Strigolactones GR-24 and Nijmegen Applications Result in Reduced Susceptibility of Tobacco and Grapevine Plantlets to Botrytis cinerea Infection

Abstract

Priming agents are plant defence-inducing compounds which can prompt a state of protection but may also aid in plant growth and interactions with beneficial microbes. The synthetic strigolactones (±)-GR24 and Nijmegen-1 were evaluated as potential priming agents for induced resistance against Botrytis cinerea in tobacco and grapevine plants. The growth and stress response profiles of B. cinerea to strigolactones were also investigated. Soil drench treatment with strigolactones induced resistance in greenhouse-grown tobacco plants and restricted lesion development. The mode of action appeared to function by priming redox-associated compounds to produce an anti-oxidant protective response for limiting the infection. The results obtained in the in vitro assays mirrored that of the greenhouse-grown plants. Exposure of B. cinerea to the strigolactones resulted in increased hyphal branching, with (±)-GR24 stimulating a stronger effect than Nijmegen-1 by affecting colony diameter and radial growth. An oxidative stress response was observed, with B. cinerea exhibiting increased ROS and SOD levels when grown with strigolactones. This study identified the application of strigolactones as potential priming agents to induce disease resistance in both tobacco and grapevine plants. In addition, strigolactones may alter the ROS homeostasis of B. cinerea, resulting in both morphological and physiological changes, thereby reducing virulence.

Keywords: Botrytis cinerea; hyphal branching; plant fitness; reactive oxygen species; strigolactones.

Figure 1

The effect of plant associated compounds on plant growth. (A) Plant growth and (B) lateral root and leaf development were measured in tobacco plants after 3 weeks of treatment under tissue culture conditions. Treatments were as follows: smoke water at concentrations of (i) 1:10,000, (ii) 1:100,000 and (iii) 1:200,000; lumichrome at concentrations of (i) 1 nM, (ii) 5 nM and (iii) 50 nM; (±)-GR24 at concentrations of (i) 1 × 10⁻⁷ M, (ii) 1 × 10⁻⁸ M and (iii) 1 × 10⁻⁹ M; Nijmegen-1 at concentrations of (i) 1 × 10⁻⁷ M, (ii) 1 × 10⁻⁸ M and (iii) 1 × 10⁻⁹ M; cultured in MS media (control). Error bars represent the means (±SE; n = 4) of two independent experiments. Letters a-h and numerals i-vii indicate significant differences.

Figure 2

Disease susceptibility of treated tobacco plants to B. cinerea. A decrease in susceptibility to B. cinerea was determined by measuring the (A) size of lesions (mm) produced on detached leaves of the control and treated tobacco plants at 48 and 144 h post inoculation. (B) The susceptibility to B. cinerea was expressed as a percentage of the decrease in susceptibility normalised against the tolerance of the control set, which was referenced as 0%. Error bars represent the means (±SE; n = 4) of two independent experiments. Letters a-f and i-vii represent significant differences.

Figure 3

Effect of (±)-GR24 on superoxide content and hydrogen peroxide levels in tobacco leaves infected with B. cinerea. Measurements were performed on tobacco plants that were treated with (±)-GR24 for a period of 48 h. The H₂O₂ profiles for (±)-GR24 (A) and Nijmegen-1 (B) were determined via a spectrometric reading at 560 nm. The SOD profiles for (±)-GR24 (C) and Nijmegen-1 (D) were determined via a spectrometric reading at 550 nm. Values obtained were normalized with the fresh cell weights. Error bars represent the means (±SE; n = 4) of two independent experiments. Letters a-d represent significant differences.

Figure 4

Disease symptom development of Nijmegen-1-treated tissue culture grapevine plants upon challenge with B. cinerea. Intact leaves were inoculated with a conidial suspension of B. cinerea and maintained at 22 °C at 100% humidity. Treatments consisted of root drench applications of water for (A) control sets or Nijmegen-1 at concentrations (B) 1 × 10⁻⁷ M, (C) 1 × 10⁻⁸ M and (D) 1 × 10⁻⁹ M. Images are representative of four biological repeats of two independent experiments. Disease development is indicated by the red circle.

Figure 5

Disease symptom development of (±)-GR24-treated tissue culture grapevine plants upon challenge with B. cinerea. Intact leaves were inoculated with a conidial suspension of B. cinerea and maintained at 22 °C at 100% humidity. Treatments consisted of root drench applications of water for (A) control sets or (±)-GR24 at concentrations (B) 1 × 10⁻⁷ M, (C) 1 × 10⁻⁸ M and (D) 1 × 10⁻⁹ M. Images are representative of four biological repeats of two independent experiments. Disease development is indicated by the red circle.

Figure 6

Effect of (±)-GR24 on the radial growth of B. cinerea. The radial growth of B. cinerea was assayed on MEA-YE agar (A) or in the presence of various concentrations of (±)-GR24 at (B) 1 × 10⁻⁷ M, (C) 1 × 10⁻⁸ M and (D) 1 × 10⁻⁹ M. Petri dishes were observed for 120 h post inoculation (hpi). Images are representatives of four replicates in each of two independent experiments.

Figure 7

Effect (±)-GR24 and Nijmegen-1 on the infection cushion formation of B. cinerea. The growth of B. cinerea was assayed on 1% water agar (A) or in the presence of (±)-GR24 at concentrations of (B) 1 × 10⁻⁷ M, (C) 1 × 10⁻⁸ M and (D) 1 × 10⁻⁹ M and Nijmegen-1 at (E) 1 × 10⁻⁷ M, (F) 1 × 10⁻⁸ M and (G) 1 × 10⁻⁹ M. Petri dishes were observed under a stereomicroscope after 5 days post inoculation. Images are representatives of four replicates of two independent experiments. The formation of infection cushions is indicated with red arrows.

Figure 8

Effect of (±)-GR24 and Nijmegen-1 on the H₂O₂ and SOD profiles of B. cinerea. The H₂O₂ profiles for (±)-GR24 (A) and Nijmegen-1 (B) were determined via a spectrometric reading at 560 nm at intervals from 0–48 h post inoculation (hpi). The SOD profiles for (±)-GR24 (C) and Nijmegen-1 (D) were determined via a spectrometric reading at 550 nm. Values obtained were normalized with the fresh cell weights. Error bars represent the means (±SE; n = 4) of two independent experiments.