Determination of Reactive Oxygen or Nitrogen Species and Novel Volatile Organic Compounds in the Defense Responses of Tomato Plants against Botrytis cinerea Induced by Trichoderma virens TRS 106

Abstract

In the present study, Trichoderma virens TRS 106 decreased grey mould disease caused by Botrytis cinerea in tomato plants (S. lycopersicum L.) by enhancing their defense responses. Generally, plants belonging to the 'Remiz' variety, which were infected more effectively by B. cinerea than 'Perkoz' plants, generated more reactive molecules such as superoxide (O₂^-) and peroxynitrite (ONOO^-), and less hydrogen peroxide (H₂O₂), S-nitrosothiols (SNO), and green leaf volatiles (GLV). Among the new findings, histochemical analyses revealed that B. cinerea infection caused nitric oxide (NO) accumulation in chloroplasts, which was not detected in plants treated with TRS 106, while treatment of plants with TRS 106 caused systemic spreading of H₂O₂ and NO accumulation in apoplast and nuclei. SPME-GCxGC TOF-MS analysis revealed 24 volatile organic compounds (VOC) released by tomato plants treated with TRS 106. Some of the hexanol derivatives, e.g., 4-ethyl-2-hexynal and 1,5-hexadien-3-ol, and salicylic acid derivatives, e.g., 4-hepten-2-yl and isoamyl salicylates, are considered in the protection of tomato plants against B. cinerea for the first time. The results are valuable for further studies aiming to further determine the location and function of NO in plants treated with Trichoderma and check the contribution of detected VOC in plant protection against B. cinerea.

Keywords: Botrytis cinerea; Trichoderma; reactive nitrogen species; reactive oxygen species; signaling; tomato defense responses; volatile organic compounds.

Figure 1

(a) Single cultivation scheme; (b) procedure of inoculation (Control and TRS 106 plants were inoculated with water drops). In each cultivation, three plants per variant were prepared (3 replicates × 8 variants = 24 plants). Abbreviations: Control, plants grown in the soil without T. virens TRS 106 spores; TRS 106, plants grown in the soil with T. virens TRS 106 spores; Bc, plants grown in the soil without T. virens TRS 106 spores, inoculated with B. cinerea; TRS 106 + Bc, plants grown in the soil with T. virens TRS 106 spores, inoculated with B. cinerea.

Figure 2

(a) Effect of TRS 106 on the tomato plants protection against B. cinerea. Evaluation of the disease symptoms, i.e., irregular brown lesions, dark brown blight blotches, and rot symptoms on leaves; (b) disease area on leaves, occurring on 5-week-old tomato plants caused by B. cinerea, 72 h after inoculation. Values represent the means + SE from four independent experiments with three replicates each (n = 12). Differences between cultivars within a given variant were analyzed with Student’s t-test and marked using letters A and B. Separately for each cultivation, differences between Bc and TRS 106 plants were analyzed with Student’s t-test and marked using asterisk symbol (*** p < 0.001). Data points followed by a different letter are significantly different at p ≤ 0.05. Abbreviations are as in Figure 1.

Figure 3

Effect of TRS 106 on superoxide (O₂⁻) and hydrogen peroxide (H₂O₂) content, and superoxide dismutase (SOD) activity in tomato plants (a). Values represent the means + SE from four independent experiments with three replicates each (n = 12). Separately for each parameter, differences between varieties within a given variant were analyzed with Student’s t-test and marked using letters A and B. Separately for each cultivation, differences between all variants were analyzed with one-way ANOVA (p < 0.05) with Tukey multiple range post hoc test. Data points followed by a different letter are significantly different at p ≤ 0.05. Under the graphs, there are histochemical visualizations of compounds prepared with NBT for O₂⁻ detection (blue blotches, left side of the image) and with DAB for H₂O₂ detection (brown blotches, right side of the image) (b). Abbreviations are as in Figure 1.

Figure 4

Effect of TRS 106 on nitric oxide (NO), peroxynitrite (ONOO⁻), and S-nitrosothiols (SNO) content, and S-nitrosoglutathione reductase (GSNOR) activity in tomato plants. Values represent the means + SE from four independent experiments with three replicates each (n = 12). Separately for each parameter, differences between varieties within a given variant were analyzed with Student’s t-test and marked using letters A and B. Separately for each variety, differences between all variants were analyzed with one-way ANOVA (p < 0.05) with a Tukey multiple range post hoc test. Data points followed by a different letter are significantly different at p ≤ 0.05. Abbreviations are as in Figure 1.

Figure 5

Histochemical visualizations of nitric oxide (NO) in cross sections of tomato leaves. Fluorescent confocal microscopy was used to detect NO localization. Light green fluorescence of NO was observed mainly in the cells localized in the parenchyma and epidermal tissue, and in the area of vascular bundles. DAF-2DA was used for histochemical visualization of NO. As a negative control, leaf pieces were immersed in a buffer containing cPTIO which eliminates NO (Figure S1). The monochrome images are added to confirm and facilitate the localization of the organelle. Abbreviations for variants are as in Figure 1; pa, parenchyma; ep, epidermis.

Figure 6

Subcellular visualizations of nitric oxide (NO) in cross sections of tomato leaves. Fluorescent confocal microscopy was used to detect NO localization in the parenchyma and in palisade and spongy mesophyll. Depending on the variant, the green fluorescence of NO was observed in the apoplast, chloroplast (chl), and nucleus (n). DAF-2DA was used for histochemical visualization of NO. As a negative control, leaf pieces were immersed in a buffer containing cPTIO which eliminates NO (Figure S1). The monochrome images are added to confirm and facilitate the localization of the organelle. Abbreviations are as in Figure 1.

Figure 7

Effect of TRS 106 on green leaf volatiles (GLV) emission. Values represent the means + SE from five independent experiments (n = 5). Separately for each parameter, differences between varieties within a given variant were analyzed with Student’s t-test and marked using letters A and B. Separately for each variety, differences between all variants were analyzed with one-way ANOVA (p < 0.05) with a Tukey multiple range post hoc test. Data points followed by a different letter are significantly different at p ≤ 0.05. Abbreviations are as in Figure 1.

Figure 8

Effect of TRS 106 on aromatic compounds emission. Values represent the means + SE from five independent experiments (n = 5). Separately for each parameter, differences between varieties within a given variant were analyzed with Student’s t-test and marked using letters A and B. Separately for each variety, differences between all variants were analyzed with one-way ANOVA (p < 0.05) with Tukey multiple range post hoc test. Data points followed by a different letter are significantly different at p ≤ 0.05. Abbreviations are as in Figure 1.