Sequestration of Exogenous Volatiles by Plant Cuticular Waxes as a Mechanism of Passive Associational Resistance: A Proof of Concept

Abstract

Numerous plant-derived volatile organic compounds (VOCs) induce the expression of resistance-related genes and thereby cause an "associational resistance" in neighbouring plants. However, VOCs can also be sequestered by plant cuticular waxes. In case that they maintain their biological activity, such sequestered VOCs could generate a "passive" associational resistance that is independent of any gene expression in the receiver. As a proof of concept, we used major components of the cuticular wax layers of the tree, Parkinsonia praecox, and conidia of Colletotrichum lindemuthianum, a fungal pathogen that has not been reported to infect P. praecox. Wax layers were re-constituted on glass slides and exposed to each of 20 pure VOCs for 1 d and then to ambient air for 1 d or 15 d. Gas chromatography-mass spectrometry (GC-MS) analyses showed that all 20 VOCs were sequestered by the re-constituted wax layers. Exposure to 18 of the VOCs significantly inhibited the germination of C. lindemuthianum conidia on these wax layers after 1 day of exposure to ambient air. Four of the VOCs: 4Z-heptenol, farnesene, limonene, and 2E-decenal, inhibited germination rates to less than 25% of the controls. After 15 d, all VOCs were still detectable, although at strongly reduced concentrations, and no significant inhibition of conidial germination could be detected anymore. Exogenous VOCs can be sequestered by the components of plant cuticular waxes and maintain their biological activity, at least over a certain time span: an effect that could generate a transient "passive associational resistance" to pathogens.

Keywords: antifungal volatiles; antracnosis; cuticle; defense; pathogen; plant disease.

Figure 1

Passive associational resistance: Hypothesis and experimental approach. (A) We propose that—in addition to the endogenous volatile organic compounds (VOCs) that are retained in the cuticular waxes during the emission process (upper panel right side)—also exogenous VOCs can be sequestered by the cuticular waxes of a plant (here: Parkinsonia praecox) and maintain their antimicrobial effects. Lower panel right side: transversal section of a P. praecox branch, waxes stained red with Sudan III (photograph donated by Silvia Aguilar Rodríguez). (B) Experimental design: Wax matrix layers were re-constituted on glass slides and then exposed to each out of twenty individual VOCs at a theoretical concentration of 7.142 µM in a closed atmosphere. After subsequent exposure to ambient air, the wax layers were randomly assigned to gas chromatography-mass spectrometry (GC-MS) analysis or biotests (germination of conidia of Colletotrichum lindemuthianum).

Figure 2

The epicuticular waxes matrix of Parkinsonia praecox contained mainly triterpenoids. (A) Representative chromatogram of compounds from cuticular waxes of P. praecox. Preliminary identification of compounds (for retention times and mass spectra, see Figure A , Supplementary Material ): 1) tetracosane (internal standard); 2) tocopherol (triterpenoid 1); 3) nonacosane (alkane 1); 4) germanicol (triterpenoid 2); 5), triacontane (alkane 2); 6) hopenone b (triterpenoid 3); 7) lupenone (triterpenoid 4); 8), cyclolanostanol-derivative 1 (triterpenoid 5), 9) oxolanostadienoate-derivative (triterpenoid 6); 10) betulin (triterpenoid 7); and 11) 24-methylene-cycloartenol (triterpenoid 8). (B) Relative abundance of the dominant compound classes in wax matrix. (C) Numbers of different compounds (i.e., distinct peaks in the GC-spur) that were assigned to each class of compounds found in the wax matrix (see Figure B , Supplementary Material ).

Figure 3

Proof of concept: Volatile organic compounds (VOCs) can be sequestered by epicuticular waxes and maintain their antifungal effect. (A) Chromatograms of epicuticular wax matrices of Parkinsonia praecox exposed to 2Z-decenal and subsequently to ambient air for 1 day (red) or 15 d (green) or exposed to ambient air only (control: black). Peak numbers: 1,2) not identified; 3) 2Z-decenal; 4) 4-undecanol; 5) 2,6,10,15-tetramethyl-heptadecane; 6) 2,4-bis(1,1-dimethylethyl)-phenol; 7) heneicosane. (B) The concentration of VOCs in wax matrices after exposure to at a theoretical concentration of 7.142 µM in a closed atmosphere and subsequent exposure to ambient air for 1 day (black bars) or 15 d (grey bars). Control: wax matrices exposed to ambient air only. Asterisks indicate significant differences between the concentration of sequestered VOC detected at day 1 and day 15 (p < 0.05, n=3 technical replicates, t-test. (C) Germination rate of Colletotrichum lindemuthianum conidia on VOC-exposed wax matrices. Asterisks mark germination rates significantly lower than controls (p < 0.05, Dunnett test, n=7 biological replicates). Bars in (B, C) indicate means, error bars indicate ± 1 SD.