Purification of an Antifungal Peptide from Seeds of Brassica oleracea var. gongylodes and Investigation of Its Antifungal Activity and Mechanism of Action

In this study, a 8.5-kDa antifungal peptide designated as BGAP was purified from the crude extract of the seeds of Brassica oleracea var. gongylodes by employing a protocol that comprised cation exchange chromatography on SP-Sepharose, cation exchange chromatography on Mono S and gel filtration chromatography on Superdex peptide. BGAP showed the highest amino acid sequence similarity to defensin peptides by mass spectrometric analysis. BGAP showed a broad spectrum of antifungal activity with a half maximal inhibitory concentration at 17.33 &mu
g/mL, 12.37 &mu
g/mL, 16.81 &mu
g/mL, and 5.60 &mu
g/mL toward Colletotrichum higginsianum, Exserohilum turcicum, Magnaporthe oryzae and Mycosphaerella arachidicola, respectively. The antifungal activity of BGAP remained stable (i) after heat treatment at 40&ndash
100 &deg
C for 15 min
(ii) after exposure to solutions of pH 1&ndash
3 and 11&ndash
13 for 15 min
(iii) after incubation with solutions containing K+, Ca2+, Mg2+, Mn2+ or Fe3+ ions at the concentrations of 20&ndash
150 mmol/L for 2 h
and (iv) following treatment with 10% methyl alcohol, 10% ethanol, 10% isopropanol or 10% chloroform for 2 h. Fluorescence staining experiments showed that BGAP brought about an increase in cell membrane permeability, a rise in reactive oxygen species production, a decrease in mitochondrial membrane potential, and an accumulation of chitin at the hyphal tips of Mycosphaerella arachidicola.