The Antifungal Activity of Loquat (Eriobotrya japonica Lindl.) Leaves Extract Against Penicillium digitatum

Abstract

This study was performed to determine the antifungal activity of loquat (Eriobotrya japonica Lindl) leaf extract (LLE) against the citrus postharvest pathogen Penicillium digitatum (P. digitatum). The LLE exhibited an antifungal activity against P. digitatum, with a minimum inhibitory concentration (MIC) of 0.625 mg/ml and a minimum fungicidal concentration (MFC) of 1.25 mg/ml. Significant inhibitory effects of LLE on mycelial growth and spore germination of P. digitatum were seen in a dose-dependent manner. Simultaneously, to investigate possible antifungal mechanisms by LLE, we analyzed their influence on morphological changes, cell membrane permeability, cell wall and cell membrane integrity, and adenosine phosphates (ATP, ADP, and AMP) levels. Alterations, such as sunken surface and malformation, occurred in the LLE-treated P. digitatum spores. Furthermore, intracellular inclusion content decreased after LLE treatment, indicating an increase in cell membrane permeability. Besides, the LLE treatment induced a significant decline in the level of adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) with a noticeable addition of extracellular ATP, ADP, and AMP during the entire treatment period. Overall, the results manifested that the antifungal activity of LLE against P. digitatum can be attributed to the derangement of cell membrane permeability and disordered energy metabolism. This is the first report on the mechanism of antifungal activity of LLE and could be useful in the development of targeted fungicides from natural origin.

Keywords: Penicillium digitatum; antifungal activity; energy metabolism; loquat leaves; membrane permeability.

Figure 1

Mycelial growth of P. digitatum on potato dextrose agar (PDA) exposure to loquat leaf extract (LLE) after 2 and 6 days. Different letters above the columns of the same day represent significant differences (p < 0.05) in colony diameter among the LLE concentrations.

Figure 2

Scanning electron microscopy (SEM) observation (× 25,000 magnification) of the morphology alteration of P. digitatum spores exposed to 1.25 mg/ml (minimum fungicidal concentration, MFC) loquat leaf extract (LLE): (A) untreated spores and (B) LLE-treated spores.

Figure 3

Effect of loquat leaf extract (LLE) on cell membrane permeability of P. digitatum. (A) Change in extracellular conductivity of P. digitatum exposed to LLE treatment; (B) change in cell lysis rate of P. digitatum exposed to LLE treatment; (C) change in nucleic acid leakage of P. digitatum exposed to LLE treatment; and (D) change in protein leakage of P. digitatum exposed to LLE treatment. Different letters above the lines represent significant differences (p < 0.05) among treatments simultaneously.

Figure 4

Effect of loquat leaf extract (LLE) on cell membrane permeability and lipid peroxidation of P. digitatum. (A) Propidium iodide (PI) staining, a₁, a₄: control treatment (0 mg/ml LLE.); a₂, a₅: minimum inhibitory concentration (MIC treatment) (0.625 mg/ml LLE); a₃, a₆: minimum fungicidal concentration (MFC) treatment (1.25 mg/ml LLE); (B) change in malondialdehyde (MDA) content of P. digitatum exposed to LLE treatment; (C) change in the β-1,3-glucanase (β-Glu) activity of P. digitatum exposed to LLE treatment; (D) change in the alkaline phosphatase (AKP) activity of P. digitatum exposed to LLE treatment. Different letters above the lines represent significant differences (p < 0.05) among treatments at the same time after treatment.

Figure 5

Effect of loquat leaf extract (LLE) on intracellular constituents of P. digitatum. (A) Change in reducing sugar content of P. digitatum exposed to LLE treatment; (B) change in protein content of P. digitatum exposed to LLE treatment; (C) change in total lipid content of P. digitatum exposed to LLE treatment; and (D) change in ergosterol content of P. digitatum exposed to LLE treatment. Different letters above the columns represent significant differences (p < 0.05) among treatments simultaneously after treatment.

Figure 6

Effect of loquat leaf extract (LLE) on adenosine triphosphate (ATP) (A,D), adenosine diphosphate (ADP) (B,E), and adenosine monophosphate (AMP) (C,F) contents in intracellular (left) and extracellular (right) P. digitatum. Different letters above the lines represent significant differences (p < 0.05) among treatments at the same time after treatment.