mediterranea. Sci. World J. 2007;7:222–230. doi: 10.1100/tsw.2007.22. - DOI - PMC - PubMed
    1. Costa D., Tavares R.M., Baptista P., Lino-Neto T. The Influence of Endophytes on Cork Oak Forests Under a Changing Climate. In: Hodkinson T.R., Doohan F.M., Saunders M.J., Murphy B.R., editors. Endophytes for a Growing World. 1st ed. Cambridge University Press; Cambridge, UK: 2019. pp. 250–274.
    1. Moricca S., Linaldeddu B.T., Ginetti B., Scanu B., Franceschini A., Ragazzi A. Endemic and Emerging Pathogens Threatening Cork Oak Trees: Management Options for Conserving a Unique Forest Ecosystem. Plant Dis. 2016;100:2184–2193. doi: 10.1094/PDIS-03-16-0408-FE. - DOI - PubMed

LinkOut - more resources