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Article

The Status of Esca Disease and the Disinfection of the Scion Prior to Grafting Affect the Phenolic Composition and Phenylpropanoid-Related Enzymes in the Callus of Vine Hetero-Grafts

by
Saša Krošelj
1,*,
Maja Mikulic-Petkovsek
1,
Matevž Likar
2,
Andreja Škvarč
3,
Heidi Halbwirth
4,
Katerina Biniari
5 and
Denis Rusjan
1
1
Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
2
Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia
3
Agriculture and Forestry Institute Nova Gorica, Chamber of Agriculture and Forestry of Slovenia, SI-5101 Nova Gorica, Slovenia
4
Institute for Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Getreidemarkt 9, A-1060 Vienna, Austria
5
Laboratory of Viticulture, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(4), 371; https://doi.org/10.3390/horticulturae11040371
Submission received: 10 February 2025 / Revised: 10 March 2025 / Accepted: 17 March 2025 / Published: 30 March 2025
(This article belongs to the Special Issue Sustainable Management of Pathogens in Horticultural Crops)
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Abstract

:
Vegetative propagation of European grapevine (Vitis vinifera L.) requires grafting onto American rootstocks due to susceptibility to phylloxera. However, the grafting yield is compromised by the presence of grapevine trunk diseases (GTDs) such as Esca. This study investigates the phenolic response and enzyme activity in grapevine callus from grafts obtained by scions with different GTD status (healthy, asymptomatic, and symptomatic) treated with different disinfection methods (Beltanol, Beltanol in combination with thermotherapy, Serenade® ASO, Remedier, BioAction ES, and sodium bicarbonate). Twenty-three phenolic compounds were identified in the graft callus, with flavanols, stilbenes, and condensed tannins predominating. Scion disinfection with BioAction ES led to a significant increase in total phenolic content in the callus, especially in symptomatic scions, for on average 510.3 µg/g fresh weight (FW) higher total phenolic content, compared to grafts where scions were treated with Beltanol. Phenolics such as epicatechin gallate, procyanidin derivatives, and resveratrol hexoside were significantly increased, indicating a strong elicitor effect of BioAction ES. Enzymatic activity analysis showed that the disinfection methods affected the activity of key enzymes involved in the phenylpropanoid metabolic pathway. In particular, BioAction ES significantly increased phenylalanine ammonia lyase (PAL) activity in callus from grafts with healthy scions by 3.4-fold and flavanone 3β-hydroxylase (FHT) activity in callus from grafts with infected scions by 4.9-fold (asymptomatic) and 6.9-fold (symptomatic) compared to callus from grafts with Beltanol-treated scions. The results highlight the potential of environmentally friendly disinfection methods, particularly BioAction ES, in influencing phenolic content and enzymatic activity in graft callus, potentially affecting the success of grapevine grafting.

Graphical Abstract

1. Introduction

Vegetative propagation of European grapevine (Vitis vinifera L.) cultivars requires grafting onto American rootstocks, such as SO4, Kober 5BB, 1103P, 110R, etc. [1], due to the susceptibility of Vitis vinifera L. to phylloxera—a sap-sucking insect known to devastate its root system [2]. Bench–omega type is the most common grafting method due to its high productivity and relatively low skill requirements [3]. Conversely, grafting at high speed with relatively unskilled labour can result in poor vine graft quality [4]. In particular, the symptoms of various grapevine trunk diseases (GTD) on source material can often be overlooked, leading to the spread of infected propagation material [5], which happens unintentionally due to the latent period of GTD [6,7].
Over the last 30 years, Esca—a common GTD—has emerged as one of the major challenges in viticulture. Its widespread occurrence from nurseries to mature vineyards [8] and the involvement of numerous potential fungal pathogens, contribute to its severity [9,10,11]. In addition, the asymptomatic status of infected propagation material leads to the unintentional spread of the infection [4]. Disinfection of the propagation material is therefore crucial [12], although there is a lack of effective preparations against Esca-associated fungi [13,14], as sodium arsenite was banned in 2003 due to its harmful effects on human health [9,15,16,17].
A crucial aspect of successful vine grafting is the development of good contact between the scion and the rootstock at the grafting site, which facilitates callus formation and the subsequent bonding of the vascular tissue [1,18,19,20]. Formation of callus—a mass of undifferentiated parenchymal cells at the graft site that initiates the healing process of the graft interface [1,21]—is initiated by various wound responses such as the production of reactive oxygen species (ROS), the upregulation of stress resistance genes, the synthesis of enzymes and secondary metabolites [20,22,23,24]. Prodhomme et al. [25] reported that graft union was associated with increased activity of two enzymes—neutral invertase (NI), which influences the regulation of sugar metabolism and phenylalanine ammonia lyase (PAL), a key enzyme for the synthesis of phenylpropanoids. Detailed profiling of phenylpropanoids accumulated at the graft interface has been carried out, mostly in the light of incompatibility responses between different scion/rootstock combinations [18,25,26,27]. The content of flavanols, especially epicatechin, decreases at the graft interface compared to the surrounding woody tissue, while stilbenes accumulate at the graft interface [25]. This finding was confirmed in homografts by Loupit et al. [27]. Furthermore, Assunção et al. [18] also reported the accumulation of gallic acid, ferulic acid and sinapic acid in the graft union.
To investigate the phenolic response of grapevine callus to grafting with scions exhibiting different Esca disease statuses—previously disinfected using various environmentally friendly methods—a detailed phenolic profile was analysed using the high-performance liquid chromatography–mass spectrometry (HPLC-MS) technique. Specifically, fresh callus samples were taken immediately after callus formation was analysed. In addition, to gain a deeper insight into the phenylpropanoid synthesis in grapevine callus, an analysis of key enzymes involved in the phenylpropanoid pathway was performed. To our knowledge, this is the first study that contributes to understanding the response and callus development in the phenylpropanoid pathway in relation to GTD, potentially informing future phytosanitary diagnostics.

2. Material and Methodes

2.1. Experiment Procedure

Canes of grapevine (Vitis vinifera L.) cultivar ‘Cabernet sauvignon’, sourced from a vineyard located at Brdice pri Neblem (46.004452 N, 13.509907 E; Goriška brda, Slovenia), were classified according to their Esca disease status, as reported by Rusjan et al. [28]. They were from vines divided into healthy (5 Esca- or GTD-free vines) and symptomatic (15 vines showing typical symptoms of Esca—chlorotic and necrotic spots on leaves (tiger leaves), several dead canes, and spurs). The characterization of Esca healthy or asymptomatic vines, was made according to fungi identification, detailly present in the previous research by Rusjan et al. [28], as same grapevines were included in this experiment. All vines were confirmed to be virus-free, as they were previously tested by ELISA for the possible presence of the viruses GFLV, ArMV, GFkV, GVA, GLRaV-1, and GLRaV-3.
Canes were stored separately according to their disease status and cut into scions in winter 2017. Scions were further divided into healthy (originating from Esca-free vines), symptomatic (from Esca-symptomatic vines; scions exhibiting visible necrosis of conductive tissue on cross-section; see Figure 1), and asymptomatic (from Esca-symptomatic vines; canes showing no visible necrosis of conductive tissue on cross-section; see Figure 1), and separately stored in transparent PVC bags at 2 °C until grafting.
Certified rootstock Kober 5BB (Vitis berlandieri Planch × Vitis riparia Michx) coming from Clonal selection center Ivanjkovci (Ormož, Slovenia), previously soaked for 12 h in Beltanol (70 mL/10 L water) following the national rules [29], was used for grafting with an omega grafting machine in April 2017. The scions were also previously disinfected (separately, according to the disease status) for 12 h using Beltanol (70 mL/10 L water), which served as positive control as it is the only allowed agent in Slovenia for disinfection of vine grafts in grapevine nurseries [29]. They were also disinfected for 12 h using five different environmentally friendly disinfection methods: (I) Beltanol in combination with hot water treatment (TT; first treated at 50 °C for 45 min, cooled in lukewarm water and then soaked, according to EPPO standard [30], in Beltanol (70 mL Beltanol /10 L water); (II) Remedier (1 kg/10 L water; prepared 24 h before soaking); (III) Serenade® ASO (80 mL/10 L water); (IV) BioAction ES (55 mL/10 L water); and (V) sodium bicarbonate (50 g/10 L water). Descriptions of each method and disinfectant are given in Table S1, summarized according to Gačnik [31]. For each disinfection method, 200 scions were used for each disease status. After disinfection, the scions were grafted onto the SO4 rootstock separately, according to their Esca disease status. Grafts were dipped in warm paraffin (95 °C), then cooled and placed in wooden boxes filled with moist sawdust. These boxes were then placed in a forcing room at 25–27 °C for 3 weeks, providing optimal conditions for callus formation at the grafting site. After callus formation, samples for further HPLC/MS analysis were taken from 15 grafts per treatment. All samples were stored separately according to the treatment received at −80 °C for later analysis.

2.2. Individual Phenolic Compounds HPLC-MS Analysis

2.2.1. Phenolic Compounds Extraction

Extraction procedure for further analysis of the phenolic compounds in the callus of high-quality grapevine scions was carried out using HPLC/MS (high performance liquid chromatography–mass spectrometry) according to the method described by Rusjan et al. [28]. First, the grapevine grafts were cleaned, and the callus was separated by carefully cutting the top and the bottom with scissors. The callus was then sharpened with a pencil sharpener and ground into a fine powder in a ceramic mortar using liquid nitrogen.
The powdered samples, weighing approximately 0.2 g each, were transferred to plastic tubes and mixed with an extraction solution consisting of methanol/formic acid/bidistilled water (80%/3%/18%, v/v/v). Phenolic compounds were extracted from the samples in a cooled ultrasonic bath at 0 °C for 1 h. After extraction, the samples were centrifuged in an Eppendorf 5819 R centrifuge (Eppendorf SE, Hamburg, Germany) at 10,000 rpm for 7 min at 4 °C. The supernatant was then filtered through a 0.20 µm Chromafil AO-20/25 polyamide filter (Macherey-Nagel, Düren, Germany) into vials and stored at −80 °C until HPLC analysis.

2.2.2. HPLC/MS Analysis

Quantification of the individual phenolic compounds was carried out using the Accela HPLC system (Thermo Scientific; San Jose, CA, USA) with the DAD detector adjusted to 280 nm for flavanols and 350 nm for stilbenes. Column Gemini C18 (150 × 4.6 mm; 3 µm; Phenomenex) at 25 °C was used for the analysis. Separation of phenolic compounds was performed by mixing two mobile phases—A (0.1% formic acid + 3% bidistilled water + 97% acetonitrile) and B (0.1% formic acid + 3% acetonitrile + 97% bidistilled water). Samples were eluted using a linear gradient from 5% to 20% B over the first 15 min, followed by a linear increase from 20% to 30% B over the next 5 min. This was followed by an isocratic phase for 5 min, then a linear gradient from 30% to 90% B over 5 min, and another isocratic phase for 15 min before returning to the initial conditions. The volume of the injected samples was 20 μL, and the flow rate of mobile phases was 0.6 mL/min.
The extracted phenolic compounds were identified using a mass spectrometer (Thermo Scientific, LCQ DecaXP MAX) with an electrospray ioniser (ESI) according to the method described by Rusjan et al. [28]. The volume of the injected samples was 10 μL, and the flow rate of the mobile phase was 0.6 mL/min. The ion spectrum was recorded in the range of m/z 115 to 1500. Spectral data were recorded using Excalibur 2.2 software (Thermo Scientific). Peak identification was confirmed by comparison with the retention times of the corresponding standards and based on fragmentation ions of each phenolic compound. The content of phenolics was expressed in μg/g callus.

2.3. Analysis of Phenylpropanoid-Pathway-Related Enzymes

After callus formation, the activity of the key enzymes of the phenylpropanoid-pathway—phenylalanine ammonia lyase (PAL; EC 4.3.1.5); chalcone synthase (CHS; EC 3.2.1.74); chalcone isomerase (CHI; EC 3.2.1.14); flavanone-3β-hydroxylase (FHT; EC 1.14.11.9); and dihydroflavonol 4-reductase (DFR; EC 1.1.1.219)—were analysed in the callus of grapevine grafts grafted with scions showing different disease states related to Esca and previously subjected to different disinfection methods (Beltanol, Beltanol + TT, Serenade® ASO, BioAction ES, Remedier, and sodium bicarbonate).

2.3.1. Crude Extract Preparation

Plant material was prepared as reported by Halbwirth et al. [32] (protocol 2), optimised for grapevine callus. Approximately 0.5 g of callus frozen in liquid nitrogen was homogenised in a pre-cooled ceramic mortar with 0.5 g of Polyclar AT (Serva Electrophoresis, Heidelberg, Germany) and transferred to a falcon tube containing 0.5 g of Dowex (1 × 2) in 1.8 mL extraction buffer (0.7 M KH2PO4/K2HPO4 (KPi) at pH 8.0 containing 0.4 M sucrose, 0.4 M sodium ascorbate, 1 mM CaCl2, 30 mM EDTA, 50 mM cysteine, 50 mM DIECA, 1.5% PEG 20000, and 0.1% BSA). To remove dissolved oxygen, the mixture was kept at 100 °C for 10 min and cooled under a N2 atmosphere before the addition of cysteine, DIECA, PEG 20000, and BSA.
To prepare crude extracts for the enzyme assay, homogenates were filtered through glass wool and centrifuged for 20 min at 4 °C and 38,000× g. To remove low molecular weight compounds, 400 µL of the supernatant was passed through gel chromatography columns packed with Sephadex G25 (Sigma-Aldrich, Steinheim, Germany) and eluted with 400 µL of the enzyme-specific buffer: (I) PAL—0.1 M KPi + 0.4% ascorbate, pH 8.5; (II) CHS/CHI—0.1 M KPi + 0.4% ascorbate, pH 7.5; (III) FHT—0.1 M Tris/HCl + 0.4% ascorbate, pH 7.5; and (IV) DFR—0.1 M KPi + 0.4% ascorbate, pH 6.0. Pasteur pipettes were used as columns with a bed volume of 1 mL and equilibrated with 5 mL of the enzyme-specific buffer. Each eluate was used immediately in a further enzyme-specific assay.

2.3.2. Enzyme Activities

Enzyme assays were performed according to the method described by Halbwirth et al. [32], with slight modifications as described here. For the PAL activity analysis, 40 µL of crude extract was combined with 55 µL of (I) buffer and 5 µL of (14C)-phenylalanine (0.027 nmol, 27.750 dpm). Similarly, for the analysis of CHS/CHI activity, 40 µL of crude extract was mixed with 50 µL of (II) buffer containing 5 µL of 1 nmol p-CuCoA and 5 µL of (14C)-malonyl-CoA (1.5 nmol, 1300 Bq). For the FHT assay, 40 µL of crude extract was used, to which 50 µL of (III) buffer containing 5 µL of 2-oxoglutarate (1.46 mg/mL H2O), 5 µL of FeSO4 × 7H2O (0.56 mg/mL H2O), and 0.036 nmol of (14C)-naringenin (100 Bq) were added. For the DFR assay, 20 µL of crude extract, to which 25 µL of (IV) buffer, 5 µL NADPH (4.186 mg/100 µL H2O), and 0.036 nmol (14C)-dihydrokaempferol, (14C)-dihydroquercetin or (14C)-dihydromyricetin were added.
The prepared mixtures were transferred to 1.5 mL Eppendorf tubes and incubated at 30 °C. After 30 min, the PAL and CHS/CHI reactions were stopped by adding 200 µL of ethyl acetate and 10 µL of acetic acid, while the FHT and DFR reactions were stopped by adding 70 µL of ethyl acetate and 10 µL of acetic acid. The samples were then centrifuged for 3 min at 24 °C and 10,000 rpm. For PAL and CHS/CHI assays, 100 µL of the upper supernatant phases were transferred to scintillation vials, to which 4 mL of scintillation cocktail (Rotiszint® eco plus LSC-universal cocktail) was added and quantified using a scintillation counter. For FHT and DFR analysis, 100 µL of the supernatant phase was applied to thin layer chromatography (TLC) plates (Merck, Darmstadt, Germany) and developed in a container with a CAW (chloroform/acetic acid/H2O) solvent system (10:9:1, v/v/v) overnight. The conversion rates were determined the following morning using a TLC linear analyser (Berthold, Bad Wildbad, Germany).
The activity of the enzymes mentioned was calculated in nanokatals per g protein, quantified according to a protocol described by Sandermann and Strominger [33] using BSA as a standard.

2.4. Statistic Analysis

The experiment incorporated two factors. The first factor was the Esca disease status of scions, which comprised three levels: healthy, asymptomatic, and symptomatic. The second factor encompassed various disinfection methods, including Beltanol, Beltanol + TT, Serenade® ASO, BioAction ES, Remedier, and sodium bicarbonate. Beltanol, the only standard method for disinfecting graft parts in Slovenian nurseries, served as a positive control.
Principle component analysis (PCA), heatmap, and statistical tests were performed using 4.2.2 R-commander statistical software (R Formation for Statistical Computing, Auckland, New Zealand, 2021) using ggplot2, FactoMineR, multcomp, and agricolae packages. Differences among treatments for phenolic content and enzymatic activity of the phenylpropanoid-pathway-related enzymes in callus, were determined with two-way ANOVA. Residual analysis was conducted to test the assumptions of ANOVA. Outliers were evaluated using the box plot method, normality was assessed using the Shapiro–Wilk normality test, and homogeneity of variances was examined using Levene’s test. Where necessary, data were logarithmically transformed to reach the assumptions of ANOVA. All tests were performed with 95% confidence. If a significant interaction was observed, statistical differences were analysed using user-defined contrasts with the glht (General linear hypotheses) function. All comparisons were made relative to the positive control. If the interaction was not significant, pairwise comparisons between the different disinfection methods used were conducted using the emmeans_test() function. A grouping based on the disinfection method used was also generated using Ward’s method based on Euclidean squared distance for mean contents of analysed phenolic groups and enzymatic activity.

3. Results

3.1. Phenolic Profile of Vine Graft Callus

The phenolic profile of fresh grapevine graft callus comprises twenty-three identified phenolic compounds from five phenolic groups—hydroxybenzoic acids (only gallic acid identified), gallotannins (only trigalloylhexose identified), nine flavanols, seven stilbenes, and five condensed tannins (Table S1; Figure 2). Disinfection of scions before grafting and their previous disease status with regard to Esca did not alter the phenolic composition of grapevine callus after grafting, but variations in the levels of individual metabolites were observed.
Principal component analysis (PCA; Figure 3), with the first and second components accounting for 58.9% and 13.6% of the total variance, respectively, revealed a clear distinction in the phenolic profile of callus from grafts with healthy and symptomatic scions pre-treated with BioAction ES, as well as across other treatments. Phenolic compounds distinguishing callus from grafts with symptomatic scions pre-treated with BioAction ES were located on the negative side of PC1 and showed a strong association with resveratrol derivative 2 and procyanidin trimer 1. In contrast, callus from grafts with healthy scions pre-disinfected with BioAction ES correlated strongly with catechin, procyanidin tetramer, and procyanidin trimer 3. Callus from grafts with other combinations of scion disease status and disinfection methods had lower metabolite contents, although callus from grafts treated with Remedier correlated strongly with higher theaflavin content.
Clustering by different disinfection methods used before grafting on scions with different Esca disease status, based on the content of each phenolic group in the graft callus, was generated using Ward’s method and Euclidean squared distance. This clustering is represented by a dendrogram displayed above the legend of the heat map for phenolic compound content (Figure 4). According to the content of individual phenolic groups, the graft callus is clearly distinguished from grafts with scions that were previously disinfected with BioAction ES and other disinfectants.
The total analysed phenolic (TAP; Figure 5) content of graft callus was significantly influenced by both Esca disease status (p < 0.01), disinfection methods (p < 0.001), and their interaction (F(10, 54) = 6.68, p < 0.001). With a confidence level of 99%, the TAP content of the graft callus was only significantly influenced by prior disinfection of healthy and symptomatic scions with BioAction ES, while no disinfectant was significant for asymptomatic scions. Disinfection of healthy scions with BioAction ES increased the TAP content of the callus by an average of 339.5 (CL: 267.7—411.2) µg/g FW, while callus from grafts with symptomatic scions showed an average increase of 510.3 (438.5–582.1) µg/g FW.
Gallic acid (Figure 2) was the only hydroxybenzoic acid derivative identified in graft callus, ranging from 2.11 to 14.43 µg/g FW, representing 1.1% to 4.2% of TAP. Two-way ANOVA showed a significant effect of both factors—disease status (p < 0.01) and disinfection methods (p < 0.001), as well as their interaction on gallic acid content in vine graft callus (F(10, 54) = 7.30, p < 0.001). User-defined contrasts showed different results when comparing different disinfection methods under different disease status of scions. Beltanol disinfection combined with thermotherapy of asymptomatic scions prior to grafting resulted in an average gallic acid content of 3.28 (CL: 1.15–5.30) µg/g FW, which is lower compared to the positive control (Beltanol). Similarly, disinfection of symptomatic scions with Serenade® ASO resulted in an average gallic acid content of 5.61 (CL: 3.49–7.74) µg/g FW, which is lower compared to Beltanol, while disinfection of healthy scions with Remedier resulted in an average gallic acid content of 5.89 (CL: 3.76–8.01) µg/g FW, the highest gallic acid content in the callus.
One gallotannin-trigalloylhexose (Figure 2) was identified in callus of vine graft with highest content in callus from grafts with healthy and symptomatic scions, previously disinfected with BioAction ES (healthy: 4.62 ± 0.88; symptomatic: 6.02 ± 1.37 µg/g FW).
Flavanols (Figure 2 and Figure 4) were the most represented phenolic group in vine graft callus, accounting for 48.5% to 79.2% of TAP, and were influenced by disease status (p < 0.001), disinfection methods (p < 0.001), and their interaction (F(10, 54) = 10.12, p < 0.001). A further analysis showed that callus from grafted vines with symptomatic and healthy scions treated with BioAction ES showed significantly increased flavanol synthesis compared to the positive control (p < 0.001). In grafts with healthy scions previously treated with BioAction ES, total flavanol content in graft callus increased on average by 317.0 µg/g FW (CL: 270.5–363.5 µg/g FW), and in grafts with symptomatic scions previously treated with BioAction ES, the increase was on average by 386.3 µg/g FW (CL: 339.8–432.8 µg/g FW). Total flavanol content in callus also increased in grafts with healthy scions when they were previously disinfected with Beltanol in combination with thermotherapy (on average by 107.7 µg/g FW, CL: 57. 5–157.9 µg/g FW), sodium bicarbonate (on average by 88.8 µg/g FW, CL: 42.3–135.3 µg/g FW), and Remedier (on average by 63.3 µg/g FW, CL: 16.8–109.8 µg/g FW) compared to scions treated with Beltanol.
Two-way ANOVA also showed similar results for individual flavanols (Figure 2), as the content of all flavanols was significantly increased in callus from grafts with healthy and symptomatic scions previously disinfected with BioAction ES (except theaflavin in callus from grafts with healthy scions). In particular, the content of epicatechin gallate (Figure 2), the most abundant flavanol representative with 15.8–44.2% TAP in the callus of grafts with scions previously treated with BioAction ES, increased on average 3.46-fold (CL: 2.35–5.10-fold) in grafts with healthy scions and on average 2.67-fold (CL: 1.82–3.96-fold) in grafts with symptomatic scions compared to the content in callus of grafts with Beltanol-treated scions. Previous disinfection of healthy scions with sodium bicarbonate also influenced the increased content of catechin, epicatechin gallate, and some procyanidin derivatives (Figure 2) in graft callus (p < 0.05). Compared to Beltanol-treated scions, the catechin content in the callus of scions treated with sodium bicarbonate was increased on average 1.53-fold (CL: 1.00–2.19-fold), the epicatechin gallate content on average 1.48-fold (CL: 1.00–2.19-fold), and the procyanidin derivatives content was increased on average 1.61-fold (1.30–2.52-fold).
Stilbenes (Figure 2 and Figure 4) represented 4.8–11.3% TAP in graft callus and were strongly influenced by disease status (p < 0.001), disinfection methods (p < 0.001), and their interaction (F(10, 54) = 25.9, p < 0.001). Further analysis showed that prior disinfection of healthy and symptomatic scions with BioAction ES significantly increased total stilbene content (Figure 4; p < 0.001) compared to the content in the callus of grafts with Beltanol-treated scions. Specifically, by an average of 13.72 (CL: 17.24–30.19) µg/g FW in callus from grafts with healthy scions and by an average of 56.75 (CL: 50.28–63.22) µg/g FW in callus from grafts with symptomatic scions. Thermotherapy had a negative effect on total stilbene content in callus from grafts with healthy scions, as the content was on average for 9.02 (CL: 2.55–15.49) µg/g FW lower than in callus from grafts with healthy Beltanol-treated scions. On the other hand, thermotherapy increased total stilbenes content/g FW on average for 7.61 (CL: 1.14–14.08) µg in the callus from grafts with symptomatic scions, when compared to Beltanol-treated scions. In the callus from grafts with symptomatic scions, the content of total stilbenes was increased not only with the use of BioAction ES and Beltanol in combination with thermotherapy, but also with the use of other environmentally friendly measures, when compared to the positive control–using Remedier increased it on average by 12.67 (CL: 6.20–19.14) µg/g FW and using sodium bicarbonate on average by 10.22 (CL: 3.75–16.69) µg/g FW.
Looking more closely at the profile of stilbenes (Figure 2) in the callus of the grafts, it is noticeable that both picetanol derivatives, resveratrol hexoside, and both resveratrol derivatives increased (p < 0.01) in the callus of grafts with healthy and symptomatic scions when disinfected with BioAction ES compared to disinfection with Beltanol.
On the other hand, in grafts with asymptomatic scions, the contents mostly decreased when treated with BioAction ES (exception: resveratrol derivative 2). Resveratrol hexoside, the most abundant stilbene representative (1.3–6.5% TAP), increased to an average of 20.33 (CL: 16.41–24.26) µg/g FW in callus from grafts with healthy scions treated with BioAction ES and to an average of 31.42 (CL: 27.50–35.35) µg/g FW in callus from grafts with symptomatic scions. On the other hand, in callus from grafts with asymptomatic scions treated with BioAction ES, the content decreased by an average of 4.19 (CL: 0.27–8.12) µg/g FW compared to Beltanol-treated scions. With the exception of picetannol derivative 1, all stilbene representatives increased (p < 0.05) in the callus of grafts with asymptomatic scions previously disinfected with Remedier. A similar trend can also be observed for the content of picetannol derivative 2, resveratrol hexoside, and both resveratrol derivatives in callus from grafts with symptomatic scions.
Three monogalloyl procyanidin dimers and two digalloyl procyanidin dimers belonging to condensed tannins (Figure 2 and Figure 4) were identified and quantified in the callus of vine grafts, representing 11.2–44.5% TAP. They were strongly influenced by disinfection methods (p < 0.001) and by the interaction between disease status and disinfection methods (F(10, 54) = 17.8, p < 0.001). Further analysis showed that the synthesis of condensed tannins was enhanced in callus from grafts with infected scions previously disinfected with BioAction ES, when compared to Beltanol-treated scions. Specifically, callus from grafts with asymptomatic scions treated with BioAction ES had an average of 20.01 (CL: 3.71–36.31) µg/g FW and callus of grafts with symptomatic scions an average of 68.11 (CL: 51.81–84.41) µg/g FW when compared to Beltanol-treated scions. Similar behaviour was also observed in individual representatives, especially when callus was from grafts with symptomatic scions. The most abundant condensed tannin in callus was monogalloyl procyanidin dimer 3, whose content in callus was strongly increased (p < 0.001) in grafts with infected scions previously disinfected with mostly all environmentally friendly measures, when compared to the Beltanol (the use of Beltanol + thermotherapy and sodium bicarbonate for asymptomatic scions was not significant). On the other hand, there was a negative trend for most of the condensed tannins (except digalloyl procyanidin dimer 1) when the callus was obtained from grafts with healthy scions treated with the thermotherapy. In particular, the content of monogalloyl procyanidin dimer 3 was on average 17.44 (CL: 7.90–26.98) µg/g FW lower in callus from grafts with healthy scions treated with thermotherapy than in callus from grafts with scions treated with Beltanol.

3.2. Phenylpropanoid-Related Enzymes

The activities of four key phenylpropanoid-related enzymes—PAL, CHS/CHI, DFR, and FHT—were studied in the fresh callus of vine hetero-grafts, previously grafted with scions, categorised by different Esca disease status and disinfected before grafting with different disinfection procedures. Each heatmap in Figure 4 corresponds to one of the enzymes and it is positioned at the corresponding regulatory position.
A separate clustering was generated based on the activities of all four analysed phenylpropanoid-related enzymes, considering the different disinfection methods used for disinfection of scions with different Esca disease status prior to grafting. This clustering, carried out using Ward’s method and Euclidean squared distance, is represented by the dendrograms shown above the legend of the enzyme activity heatmap (Figure 4). Callus from grafts with scions, previously disinfected with Beltanol in combination with thermotherapy differed the most from the other disinfection methods used. In general, the activity of CHS/CHI and DFR enzymes in the callus of grafts additionally treated with thermotherapy was significantly reduced compared to the positive control (Beltanol), while the activity of PAL and FHT enzymes was increased.
The activity of PAL enzyme in graft callus (Figure 4) was strongly influenced by both Esca disease status (p < 0.001) and disinfection methods (p < 0.001), and by their interaction (F(10, 54) = 5.53, p < 0.001). Further analysis showed that prior disinfection of healthy scions with all environmentally friendly preparations increased the activity of the PAL enzyme in graft callus (p < 0.001) compared to Beltanol-treated scions. On the other hand, the activity of the PAL enzyme in the callus of grafts with infected scions was only increased if the scions had previously been disinfected with Beltanol in combination with thermotherapy and with Serenade® ASO (p < 0.001). In particular, compared to the callus of grafts previously treated with Beltanol, the callus of grafts treated with Serenade® ASO showed the greatest increase in PAL enzyme activity—an average of 9.54-fold (CL: 5.46–16.67-fold) in grafts with asymptomatic scions and an average of 6.37-fold (CL: 3.64–11.13-fold) in grafts with asymptomatic scions.
The activity of the CHS/CHI enzymes in callus (Figure 4) was influenced by the disinfectant used (p < 0.001) and by the interaction between the Esca disease status of the scions and the disinfectant (p < 0.05). Disinfection with a combination of Beltanol and thermotherapy or with Serenade® ASO reduced the CHS/CHI activity compared with Beltanol-treated scions, regardless of the Esca disease status of the scion. In callus from grafts with asymptomatic scions, CHS/CHI activity was reduced on average by 87.5% (CL: 70–99%) with Beltanol and thermotherapy, and by 83.9% (CL: 75–93%) with Serenade® ASO treatment. In the callus from grafts with healthy scions, disinfection with thermotherapy reduced the activity by an average of 98.4% (CL: 82–95%) and with Serenade® ASO by 57.6% (CL: 48–85%). In the callus with symptomatic scions, disinfection with thermotherapy reduced the activity of CHS/CHI enzyme by an average of 92.4% (CL: 85–96%) and Serenade® ASO by 79.7% (CL: 61–89%).
Esca disease status (p < 0.05), the use of different methods of scion disinfection before grafting (p < 0.001), and their interaction (F(10, 54) = 5.10, p < 0.001) influenced the activity of the FHT enzyme (Figure 4), which was improved in callus from grafts with infected scions with all disinfection methods used compared to Beltanol. The highest FHT enzyme activity in callus was obtained by prior scion disinfection with BioAction ES. Specifically, callus from grafts with asymptomatic scions had on average a 4.97-fold higher activity of FHT enzyme (CL: 3.14–7.87-fold) and callus from grafts with symptomatic scions had on average a 6.91-fold higher activity (CL: 4.37–10.95-fold). In callus from grafts with healthy scions, FHT activity was increased only by scion disinfection with Serenade® ASO (on average by 1.62-fold; CL:1.02–2.56-fold), Remedier (on average by 1.81-fold; CL:1.14–2.87-fold), and sodium bicarbonate (on average by 2.38-fold; CL: 1.5–3.77-fold).
DFR enzyme activity (Figure 4) in graft callus was only affected by previous scion disinfection (p < 0.001) and only in the case of thermotherapy treatment (p < 0.001). Compared to control grafts, disinfection of scions with thermotherapy reduced the activity of the FHT enzyme in the callus by an average of 1.06 nkat/g (CL:0.59–1.54 nkat/g) regardless of Esca disease status.

4. Discussion

A crucial aspect of successful grafting is the establishment of stable contact between the scion and the rootstock at the grafting site, which facilitates callus formation and vascular tissue connection [1,18,19,20]. Callus formation is driven by a variety of wound responses, including the production of reactive oxygen species, the upregulation of stress resistance genes, and the synthesis of enzymes and secondary metabolites [1,20,22,23,24].
Secondary metabolites, especially phenolic compounds among others, are crucial for the defence mechanisms of grapevine against pathogen infections [28,39].
In this study, a comprehensive phenolic profile was generated using HPLC-MS, focusing on the enzymes of phenylpropanoid pathway, which provided insights into the biochemical responses during the grafting processes. Phenolic profiling revealed that the phenolic composition of grapevine callus is not altered by the prior Esca disease status of the scion or by disinfection methods, although variations in individual phenolic contents were observed (Figure 2). Principal component analysis (PCA; Figure 3) distinguished the phenolic profiles of callus from grafts with healthy and symptomatic scions pretreated with BioAction ES foliar fertiliser. In particular, grafts with healthy scions pretreated with BioAction ES showed strong associations with specific phenolic compounds such as catechin, procyanidin tetramer, and procyanidin trimer 3. Clustering based on phenolic group content also highlighted the particular profile of callus from graft disinfected with BioAction ES compared to other disinfectants (Figure 4). BioAction ES, which contains a 3% concentration of natural extracts of clove, lemon juice, garlic oil and peppermint, together with 4.5% copper and MicroSap® microcrystals (15%), can, according to the manufacturer, penetrate through the trunk and reach the conducting tissues, thereby boosting the plant’s immune system and promoting natural resistance to disease [40]. If we compare it with the effect of Beltanol, this cannot be denied, since the previous disinfection of healthy scions with BioAction ES acts as an elicitor for the activation of the callus defence mechanism [41,42], increasing the synthesis of almost all representatives of the flavanols (with the exception of theaflavin) and some stilbenes (Figure 2) in graft callus compared to callus from grafts with healthy scions previously treated with Beltanol. When compared to callus from healthy and symptomatic scions treated with Beltanol, a few individual phenolics were also significantly increased when the scions were previously disinfected with BioAction ES. These were gallotannin-trigalloylhexose, flavanols-epicatechin gallate and procyanidin derivative, stilbene-resveratrol hexoside and, only in callus from symptomatic scions, a condensed tannin-monogalloyl procyanidin trimer (Figure 2). These phenolics were also the most abundant in each group. Consequently, the total analysed phenolic content (TAP, Figure 5) was also increased in callus from grafts treated with BioAction ES in healthy and symptomatic scions. This was also confirmed by analysing the effect of BioAction ES on the increased activity of the main enzyme of the phenylpropanoid pathway—PAL—in the callus of grafts with healthy scions (Figure 4). A similar trend of increased activity is also shown for the DFR enzyme, which catalyses the reduction of dihydroflavonols into their respective leucoanthocyanidins (Figure 4). These are common precursors for anthocyanin and proanthocyanidin biosynthesis [43], but the increase was not significant. Prodhomme et al. [25] already reported that graft union was associated with increased activity of PAL enzyme, which is crucial in the process of callus formation for converting phenylalanine into trans-cinnamic acid (Figure 4), a precursor for synthesis of flavonoids and lignin [44] necessary for the xylem formation [25]. The formation of vascular tissue (xylem and phloem) is considered the final stage of successful grafting, which begins once cambial continuity and a strong connection between the scion and rootstock are established [21,45,46]. Failure to achieve cambial continuity on the grafting site can lead to scion desiccation and graft failure [47]. This could be one of the main reasons why the grafting success rate (measured in the autumn after graft ranking (data available to authors) was the highest (75%) in healthy scions previously treated with BioAction ES, along with those treated with Serenade® ASO when compared to grafts with Beltanol-treated scions. PAL activity (Figure 4) was also particularly increased in callus from grafts with infected scions previously treated with Serenade® ASO, probably indicating a slightly higher grafting success for grafts with symptomatic scions also in the autumn graft ranking (4.5%) compared to other grafts (data available to authors)). Due to the complex composition of BioAction ES, it is difficult to determine the main cause of the elicitor effect of this disinfectant, but it has been reported in the literature that individual compounds contained in disinfectants, such as copper treatment, increase the synthesis of phenolic compounds in buckwheat seedlings [48]. In wine it has also been shown that the concentration of polyphenols increases with low copper pollution of the soil [49]. A more detailed analysis of the effect of this disinfectant would require further studies to verify the effects of the individual components of the disinfectant at the biochemical level.
It is interesting to note that BioAction ES did not induce a phenolic response in callus with asymptomatic scions (Figure 2, Figure 4 and Figure 5). Although asymptomatic and symptomatic scions have the same genetic predisposition, their immunity level, achieved by phytoalexins and especially phenolic compounds, may be different as several studies have reported [28,39,50,51]. Goufo et al. [51] suggested that the absence of Esca-related symptoms in asymptomatic vines is due to constitutive defences, with preformed compounds effectively inhibiting fungal proliferation and metabolite translocation to leaves, and phenolic compound production only being induced when metabolites reach leaves at symptom-inducing doses. Also, in this case, it could mean that the asymptomatic vines already have pre-arranged defence mechanisms that reduce the need for a strong phenolic response in the callus of scions treated with BioAction ES, which could explain why no phenolic response was observed in the callus of these grafts.
Assunção et al. [18] reported that phenolic acids—gallic acid, ferulic acid, and sinapic acid—were accumulated in the graft union. In this case, only gallic acid was identified in the graft callus after the process of callusing (Figure 2), while no gallic acid was identified in the graft after classification, not even in the other graft parts (data available to authors). De Cooman et al. [52] reported that gallic acid was one of the most abundant phenolics accumulated in the graft interface zone, possibly representing the wound-induced response of incompatible micrografted Eucalyptus gunnii Hook. f. Similarly, Canas et al. [26] found higher levels of gallic acid in highly incompatible combinations of grafts. Loupit et al. [27] also observed increased content of gallic acid, together with epicatechin and catechin, in the graft vine interface of rather incompatible graft vine scion/rootstock combinations at the rooting stage.
In the callus of grafts with healthy and Remedier-treated scions, gallic acid was strongly increased (Figure 2), which could be one of the reasons for the lower final grafting success (55%, data available to authors) compared to BioAction ES and Serenade® ASO, although this was not observed in grafts with Beltanol and Beltanol in combination with thermotherapy-treated scions.

5. Conclusions

This study showed the complexity of phenolic responses and enzyme activity in grapevine callus after grafting with scions with different Esca disease status previously disinfected with different environmentally friendly disinfection methods. The results highlight the potential of environmentally friendly disinfection methods, particularly BioAction ES, in modulating phenolic content and enzymatic activity in graft callus, thereby potentially influencing the success of grapevine grafting. Twenty-three phenolic compounds were identified, with flavanols, stilbenes, and condensed tannins predominating in the graft callus. Disinfection with BioAction ES had the most significant effect on increasing the phenolic content in the callus, especially in symptomatic scions. BioAction ES also increased the synthesis of important phenolic compounds such as epicatechin gallate, procyanidin derivatives, and resveratrol hexoside, suggesting that it could act as a strong elicitor that enhances the defence mechanisms of grapevine graft callus. This was confirmed with the enzymatic activity analysis, which showed that the disinfection methods affected the activity of four key enzymes in the phenylpropanoid pathway: PAL, CHS/CHI, DFR, and FHT. Disinfection with thermotherapy combined with Beltanol reduced the activity of CHS/CHI, and DFR, while increasing the activity of PAL and FHT. BioAction ES significantly increased the activity of PAL and FHT in the graft callus. Further research is needed to investigate the long-term effects of these biochemical changes on grafting success and grapevine health status.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11040371/s1, Table S1: Methods of disinfection and preparation of disinfectant suspensions for grapevine scions’ disinfection before grafting in 2017.

Author Contributions

Conceptualization: D.R., A.Š., K.B., M.L. and M.M.-P.; data curation: S.K.; formal analysis: S.K., A.Š., H.H. and M.M.-P.; funding acquisition: D.R. and H.H.; investigation: S.K., A.Š., H.H. and M.M.-P.; methodology: D.R., M.M.-P. and H.H.; project administration: D.R., H.H. and K.B.; resources: D.R., H.H. and K.B.; software: S.K.; supervision, D.R. and M.M.-P.; validation: M.M.-P. and H.H.; visualization: D.R., A.Š., K.B., M.M.-P. and M.L.; writing—original draft: S.K.; writing—review and editing: D.R., M.M.-P. and H.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Slovenian Research and Innovation Agency (ARIS) (Horticulture Program no. P4-0013-0481) and Austrian Science Fund FWF (Project I 6939-B).

Data Availability Statement

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Horticulturae 11 00371 g001
Figure 1. Left—scions exhibiting visible necrosis of conductive tissue on cross-section. Right—scions showing no visible necrosis of conductive tissue on cross-section (foto: personal communication, Rusjan, 2017).
Figure 1. Left—scions exhibiting visible necrosis of conductive tissue on cross-section. Right—scions showing no visible necrosis of conductive tissue on cross-section (foto: personal communication, Rusjan, 2017).
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Figure 2. Heatmap depicting the average contents (µg/g FW) of phenolic compounds in callus of grafts, initially grafted with scions of varying Esca disease status (healthy, asymptomatic and symptomatic), and disinfected before grafting using different disinfectants—Bel: Beltanol; Bel + TT: Beltanol + thermotherapy; BA: BioAction ES; Rem: Remedier; Ser: Serenade® ASO; and SB: sodium bicarbonate. The results of a two-factor ANOVA are presented on the left side. Significance levels are denoted as follows: “***” for p < 0.001, “**” for p < 0.01, “*” for p < 0.05, and “ns” for not significant. Stars within the heatmap indicate significant comparisons between the phenolic content in callus from grafts with scions treated with Beltanol and those treated with other disinfectant methods across different Esca disease statuses, as determined by two-way contrasts. Abbreviations: procy: procyanidin; dim: dimer; der: derivative; monogall: monoalloyl; digall: digalloyl; hex: hexoside.
Figure 2. Heatmap depicting the average contents (µg/g FW) of phenolic compounds in callus of grafts, initially grafted with scions of varying Esca disease status (healthy, asymptomatic and symptomatic), and disinfected before grafting using different disinfectants—Bel: Beltanol; Bel + TT: Beltanol + thermotherapy; BA: BioAction ES; Rem: Remedier; Ser: Serenade® ASO; and SB: sodium bicarbonate. The results of a two-factor ANOVA are presented on the left side. Significance levels are denoted as follows: “***” for p < 0.001, “**” for p < 0.01, “*” for p < 0.05, and “ns” for not significant. Stars within the heatmap indicate significant comparisons between the phenolic content in callus from grafts with scions treated with Beltanol and those treated with other disinfectant methods across different Esca disease statuses, as determined by two-way contrasts. Abbreviations: procy: procyanidin; dim: dimer; der: derivative; monogall: monoalloyl; digall: digalloyl; hex: hexoside.
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Figure 3. Principal component analysis (PCA) of phenolic content in vine graft callus, categorized by different health statuses (healthy: HLT, asymptomatic: ASYM, and symptomatic: SYM), disinfected prior to grafting using various disinfectant methods (n = 4)—Bel: Beltanol; Bel + TT: Beltanol in combination with thermotherapy; BA: BioAction ES; Ser: Serenade® ASO; Rem: Remedier; SB: sodium bicarbonate; Dim1: Dimension 1, Dim2: Dimension 2. Blue arrows represent PCA loadings plot of the 11 most contributing metabolites. The size of the arrows indicates the contribution strength of each metabolite.
Figure 3. Principal component analysis (PCA) of phenolic content in vine graft callus, categorized by different health statuses (healthy: HLT, asymptomatic: ASYM, and symptomatic: SYM), disinfected prior to grafting using various disinfectant methods (n = 4)—Bel: Beltanol; Bel + TT: Beltanol in combination with thermotherapy; BA: BioAction ES; Ser: Serenade® ASO; Rem: Remedier; SB: sodium bicarbonate; Dim1: Dimension 1, Dim2: Dimension 2. Blue arrows represent PCA loadings plot of the 11 most contributing metabolites. The size of the arrows indicates the contribution strength of each metabolite.
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Figure 4. Simplified scheme of the phenylpropanoid pathway with key intermediates, enzymes and end products. Heat maps showing mean values of different phenolic group contents identified in grapevine graft callus and enzymatic activities of key enzymes involved in the phenylpropanoid pathway: PAL: phenylalanine ammonia lyase; CHS/CHI: chalcone isomerase/chalcone synthase; FHT: flavanone 3-hydroxylase; and DFR: dihydroflavonol 4-reductase. The rows of each heat map indicate Esca disease status—the first row shows data for callus from grafts with asymptomatic scions, the second row shows data for callus from grafts with healthy scions, and the third row shows data for callus from grafts with symptomatic scions. The columns correspond to the data for each disinfection method used—Bel: Beltanol; Bel+TT: Beltanol combined with thermotherapy; BA: BioAction ES; Ser: Serenade® ASO; Rem: Remedier; and SB: sodium bicarbonate. A grouping based on the disinfection method used was also generated using Ward’s method and Euclidean squared distance. It is presented with dendrograms above the heatmap legends, separately for enzymatic activity and phenolic group content. Stars (*) within the heat map indicate significant comparisons between the content of phenolic groups or enzymatic activity in callus from scions treated with Bel and those treated with other disinfectant methods across different health statuses, determined by two-way contrasts with 95% confidence interval (where the interaction between health status and disinfectant was significant; p < 0.05). Stars above DFR heat map indicate significant difference in enzymatic activity between callus from grafts with Bel-treated scions and those treated with other disinfectant methods (interaction not significant; p > 0.05). Abbreviations: C4H, cinnamate 4-hydroxylase; C3H, ρ-Coumarate 3-hydrolase; 4CL,4-coumarate-CoA ligase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; GT, glucosyl transferase; ANS, anthocyanidin synthase; STS, stilbene synthase; UGT, UDP-glucosyltransferase; CoA, coenzyme A. Scheme modified according to [34,35,36,37,38].
Figure 4. Simplified scheme of the phenylpropanoid pathway with key intermediates, enzymes and end products. Heat maps showing mean values of different phenolic group contents identified in grapevine graft callus and enzymatic activities of key enzymes involved in the phenylpropanoid pathway: PAL: phenylalanine ammonia lyase; CHS/CHI: chalcone isomerase/chalcone synthase; FHT: flavanone 3-hydroxylase; and DFR: dihydroflavonol 4-reductase. The rows of each heat map indicate Esca disease status—the first row shows data for callus from grafts with asymptomatic scions, the second row shows data for callus from grafts with healthy scions, and the third row shows data for callus from grafts with symptomatic scions. The columns correspond to the data for each disinfection method used—Bel: Beltanol; Bel+TT: Beltanol combined with thermotherapy; BA: BioAction ES; Ser: Serenade® ASO; Rem: Remedier; and SB: sodium bicarbonate. A grouping based on the disinfection method used was also generated using Ward’s method and Euclidean squared distance. It is presented with dendrograms above the heatmap legends, separately for enzymatic activity and phenolic group content. Stars (*) within the heat map indicate significant comparisons between the content of phenolic groups or enzymatic activity in callus from scions treated with Bel and those treated with other disinfectant methods across different health statuses, determined by two-way contrasts with 95% confidence interval (where the interaction between health status and disinfectant was significant; p < 0.05). Stars above DFR heat map indicate significant difference in enzymatic activity between callus from grafts with Bel-treated scions and those treated with other disinfectant methods (interaction not significant; p > 0.05). Abbreviations: C4H, cinnamate 4-hydroxylase; C3H, ρ-Coumarate 3-hydrolase; 4CL,4-coumarate-CoA ligase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; GT, glucosyl transferase; ANS, anthocyanidin synthase; STS, stilbene synthase; UGT, UDP-glucosyltransferase; CoA, coenzyme A. Scheme modified according to [34,35,36,37,38].
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Figure 5. Content (µg/g FW) of TAP—total analysed phenolic content in fresh callus from grafts with scions, categorized by different Esca disease statuses (healthy, asymptomatic, and symptomatic) and disinfected prior to grafting using various disinfectant methods (Beltanol, Beltanol + Thermotherapy (TT), BioAction ES, Remedier, Serenade® ASO, sodium bicarbonate). Black dots representing outliers.
Figure 5. Content (µg/g FW) of TAP—total analysed phenolic content in fresh callus from grafts with scions, categorized by different Esca disease statuses (healthy, asymptomatic, and symptomatic) and disinfected prior to grafting using various disinfectant methods (Beltanol, Beltanol + Thermotherapy (TT), BioAction ES, Remedier, Serenade® ASO, sodium bicarbonate). Black dots representing outliers.
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MDPI and ACS Style

Krošelj, S.; Mikulic-Petkovsek, M.; Likar, M.; Škvarč, A.; Halbwirth, H.; Biniari, K.; Rusjan, D. The Status of Esca Disease and the Disinfection of the Scion Prior to Grafting Affect the Phenolic Composition and Phenylpropanoid-Related Enzymes in the Callus of Vine Hetero-Grafts. Horticulturae 2025, 11, 371. https://doi.org/10.3390/horticulturae11040371

AMA Style

Krošelj S, Mikulic-Petkovsek M, Likar M, Škvarč A, Halbwirth H, Biniari K, Rusjan D. The Status of Esca Disease and the Disinfection of the Scion Prior to Grafting Affect the Phenolic Composition and Phenylpropanoid-Related Enzymes in the Callus of Vine Hetero-Grafts. Horticulturae. 2025; 11(4):371. https://doi.org/10.3390/horticulturae11040371

Chicago/Turabian Style

Krošelj, Saša, Maja Mikulic-Petkovsek, Matevž Likar, Andreja Škvarč, Heidi Halbwirth, Katerina Biniari, and Denis Rusjan. 2025. "The Status of Esca Disease and the Disinfection of the Scion Prior to Grafting Affect the Phenolic Composition and Phenylpropanoid-Related Enzymes in the Callus of Vine Hetero-Grafts" Horticulturae 11, no. 4: 371. https://doi.org/10.3390/horticulturae11040371

APA Style

Krošelj, S., Mikulic-Petkovsek, M., Likar, M., Škvarč, A., Halbwirth, H., Biniari, K., & Rusjan, D. (2025). The Status of Esca Disease and the Disinfection of the Scion Prior to Grafting Affect the Phenolic Composition and Phenylpropanoid-Related Enzymes in the Callus of Vine Hetero-Grafts. Horticulturae, 11(4), 371. https://doi.org/10.3390/horticulturae11040371

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