New plant immunity elicitors from a sugar beet byproduct protect wheat against Zymoseptoria tritici

Abstract

The current worldwide context promoting agroecology and green agriculture require the discovery of new ecofriendly and sustainable plant protection tools. Plant resistance inducers, called also elicitors, are one of the most promising alternatives fitting with such requirements. We produced here a set of 30 molecules from pyroglutamic acid, bio-sourced from sugar beet byproducts, and examined for their biological activity on the major agro-economically pathosystem wheat-Zymoseptoria tritici. Foliar application of the molecules provided significant protection rates (up to 63% disease severity reduction) for 16 among them. Structure-activity relationship analysis highlighted the importance of all chemical groups of the pharmacophore in the bioactivity of the molecules. Further investigations using in vitro and in planta antifungal bioassays as well as plant molecular biomarkers revealed that the activity of the molecules did not rely on direct biocide activity towards the pathogen, but rather on the activation of plant defense mechanisms dependent on lipoxygenase, phenylalanine ammonia-lyase, peroxidase, and pathogenesis-related protein pathways. This study reports a new family of bio-sourced resistance inducers and provides new insights into the valorization of agro-resources to develop the sustainable agriculture of tomorrow.

Figure 1

Chemical structure of γ-aminobutyric acid (GABA), pyroglutamic acid (PGA), the molecule M1 derived from PGA, and the 28 molecules (from M2 to M29) derived from M1.

Figure 2

Pharmacophore of the molecule M1, showing the structure modifications performed on the different positions of the main nucleus of M1. All changes were done separately on all positions of the nucleus, except on position 4. Some molecules were modified in several positions simultaneously.

Figure 3

Disease severity on wheat plants (cultivar Alixan) against Zymoseptoria tritici (strain T02596) treated with water (control), Cantor (0.15%), γ-aminobutyric acid (GABA), and M1 and M2 molecules at 5 mM. A stands for representative third leaves from the control and each treatment, while B strands for the means scored on 36 third leaves from the control and each treatment. Disease symptoms (in A and B) were recorded 21 days post-inoculation by scoring the percentage of the third leaf area covered with lesions bearing pycnidia. Asterisks indicate significant differences according to the Tukey test at P = 0.001. Bars indicate standard deviation (SD).

Figure 4

In planta effect of γ-aminobutyric acid (GABA), M1, M2 and Cantor treatments on the epiphytic hyphal growth of Zymoseptoria tritici (strain T02596) on the wheat leaves (cultivar Alixan), compared to the water-treated control. Three third-leaf segments with 100 fungal spores monitored on each were used as replicates in each condition. Four different classes of Calcofluor-stained germinated spores were assessed five days post-inoculation (class 1, non-geminated spores; Class 2, geminated spores with small germ tube; class 3, geminated spores with developed germ tube; class 4, geminated spores with a strongly developed germ tube). Within each class, bars with common letters are not significantly different using the Tukey test at P = 0.05. Scale bar = 10 µ.

Figure 5

Relative expression of four selected defense-related genes in wheat leaves (cultivar Alixan) treated or not with γ-aminobutyric acid GABA, M1 and M2 and inoculated or not with Zymoseptoria tritici (strain T02596). (A) non-infectious conditions (ni) where GABA, M1 and M2-treated and non-inoculated plants are compared to Cantor-treated and non-inoculated plants; (B) Cantor-treated and inoculated plants compared to Cantor-treated and non-inoculated control plants; (C) GABA-, M-1 and M2-treated and inoculated plants compared to Cantor-treated and inoculated control plants. The data are resulting from two independent experiments, with three biological replicates and two technical replicates (six replicates) each. Values of relative gene expression were converted to Log 2 and represented in the form of a heatmap. Over-expression is highlighted with shades of green to yellow, while down-expression is presented with shades of purple to blue. The genes were considered significantly down-regulated (white asterisks) or up-regulated (black asterisks) compared to the corresponding controls when changes in their expression were < 0.5 × or > 2 x, respectively. The numbers on the heatmap indicate days post-treatment (dpt).