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

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.


Introduction
Trichoderma spp., saprophytic fungi, are among the extensively studied biocontrol agents (BCA), i.e., microorganisms that are able to promote plant growth and/or protect plants against pathogens [1]. Some Trichoderma strains exhibit antagonistic strategies against biotrophs and necrotrophs, such as antibiosis, mycoparasitism, and competition for niches and nutrients. Other strains induce local and systemic defense responses and resistance in plants against different pathogens [2,3]. The biochemical and molecular pathways of defense responses induced in plants by Trichoderma are still not fully explained and arouse controversy, as they may strongly vary depending on the Trichoderma strain, plant species, and pathogen, as well as soil and climate conditions [4,5]. During plant-Trichoderma interaction, elicitors released by the fungi, after detection and recognition, induce various cellular and molecular responses in plants [1]. The plant responses include enhanced synthesis of signaling molecules, upregulation of expression of defense-related genes as well as enhancement of synthesis of compounds with antimicrobial activity or ability to reinforce mechanical barriers in plant tissues [1,4]. Trichoderma spp. were the generation and location of compounds, many of which were not studied extensively as signaling molecules of tomato protection against B. cinerea. They are especially valuable for further analysis towards more specifically determining the location and function of NO in plants treated with Trichoderma and checking the contribution of detected VOC in plant protection against B. cinerea. The obtained knowledge may confirm the role of TRS 106 as BCA of B. cinerea, and be useful for scientific research focusing on the development of eco-friendly methods of protecting plants against this pathogen as alternatives to chemicals for crop protection, the use of which is being subjected to increasing law restrictions.

Trichoderma virens and Botrytis cinerea Inoculum Preparation
T. virens TRS 106 was obtained from the bank collection of Microbiology Laboratory Department of Vegetable Plant Protection of Research Institute of Horticulture (Skierniewice, Poland). The morphological identification and molecular classification of TRS 106 were described previously and deposited at the NCBI GenBank [42,43]. TRS 106 isolate was previously characterized as able to induce plant resistance against different pathogens and promote plant growth [42]. The positive influence of TRS 106 on the growth of tomato plants (S. lycopersicum L.) belonging to two varieties, i.e., 'Perkoz' and 'Remiz', has been proven (Tables S1 and S2).
Before being used for tomato treatment, TRS 106 was grown on Malt Extract Agar medium in Incubator-incu Cell-v for 10 days at 25 • C and exposed every 24 h to daylight for 20 min. Such conditions were the most optimal for efficient sporulation of TRS 106. To obtain TRS 106 inoculum, the spores of the fungus from one Petri plate were washed off the surface with 10 mL of 0.85% NaCl solution. B. cinerea isolate 1631, an effective pathogen of tomato plants, was obtained from the Bank of Plant Pathogens (Poznań, Poland) and was maintained in stock culture on potato dextrose agar (PDA) in the dark at 24 • C for 14 days. The conidial suspension was prepared by washing PDA cultures with tap water supplemented with 0.3 mM H 2 KPO 4 and 2.2 mM glucose and diluted to obtain 1 × 10 6 of the spores per 1 mL.

Plant Material, Growth Conditions, and Treatment of Plants with T. virens TRS 106 and B. cinerea
Six-week-old tomato plants (S. lycopersicum L.) belonging to two plant varieties susceptible to B. cinerea, i.e., 'Perkoz'and 'Remiz', were used in the experiment. Both varieties are F 1 hybrids with functional sterility controlled by the ps gene, registered in the Polish plant variety database. They are used commercially and recommended for cultivation in greenhouses [44]. The plants were grown under controlled conditions (one plant per pot with sowing potting soil and perlite 1:0. 25 (v:v)). Ten days after sowing, the growing substrate of half of the tomato seedlings was supplemented with prepared TRS 106 spore suspension to obtain 10 6 spore density per 1 g of the substrate. The plants were cultivated for six weeks in a chamber at a temperature of 25/20 • C with a 14/10 h day/night photoperiod at 80% relative humidity. The light was supplied by white fluorescent lamps (type 36W, Philips TDL 36/84) at 150 µEm −2 s −1 intensity. After six weeks, the third fully expanded leaves on half of the control plants and half of the plants grown in the soil with TRS 106 were inoculated with a spore suspension of B. cinerea (1 × 10 6 of the spores per 1 mL) (Figure 2b). B. cinerea suspension was placed in 30 µLdrops on the adaxial leaf surface, one drop per 2 cm 2 of the leaf. After inoculation, the tomato plants were placed into the chamber at a temperature of 20 • C and 95% relative humidity, conditions favorable to the B. cinerea infection development. After 72 h, the disease area on the leaves of tomato plants was evaluated using photographs processed in a Motic Images Plus 2.0ML program (Motic China Group, Asia) according to the manufacturer's instruction. The disease area of each inoculated leaf was presented as % of the total area of the leaf. The experiment was prepared four times under the same conditions. In each experiment, the samples for disease area determination were prepared in triplicate per variant. In both tomato the chamber at a temperature of 20 °C and 95% relative humidity, conditions favorable to the B. cinerea infection development. After 72 h, the disease area on the leaves of tomato plants was evaluated using photographs processed in a Motic Images Plus 2.0ML program (Motic China Group, Asia) according to the manufacturer's instruction. The disease area of each inoculated leaf was presented as % of the total area of the leaf. The experiment was prepared four times under the same conditions. In each experiment, the samples for disease area determination were prepared in triplicate per variant. In both tomato varieties, four experimental groups (variants) of plants were tested: (I) plants grown in the soil without T. virens TRS 106 spores (Control), (II) plants grown in the soil with T. virens TRS 106 spores (TRS 106), (III) plants grown in the soil without T. virens TRS 106 spores, inoculated with B. cinerea (Bc), and (IV) plants grown in the soil with T. virens TRS 106 spores, inoculated with B. cinerea (TRS 106 + Bc) ( Figure 1).

Determination of Superoxide (O2 − ) and Hydrogen Peroxide (H2O2) Content and Histochemical Visualization
O2 − and H2O2 contents were determined according to the modified method of Doke [45] and Capaldi and Taylor [46], respectively, as we described previously [12]. The O2 − content was determined indirectly by measurement of an increase in the absorbance at 580 nm related to nitrotetrazolium blue chloride stain (NBT) reduction, and expressed as A580 g −1 of fresh weight (FW). The H2O2 content was determined indirectly by measurement of an increase in the absorbance at 630 nm related to oxidative coupling MBTH in the presence of this compound. The H2O2 content was calculated based on the standard curve of H2O2 and expressed in µmol per g of FW.
Histochemical visualizations of O2 − and H2O2 were performed according to the modified method of Romero-Puertas et al. [47] and Thordal-Christensen et al. [48], respectively, as we described previously [12]. Regarding O2 − detection, tomato leaves were incubated in a solution of NBT, and, to detect H2O2, in a solution of 3,3′-diaminobenzidinetetrahydrochloride stain (DAB). After staining, all the leaves were discolored with 95% ethanol, leaving only places with detected O2 − and H2O2. The leaves were preserved at RT in ethanol and photographed.   [45] and Capaldi and Taylor [46], respectively, as we described previously [12]. The O 2 − content was determined indirectly by measurement of an increase in the absorbance at 580 nm related to nitrotetrazolium blue chloride stain (NBT) reduction, and expressed as A 580 g −1 of fresh weight (FW). The H 2 O 2 content was determined indirectly by measurement of an increase in the absorbance at 630 nm related to oxidative coupling MBTH in the presence of this compound. The H 2 O 2 content was calculated based on the standard curve of H 2 O 2 and expressed in µmol per g of FW.

Determination of Nitric Oxide (NO), Peroxynitrite (ONOO − ), and S-Nitrosothiols (SNO) Content and Histochemical Visualization of NO
Histochemical visualizations of O 2 − and H 2 O 2 were performed according to the modified method of Romero-Puertas et al. [47] and Thordal-Christensen et al. [48], respectively, as we described previously [12]. Regarding [49], as we described previously [12]. The reaction mixture contained the leaf extract and its equivalent volume of Griess reagent (Sigma-Aldrich, St. Louis, MO, USA). Absorbance related to the reaction of NO ions with Griess reagent to form a chromophoric azo product was measured at 540 nm. Indirectly, NO content was calculated by comparison to a standard curve using NaNO 2 and expressed in nmol per g of FW.
Determination of the ONOO − content was performed according to the modified method of Huang et al. [50]. Leaf discs (500 mg) were immersed in an incubation mixture containing a barbital buffer solution (pH 9.4) and folic acid (10 −5 M). After 30 min incubation at RT, the fluorescence intensity of the solution, related to scavenging of ONOO − by folic acid, was recorded at 460 nm with the excitation wavelength set at 380 nm. The ONOO − content was calculated by comparison to a standard curve using SIN-1, ONOO − donor, and expressed in nmol per g of FW.
Determination of the SNO content was performed according to the modified method of Rustérucci et al. [51], as we described previously [12]. The extracts were incubated with an equivalent volume of 1% sulfanilamide solution or with that of 1% sulfanilamide with HgCl 2 , which by hydrolyzing SNO allows for the formation of the diazonium salt. Absorbance related to the formation of the azo dye product, obtained through the reaction of diazonium salts with N-(1-naphthyl)ethylenediamine dihydrochloride (NEA), was read at 550 nm. The SNO content was calculated by comparison to a standard curve made from the solution of S-nitrosoglutathione (GSNO). The results were expressed in nmol SNO per mg of protein.
Histochemical visualization of NO was performed according to the modified method of Corpas et al. [52] and Piterková et al. [53], as we described previously [12]. Sections of tomato leaves were incubated in a solution of 4,5-diaminofluorescein diacetate (DAF-2DA). and examined under a confocal laser scanning microscope system (Leica TCS SP8; Leica Microsystems, Mannheim, Germany), using standard filters and collection modalities for DAF-2 green fluorescence (excitation 488 nm; emission 530 nm). The production of green fluorescence under the presented conditions was attributed to the presence of NO. As negative controls, some leaf pieces, before staining with DAF-2DA, were immersed in a solution of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (Sigma-Aldrich), which eliminates NO. The slides were scanned using the Leica LAS-AF program, version 3.3.0. Determination of SOD and GSNOR activities were performed following the method of Beauchamp and Fridovich [54] and Sakamoto et al. [55], respectively, as we described previously [12]. SOD activity analysis was based on monitoring its ability to inhibit the photochemical reduction of NBT, resulting in changes in the absorbance at 560 nm. The SOD ability to inhibit 50% of the photochemical reduction of NBT as compared to the control was used as one unit of its activity normalized per mg of protein. GSNOR activity analysis was based on monitoring its ability to reduce GSNO by using NADH, resulting in changes in the absorbance at 340 nm. GSNOR activity was calculated based on the millimolar extinction coefficient of NADH ε = 6.22 mM cm −1 and presented in enzymatic units of NADH min −1 per mg of protein.
Protein was determined by the method of Bradford [56], as we described previously [12], with standard curves prepared using bovine serum albumin (Sigma-Aldrich).

Determination of Volatile Organic Compounds (VOC) Emission, including Green Leaf Volatiles (GLV) and Aromatic Compounds
Solid-phase microextraction (SPME) used for the determination of VOC was carried out with the method of Carlin et al. [57], as we described previously [8]. All samples were analyzed by GCxGC TOF-MS. Analysis was performed using a Pegasus 4D mass spectrometer equipped with a consumable-free dual-stage, quad-jet thermal modulator (LECO Corp.). A BPX5 (30 m, 0.25 mm, 0.25 µm) was used as a first-dimension (1D) column, and a BPX50 (2 m, 0.1 mm, 0.1 µm) was used as a second-dimension (2D) column. The mass spectra of detected compounds were identified with the aid of the National Institute of Standards and Technology Mass Spectral Library (version 2.0g). Each VOC content was presented as a relative peak area.

Statistical Analyses
After confirmation of the data normality and homogeneity of variances, the effect of TRS 106 and B. cinerea treatments on each parameter were checked with: (i) Student's t-test, where-separately for each tomato variety-only two variants were compared, e.g., control and TRS 106 or Bc and TRS 106 + Bc plants, or (ii) one-way ANOVA and Tukey's HSD post-hoc test, when-separately for each variety-all four variants were compared (small letters, a, b, c, etc., in the Figures). Differences between varieties within a given treatment were analyzed with Student's t-test (capital letters A and B in the Figures). The influence of the variety ('Perkoz' and 'Remiz'), TRS 106, and B. cinerea treatment and their interaction on the investigated traits were checked with two-way ANOVA. For each test, differences were accepted as significant at the value of p < 0.05. All statistical evaluations were conducted using Statistica 13.0 software (StatSoft Inc., Palo Alto, CA, USA).

T. virens TRS 106 Suppresses B. cinerea Infection of Tomato Plants
To study whether TRS 106 protects tomato plants against B. cinerea, the disease symptoms were observed on the leaves of Bc and Bc + TRS 106 plants. The successful infection of Bc plants by B. cinerea began within 48 h and was optimal for biochemical analyses 72 h after inoculation. The grey mould symptoms caused by the pathogen were identified as brown lesions, dark brown blight blotches, and rot symptoms on leaves, spreading irregularly from the pathogen inoculation sites (Figure 2a).   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 2.
Comparing the plants belonging to two tomato varieties, the area of infected leaves was greater in 'Remiz' than in 'Perkoz' plants. The most extensive disease area development was observed in Bc 'Remiz' plants, reaching on average 45 % of the leaf area ( Figure 2b). The disease symptoms were significantly suppressed in plants belonging to both varieties when spores of TRS 106 were added to the plant soil, as indicated by a significant reduction of the disease area on the leaves of TRS 106 + Bc plants as compared to the respective Bc plants. As compared to Bc plants, TRS 106 caused a pronounced disease area decrease on the leaves of TRS 106 + Bc plants from on average 45 to 29% and 27 to 15% in 'Remiz' and 'Perkoz' plants, respectively, with the latter being the weakest observed symptoms of the disease. Considering two-way ANOVA, both variety and treatment significantly affected O2 − and H2O2 content in tomato plants, whereas SOD activity was significantly affected by the treatment and interaction of variety and treatment (Table S2). The content of O2 − in 'Remiz' plants was significantly higher as compared to 'Perkoz' plants in all tested variants, while the content of H2O2 in 'Perkoz' plants was significantly higher as compared to 'Remiz' plants in Bc and TRS 106 + Bc plants (Figure 3a). 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.

RNS Content and GSNOR Activity
Two-way ANOVA test showed that variety, treatment, and interaction between them significantly affected NO, ONOO − and SNO contents in the tomato plants, whereas GSNOR activity was significantly affected by variety and treatment (Table S2). The contents of NO and ONOO − in 'Remiz' plants were significantly higher as compared to 'Perkoz' plants in all tested variants (Figure 4). Excluding Bc plants, the activity of S-nitrosoglutathione reductase (GSNOR) was significantly higher in 'Remiz' plants as compared to 'Perkoz' plants, and excluding TRS 106 + Bc plants, the S-nitrosothiols (SNO) content was significantly higher in 'Perkoz' plants as compared to 'Remiz' plants. In   . 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.
The histochemical visualization of NO in cross sections of tomato leaves showed that fluorescence emitted by NO was observed systemically throughout leaf tissues in plants belonging to both varieties, however, stronger fluorescence signals of NO were detected in 'Remiz' plants as compared to 'Perkoz' plants ( Figure 5). In TRS 106 and TRS 106 + Bc plants of both varieties, stronger fluorescence signals of NO were detected as compared . 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 2.
The histochemical visualization of NO in cross sections of tomato leaves showed that fluorescence emitted by NO was observed systemically throughout leaf tissues in plants belonging to both varieties, however, stronger fluorescence signals of NO were detected in 'Remiz' plants as compared to 'Perkoz' plants ( Figure 5). In TRS 106 and TRS 106 + Bc plants of both varieties, stronger fluorescence signals of NO were detected as compared to the respective control and Bc plants. NO signals were observed mainly in the parenchyma tissue, in the area of vascular bundles, and to a smaller extent in the epidermal tissues ( Figure 5), as well as in palisade mesophyll and to some extent in the spongy mesophyll ( Figure 6). In the control and Bc plants of both varieties, NO was accumulated as punctate distribution patterns mainly inside the cells, in the cytoplasm, close to the cell walls, and in the case of Bc plants, in chloroplasts. In TRS 106 plants the NO location was mainly apoplastic and to a smaller extent symplastic, where NO was detected in the nuclei of parenchyma and palisade mesophyll cells. Regarding TRS 106 + Bc variant, in 'Perkoz' plants, NO was detected in the cell nuclei of palisade mesophyll cells, and in 'Remiz' plants, NO was observed both in the nuclei and some chloroplasts in the parenchyma and palisade mesophyll cells.

GLV and Aromatic Compounds Emission
SPME coupled with GCxGC TOF-MS analysis detected 24 VOC belonging to two volatile groups, i.e., GLV and aromatic compounds, emitted by tomato plants. Two-way ANOVA test showed that variety, treatment, and interaction of both, significantly affected 10 GLV being volatile aldehydes and alcohols as well as 12 aromatic compounds belonging to volatile aldehydes, ketones, salicylates, and alcohols (Table S2).
Regarding GLV, the emission of 2-and 3-hexenal, hexanal, 1-hexanol, and 2-ethyl-1hexanol in 'Perkoz' plants was significantly higher as compared to 'Remiz' plants in all tested variants, and 2-hexen-1-ol was significantly higher in 'Remiz' plants as 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 S2). 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.

GLV and Aromatic Compounds Emission
SPME coupled with GCxGC TOF-MS analysis detected 24 VOC belonging to two volatile groups, i.e., GLV and aromatic compounds, emitted by tomato plants. Two-way ANOVA test showed that variety, treatment, and interaction of both, significantly affected 10 GLV being volatile aldehydes and alcohols as well as 12 aromatic compounds belonging to volatile aldehydes, ketones, salicylates, and alcohols (Table S2).
Regarding GLV, the emission of 2-and 3-hexenal, hexanal, 1-hexanol, and 2-ethyl-1hexanol in 'Perkoz' plants was significantly higher as compared to 'Remiz' plants in all tested variants, and 2-hexen-1-ol was significantly higher in 'Remiz' plants as compared to 'Perkoz' plants in all tested variants (Figure 7). Excluding 2,4-hexadienal (TRS 106 plants), 2-hexanol (Bc plants), and 4-methyl-3-hexanol (control plants), the emission of these compounds was significantly higher in 'Perkoz' plants as compared to 'Remiz' plants. As compared to the control, B. cinerea inoculation increased the emission of four 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 2.
Additionally, in TRS 106 'Remiz' plants, 2,4-hexadienal emission increased as compared to the control and Bc plants, and 2-hexen-1-ol and hexanal emission increased as compared to the control, while in TRS 106 + Bc 'Remiz' plants, 2-hexenal and 2-hexyn-1-ol emission increased as compared to the control, Bc, and TRS 106 plants. Two compounds were detected only in the plants treated with Trichoderma, 4-ethyl-2-hexynal in TRS 106 + Bc 'Perkoz' plants, and 1,5-hexadien-3-ol in TRS 106 + Bc 'Perkoz' and 'Remiz' plants (Figure 7). 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 2.
Regarding aromatic compounds, the emission of benzaldehyde, 4-ethylbenzaldehyde, and 2-methylphenol in 'Perkoz' plants were significantly higher as compared to 'Remiz' plants in all tested variants (Figure 8). Excluding acetophenone and methyl salicylate (control plants), 2-hydroxyacetophenone and isoamyl salicylate (control and Bc plants), 4-methylbenzaldehyde (TRS 106 plants), ethyl salicylate (Bc plants), and 4-hepten-2-yl salicylate (control and TRS 106 + Bc plants), the emission of these compounds was significantly higher in 'Remiz' plants as compared to 'Perkoz' plants. As compared to the control, B. cinerea inoculation increased the emission of three aromatic compounds in Bc 'Perkoz' plants and six in Bc 'Remiz' plants. In Bc 'Perkoz' plants, these were 2-hydroxyacetophenone, ethyl salicylate, and phenol, and in 'Remiz' plants 2-methylbenzaldehyde, acetophenone, methyl salicylate, 4-hepten-2-yl salicylate, phenol, and 2-methylphenol. As compared to the control, Trichoderma increased the emission of six and three aromatic compounds in TRS 106 'Perkoz' and TRS 106 + Bc 'Perkoz' plants, respectively, as well as of nine aromatic compounds in both TRS 106 'Remiz' and TRS 106 + Bc 'Remiz' plants. In 'Perkoz' plants, 4-ethylbenzaldehyde and methyl salicylate emission increased in TRS 106 and TRS 106 + Bc as compared to the control and Bc plants, and 2-Hydroxyacetophenone emission as compared to the control. Simultaneously, in TRS 106 'Perkoz' plants, significant increases in benzaldehyde, 4-methylbenzaldeyde, and ethyl salicylate emission, as compared to the control, Bc, and TRS 106 + Bc plants, were observed. Regarding TRS 106 and TRS 106 + Bc 'Remiz' plants, the significant increase in 2-hydroxyacetophenone, 2-methoxyphenol emission as compared to the control and Bc plants, and the significant increase in acetophenone, methyl salicylate, 4-hepten-2-yl salicylate, and phenol as compared to the control, was observed. Additionally, in TRS 106 'Remiz' plants, 4-ethylbenzaldehyde and 2-methylphenol emissions increased as compared to the control, Bc, and TRS 106 + Bc plants, and 4-methylbenzaldeyde emission increased as compared to the control and TRS 106 + Bc plants. In TRS 106 + Bc 'Remiz' plants, the increases in benzaldehyde, ethyl salicylate, and 4-hepten-2-yl salicylate emission as compared to the control, Bc, and TRS 106 plants, were observed. Additionally, one compound, isoamyl salicylate was detected only in 'Remiz' plants treated with Trichoderma ( Figure 8).

Discussion
In this study, we focus on the innate and induced defense responses of tomato plants belonging to two greenhouse varieties 'Perkoz' and 'Remiz', which according to the unofficial farmers' observations show, respectively, weaker and stronger symptoms of

Discussion
In this study, we focus on the innate and induced defense responses of tomato plants belonging to two greenhouse varieties 'Perkoz' and 'Remiz', which according to the unofficial farmers' observations show, respectively, weaker and stronger symptoms of mould disease caused by B. cinerea. The innate defense may be dependent on modification of the plant cell wall, lignin and callose deposition, stomatal guard cells functioning, water and photoassimilates transport, and many other features regarding plant anatomy, physiology, biochemistry, and genetics [58,59]. In the present work, we focus on identifying biochemical differences in the generation of ROS, RNS, and VOC, which are important elements of plant defense responses against pathogens. Our results confirmed the observations of farmers that the successful infection of Bc plants was faster and more effective in the plants belonging to 'Remiz' variety. Moreover, the studies showed that when spores of T. virens TRS 106 were added to the soil, a significant decrease in the spread of the disease in both 'Perkoz' and 'Remiz' plants occured. In some inoculation sites, complete inhibition of disease development was observed. In addition to the protection against B. cinerea and R. solani, as we described previously [42], TRS 106 showed the ability to enhance tomato plant growth. Since the simultaneous growth promotion without compromising the level of plant protection against pathogens and without fitness costs for plants are highly valuable [60], TRS 106 strain shows the appropriate potential to act as a BCA of tomato grey mould and as a biostimulator of tomato growth and development.
Taking into account our preliminary studies which showed the moderate ability of TRS 106 to directly suppress B. cinerea development, and excluding direct contact of TRS 106, which was applied to the soil with B. cinerea used in foliar inoculation, the induction of defense responses as the main way of tomato plant protection against this pathogen was considered. The activation of defense responses was previously observed in other plants protected by different Trichoderma strains, i.e., in cucumber protected against B. cinerea, Pseudoperonospora cubensis, Sclerotinia sclerotiorum, Sphaerotheca fusca, and Pseudomonas syringae [61,62], or tomato and onion protected against F. oxysporum [63,64]. In the present study, the obtained results pointed to different molecules, including ROS, RNS, and VOC, that may be involved in divergent defense responses against B. cinerea induced by TRS 106 in 'Perkoz' and 'Remiz' plants, varying in their self-protection response to the pathogen. Some of the molecules, especially those belonging to GLV and aromatic compounds, were not detected in the defense responses induced by Trichoderma in plants until now.
ROS and RNS are important molecules involved in plant responses to biotic stress such as attacks by pathogenic bacteria, fungi, nematodes, and insects [65][66][67]. The dual, negative and positive role of ROS and RNS in plant response to pathogens is known [65,67]. In high, uncontrolled concentrations, they may cause damage to membranes, DNA, proteins, carbohydrates, and lipids in plant cells [68][69][70]. On the other hand, ROS and RNS play an important role in the development of plant defense responses and resistance, including those induced by BCA such as Trichoderma spp. [71][72][73]. In this approach, ROS are usually generated locally during a fast oxidative burst, for example in the hypersensitive response (HR), and then slowly, systemically throughout the plant tissues. The mechanism of RNS generation seems to be more complicated [70,74]. The results of the present studies suggest that ROS and RNS are generated differently during the defense responses of 'Perkoz' and 'Remiz' plants to B. cinerea. 'Remiz' plants nontreated with Trichoderma, after inoculation with B. cinerea, generated more O 2 − and ONOO − . In 'Perkoz' plants nontreated with Trichoderma, in which the disease development was slower and less intensive, stronger H 2 O 2 accumulation, lower NO accumulation, and decreased generation of SNO with simultaneously increased GSNOR activity were observed. In the previous studies, it was demonstrated that during the successful infection, B. cinerea actively contributed to the generation of an oxidative burst related to O 2 − but not H 2 O 2 accumulation in tomato and A. thaliana plants [75]. O 2 − may act as an oxidant directly, and by fast reaction with NO participates in generation of ONOO − , which, in high concentrations, promotes uncontrolled oxidation and nitration of key cellular molecules, which may negatively affect the functioning of plant cells [76,77]. Therefore, we suppose that stronger generation of O 2 − and ONOO − in 'Remiz' plants may not positively influence the defense responses of tomato and facilitate the effective spreading of B. cinerea infection.
Interestingly, in the present study, in both tomato varieties, TRS 106 seemed to direct ROS metabolism towards H 2 O 2 generation and systemic spreading of O 2 − and H 2 O 2 location within B. cinerea-inoculated plant leaf, similarly to T. virens and T. atroviride, which caused H 2 O 2 accumulation in A. thaliana [6,78]. The systemic increase in H 2 O 2 content in plants treated with TRS 106 strain seems to confirm its role as one of the signaling molecules of induced defense responses. H 2 O 2 is more stable as compared to other ROS, relatively long-lived, and freely diffusible across membranes, which enables it to act as an important player in signal transduction pathways [79,80]. Regarding RNS, the TRS 106 influence was different in 'Perkoz' and 'Remiz' plants. In 'Perkoz' plants, both uninoculated and inoculated with B. cinerea, TRS 106 enhanced the accumulation of NO, which might be partly related to the heightened activity of GSNOR and reduced content of SNO, an important reservoir of NO, as described in other pathosystems [81][82][83]. On the other hand, in 'Remiz' plants, which generated more RNS than 'Perkoz', TRS 106 lowered the ONOO − increase and seemed to influence plants to use the excess of NO to S-nitrosylationdependent SNO formation. S-nitrosylation was presented as an important process during plant defense responses against different pathogens, e.g., A. thaliana and cucumber effective defense responses against P. syringae and R. solani, respectively [12,84]. GSNOR, an enzyme involved in a reversible process of S-nitrosylation [85], is one of the key modulators of NO signaling, which is usually down-regulated in effectively infected plants [86] and upregulated in these effectively protected against different pathogens [87], which is in line with our results with regard to 'Perkoz' plants. The accumulation of different RNS, especially NO, was observed during other plant-microbe interactions. In many studies, similarly to H 2 O 2 , NO is considered an important mediator involved in plant defense responses against various pathogens [88][89][90]. Regarding the protection of plants against B. cinerea, enhanced NO generation was observed, for example, in tomato plants treated with βaminobutyric acid or abscisic acid [88], and tomato fruits treated exogenously with NO [91]. Concerning Trichoderma interaction with plants, the role of NO and RNS is currently the subject of intense research. For example, NO signaling was required in A. thaliana roots for activation of Trichoderma VOC-mediated ISR against B. cinerea [11], and in cucumber leaves for activation of local and systemic defense responses against R. solani [12]. On the other hand, it was presented that T. harzianum could alleviate oxidative and nitrosative stress by minimizing H 2 O 2 , O 2 − and NO accumulation under F. oxysporum infection in cucumber roots [92].
The use of NO by the plant as a signaling molecule of defense responses and resistance is strongly related to the timing, extent, and location of NO accumulation [93,94]. Regarding the timing of NO generation in plants treated with Trichoderma, we previously established that its long-time, systemic accumulation in cucumber plants protected them effectively against R. solani [12]. Similarly, Schlicht and Kombrink [94] showed that a short burst and then sustained and controlled NO production prevented A. thaliana leaf colonization by the biotrophic fungi, Golovinomyces orontii and Erysiphe pisi. Little information is available about the spatial accumulation of NO during Trichoderma-triggered defense responses and resistance in plants. In the present studies, the systemic location of NO mainly in the parenchyma, in the area of vascular bundles, in the palisade mesophyll, and to a smaller extent in the epidermis and spongy mesophyll, was similar for plants belonging to both 'Perkoz' and 'Remiz' variety. Regarding the subcellular NO location, B. cinerea and Trichoderma caused the same kind of changes in plants belonging to both varieties. Chloroplasts are the sites of production of ROS, NO, JA, and SA, in which NO can provoke both beneficial and harmful effects [95,96]. NO accumulation in chloroplasts, observed in Bc plants and reduced or absent in plants belonging to the other variants, suggested that this NO location was rather a marker of the stress response than an effective defense response of plants to B. cinerea. However, further studies are required to determine if NO can interfere with chloroplast functionality, for example, by influencing photosynthesis, CO 2 uptake, electron transport functioning, or ATP synthesis in the Bc plants, as suggested in other stressed plants [96,97]. On the other hand, in plants showing decreased infection with B. cinerea, TRS 106 suppressed the appearance of the NO signal in chloroplasts, and at the same time caused the emission of strong NO signals from the nuclei, and additionally from the apoplast. The location of NO in the nuclei, mainly in the parenchyma and palisade mesophyll cells, suggests its functioning at the molecular level. According to the suggestion of del Río [65], both RNS and ROS may be involved in posttranslational modifications (PTMs) of proteins, however, the range of NO influence may be dominant. The activity of NO in the nuclei of Trichoderma-treated plants needs further investigation. Although based on the other studies, we may suppose that in tomato cells, NO may cause changes at the transcriptomic level by several main processes, i.e., S-nitrosylation or nitration of nucleus-located proteins with different functions [98][99][100]. Modification of the activity of the SA signaling component, NPR1 protein [101], and of the genes encoding the ROS generating complex NADPH oxidase, AtRBOHD [16] during defense responses, are notable examples of the role of NO-mediated S-nitrosylation present in nuclei. The location of NO in the apoplast may, in turn, suggest its role as a signaling molecule of defense responses [102,103], in which NO together with H 2 O 2 may modulate the synthesis and activities of defense-related plant hormones including SA, JA, and ET [71,72,104]. Furthermore, we may speculate that as the signaling molecule, NO can participate in resistance induction, as presented in cucumber TISR induced by T. atroviride against R. solani [8,12], in resistance largely dependent on JA induced in A. thaliana by Trichoderma against B. cinerea [78], or in tomato plants protection enhanced by Trichoderma viride against F. oxysporum or R. solani [105]. However, to confirm this, additional analyses at the molecular level are necessary.
Recently, consideration has been given to VOC, which are precursors and derivatives of phytohormones, emitted by plants as a consequence of mechanical damage, an insect attack, a pathogen infection, and abiotic stresses [18], and that may play the role of signaling molecules of defense responses [12,33]. Recent studies showed that VOC emission might be the result of plant response to the BCA. As an example, T. harzianum, T. asperellum, and T. virens were shown to differentially enhance VOC production in plants, affecting a variety of biochemical pathways related to lipids and shikimate metabolism [106]. In the present study, the preliminary analyses showed no significant changes in the content of JA and ET in tomato plants (data not shown). On the other hand, in the same plants, the emission of VOC belonging to two volatile groups, i.e., GLV and aromatic compounds, which may be related to JA and SA signaling, respectively, were detected. Many of the assessed 24 VOC, belonging to aldehydes, alcohols, and ketones, have not yet been considered in plant defense responses and resistance induced by Trichoderma against B. cinerea. In general, 'Perkoz' plants released more GLV, than 'Remiz' plants. Taking  in 'Perkoz' plants, as well as benzaldehyde, 4-methylbenzaldehyde, acetophenone, 4hepten-2-yl salicylate, phenol, and 2-methoxyphenol. Among the compounds which were released only by plants treated with Trichoderma, the following VOC should be mentioned, in TRS 106 + Bc 'Perkoz' plants, 4-ethyl-2-hexynal, 1,5-hexadien-3-ol, and 4-hepten-2-yl salicylate, as well as in TRS 106 + Bc 'Remiz' plants, 1,5-hexadien-3-ol and isoamyl salicylate. It seems that despite the differences, both blends of VOC including GLV and salicylates may participate in the enhanced protection of tomato plants against B. cinerea. Regarding GLV, their close relation to JA generation was previously confirmed. The group includes unsaturated fatty acid derivatives which are generated by a biosynthetic pathway similar to that for jasmonates [107]. Even though it is still unknown how plants sense GLV, based on the previous reports, we could suppose that they may induce metabolomic, transcriptomic, and behavioral reactions in organisms. Several GLV, whose emission increased in TRS 106 treated tomato plants, were presented in other studies to directly suppress the development of pathogens. For example, E-2-hexenal and Z-3-hexenol, with antimicrobial activity, prevented microbes from invading plants as well as participated in stronger lignification of plant tissues [108,109]. The involvement of several GLV, including 2-hexenal, 3-hexenal, and 3-hexen-1-ol in the induction of defense responses in A. thaliana against B. cinerea was suggested as well [107]. The treatments of A. thaliana seedlings with 2-hexenal induced the formation of phytoalexins as well as activated defense-related genes [107,109]. Similarly, GLV induced defense responses in lima bean and corn, in which they primed higher production of JA and sesquiterpene volatiles [110,111]. 2-hexanol strongly emitted in plants, with enhanced defense responses against B. cinerea was previously presented to elicit defense responses in grapes [112]. 1-hexanol and 2-ethyl-1-hexanol released by chickpea and/or wheat were important GLV toxic to pathogens, Fusarium graminearum, and Fusarium avenaceum [113]. Hexanal induced resistance of banana (Musa acuminata L.) against Colletotrichum gloeosporioides and Lasiodiplodia theobromaemajor [114], and in combination with bacterial antagonists, Pseudomonas fluorescens and Bacillus subtilis, induced defense-related enzymes, involved in the protection of mango fruits against Lasiodiplodia theobromae [115]. In turn, 2-Hexyn-1-ol was an important component of Calotropis gigantea leaf essential oil, and 1,5-Hexadiene-3-ol was an important component of Capsicum fruit extract with antimicrobial activity against selective bacterial and fungal pathogens [116,117]. Regarding 4-ethyl-2-hexynal, detected only in the TRS 106 + Bc 'Perkoz' variant, its emission by Stevia rebaudiana increased with elevated temperature and carbon dioxide treatment [118]. Other GLV emitted in plants with enhanced defense responses against B. cinerea, such as 4-methyl-3-hexanol and 1,5-hexadien-3-ol, were not detected in other plants treated with different pathogens. Therefore, their role in the protection of plants against B. cinerea remains to be discovered. Regarding salicylates, the present results indicated that defense responses induced by TRS 106 against B. cinerea, in parallel with GLV, might be largely dependent on the derivatives of SA, as in the case of A. thaliana protected by T. asperellum against cucumber mosaic virus [119], where SA played a crucial role in the upregulation of defense genes and active resistance mechanisms. The tested TRS 106 strain significantly enhanced the emission of methyl salicylate, which is SA derivative, involved in the longdistance signaling of SAR [120]. Systemic release of methyl salicylate, accompanied by the emission of other VOC, characteristic of SAR or ISR, was observed, for example, in tomato plants treated with T. asperellum, protected against herbivory Tuta absoluta [121], or treated with T. harzianum, and protected against B. cinerea [122]. Taking into account other volatile derivatives of SA, we suppose that ethyl salicylate, 4-hepten-2-yl salicylate, and isoamyl salicylate may also affect tomato defense responses, which enhance protection against B. cinerea. However, their role needs further elucidation.
In the tested pathosystem, possible cooperation of the H 2 O 2 , NO, GLV, and salicylates in the signaling of defense responses induced by Trichoderma, is described for the first time. Based on the results of the other studies, we speculate that NO may influence the accumulation of volatile SA derivatives for instance by S-nitrosylation and inhibition of enzymes involved in H 2 O 2 and SA removal [83,123]. On the other hand, generated salicylates may modulate back the content of H 2 O 2 or NO [65,110]. The network of H 2 O 2 , NO, GLV, and different SA derivatives in tomato plants become more complicated as it is accompanied by a significant accumulation of other aromatic compounds, which recently were found to protect plants against different diseases. For example, 4-ethylbenzaldehyde, emission of which increased in 'Perkoz' plants treated with TRS 106, was shown to be emitted by Eruca sativa and to be a component of VOC blend which had a nematicidal potential against M. incognita [124]. Regarding 2-hydroxyacetophenone and acetophenone, emitted intensively mainly by 'Remiz' plants treated with TRS 106, their important roles as antimicrobial compounds were determined in conkerberry (Carissa lanceolata) roots [125]. Benzaldehyde and 4-methylbenzaldehyde showed fungistatic and antibiotic activity against different pathogens and insects, as presented in other studies [126,127]. Finally, all these compounds, together with phenol, and 2-methoxyphenol might be important precursors of different metabolites with antimicrobial activity, and, at the same time, they may play an important role in defense responses and resistance induction [128,129]. VOC present some advantages over other signaling molecules. For example, they are effective at low concentrations, they diffuse through plants easily and quickly, and they can act on pathogens without establishing actual physical contact with them [33]. VOC signals seem to be faster than vascular signal molecules and can also reach organs not directly connected through the vascular system. In this model, vascular and airborne signals may act synergistically to ensure optimal protection against pathogens in distal plant parts [108]. Therefore, the detected molecules together with NO and H 2 O 2 might be able to directly elicit or prime plant systemic defense responses against B. cinerea. Even though it was established that VOC might react with ROS and RNS in cells [107], the mechanisms of interaction between these compounds during defense responses and resistance induction by Trichoderma and other BCA are unknown. Therefore, further research will focus on temporal and spatial interactions between these molecules.

Conclusions
In summary, in the present study, the important role of T. virens TRS 106 as BCA, which significantly increases tomato plant (S. lycopersicum L.) growth and decreases grey mould disease in them, is suggested. We excluded direct contact of TRS 106, which was applied to the soil, with B. cinerea, used in foliar inoculation, and using this system we could prove that the induction of defense responses is the main method of tomato plant protection against this pathogen. Treatment of the tomato plants with TRS 106 induced oxidative, signaling and biochemical changes, which were very important during the induction of plant defense responses. In the tested plant-microbial system including TRS 106 we suggest, for the first time, cooperation of redox-active small molecules belonging to ROS and RNS, with GLV and aromatic compounds, especially salicylates, in plant defense responses. The biochemical, histochemical, and SPME-GCxGC TOF-MS analyses revealed that 'Remiz' plants, more seriously infected by B. cinerea, than 'Perkoz' plants, generated more ONOO − , O 2 − in the infection site, and NO in chloroplasts, as well as less H 2 O 2 , SNO, and GLV. Moreover, they exhibited higher activities of SOD and GSNOR. In plants belonging to both varieties, with decreased grey mould disease symptoms, TRS 106 increased the accumulation of H 2 O 2 in whole leaves and NO in the apoplast and in the cell nuclei, especially in the parenchyma and palisade mesophyll, and increased GSNOR activity. In 'Perkoz' plants, TRS 106 decreased SNO generation, and in 'Remiz' plants, it decreased O 2 − and increased SNO generation. In this case, our results suggest that in plants protected against the pathogen by Trichoderma, a rearrangement of the production, turnover, and location of ROS and RNS occurred, regulating their bioavailability toward the accumulation of their most stable forms. i.e.,H 2 O 2 and NO. In the same plants, 24 VOC belonging to GLV and aromatic compounds, including salicylates, were detected. TRS 106 increased the emission of five GLV and three aromatic compounds in 'Perkoz' plants, and five GLV and eight aromatic compounds in 'Remiz' plants. The novel compounds detected only in the Trichoderma-treated plants were 4-ethyl-2-hexynal, 1,5-hexadien-3-ol, and 4-hepten-2-yl salicylate in 'Perkoz' plants, and 1,5-hexadien-3-ol and isoamyl salicylate in 'Remiz' plants. It seems that both, quite different modes of biochemical changes induced by Trichoderma in two tomato varieties may participate in the induction of defense responses against B. cinerea, which may involve pathways similar to those presented in TISR. To be able to explain the exact relationships between the identified compounds, more precise studies of their spatial and temporal synthesis, transport, and emissions are required. The obtained knowledge may confirm the role of TRS 106 as BCA of B. cinerea, and be useful for scientific research focusing on the development of eco-friendly methods of protecting plants against this pathogen, as alternatives to chemicals for crop protection, the use of which is being the subject to increasing law restrictions.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11193051/s1, Figure S1: A negative controls for histochemical detection of NO, prepared by immersing of leaf pieces in cPTIO, which eliminate NO by the production of nitrites; Table S1: Effect of TRS 106 on tomato growth parameters. Values represent the means + SE from four independent experiments with four replicates each (n = 16). Separately for each parameter, differences between varieties within a given treatment were analyzed with Student's t-test and marked using letters A and B. Separately for each variety, differences between TRS 106 and control plants were analyzed with Student's t-test and marked using an asterisk symbol with the following manner (* p < 0.05; ** p < 0.01; *** p < 0.001). The influence of the variety (Perkoz and Remiz) and TRS treatment and their interaction on the investigated traits was checked with two-way ANOVA. Abbreviations: TRS 106, plants grown in the soil with T. virens TRS 106 spores; FW, fresh weight; DW, dry weight; Table S2: Results of two-way ANOVA investigating effects of studied treatments on the tested parameters of the studied tomato varieties.