Antifungal Activity of Glucosinolate-Derived Nitriles and Their Synergistic Activity with Glucosinolate-Derived Isothiocyanates Distinguishes Various Taxa of Brassicaceae Endophytes and Soil Fungi

Abstract

The glucosinolates of Brassicaceae plants are converted into bioactive isothiocyanates and other volatiles during a challenge by pathogens and other biotic stressors. However, the role of alternative downstream products with weaker potency (e.g., nitriles) is far from being fully understood. This study tested the possible synergistic antifungal interaction between various glucosinolate-derived nitriles and 2-phenylethyl isothiocyanate (PEITC) on 45 fungal strains, including endophytes from horseradish roots (Brassicaceae) and soil fungi, using an airtight system enabling the accurate study of extremely volatile antifungal agents. The median minimal inhibitory concentrations (MICs) were 1.28, 6.10, 27.00 and 49.72 mM for 1H-indole-3-acetonitrile (IAN), 3-phenylpropanenitrile (PPN), 4-(methylsulfanyl)-butanenitrile (MSBN) and 3-butenenitrile (BN, = allyl cyanide), respectively. Thus, nitriles were considerably weaker antifungal agents compared to PEITC with a median MIC of 0.04 mM. For the same nitriles, the median fractional inhibitory concentration indices (FICIs) of the combinations were 0.562, 0.531, 0.562 and 0.625, respectively. Altogether, 47.7%, 56.8%, 50.0% and 27.3% of tested fungal strains showed a synergistic antifungal activity (FICI ≤ 0.5) for the nitrile-isothiocyanate combinations, respectively. Hypocreales strains showed the least sensitivity towards the GSL decomposition products and their combinations. The mean MIC values for PEITC showed 0.0679 ± 0.0358, 0.0400 ± 0.0214, 0.0319 ± 0.0087 and 0.0178 ± 0.0171 mM for Hypocreales, Eurotiales, Glomerellales and Pleosporales, respectively. In addition, nitriles, especially IAN, also showed significant differences. For the same fungi, the median FICI values fell in the ranges of 0.61-0.67, 0.52-0.61, 0.40-0.50 and 0.48-0.67, respectively, depending on the nitrile. Our results suggest that glucosinolate-derived nitriles may enhance isothiocyanate antifungal activity and that they may play an active role in shaping the plant microbiome and contribute to the filtering of microbes by plants.

Keywords: ITC; antifungal activity; endophyte fungi; isothiocyanate; nitrile; soil fungi; synergy.

Figure 1

Effects of specifier proteins on the main routes of glucosinolate (GSL) decomposition. In the absence of any specifier proteins, isothiocyanates are formed. Data compiled from [5] and references therein. Abbreviation: ESP: epithiospecifier protein; TFP: thiocyanate-forming protein; ESM: epithiospecifier modifier protein; NSP: nitrile-specifier protein.

Figure 2

Biplot of principal component analysis scores of fungal sensitivities to glucosinolate decomposition products (nitriles and 2-phenylethyl isothiocyanate) and their combinations. Crosshairs show the average ± standard deviation for different taxonomic groups. The labeled vectors represent the relative contribution of loadings to the two principal components, PC1–2. Strains from various fungal orders are shown with different colors. Abbreviations for loading vectors: BN, IAN, MSBN, PEITC and PPN denote MIC values for individual agents; P_BN, P_IAN, P_MSBN and P_PPN denote FICI values for nitrile-PEITC combinations. Fungi of different origin are shown with circles and triangles. Abbreviations for origin: H, horseradish endophytes; S, soil.