Mitigating grapevine esca disease: an innovative integrated management strategy to reduce incidence and severity by enhancing plant physiology and defence mechanisms

TL;DR

This study introduces a new treatment, AF5, that reduces grapevine esca disease by improving plant health and activating defense mechanisms.

Contribution

The study presents AF5 as a novel, sustainable solution for mitigating esca disease through physiological and immune pathway activation.

Findings

AF5 reduced esca incidence and severity by 26-50% across three vineyards in northern Italy.

AF5-treated vines showed improved stomatal conductance and transpiration, indicating reduced hydraulic limitations.

RNA sequencing revealed activation of defense-related genes in AF5-treated vines, including WRKYs and FRK1.

Abstract

Esca is one of the most destructive grapevine trunk diseases and a growing challenge for viticulture worldwide, particularly in the context of climate-driven increases in disease expression. Due to the lack of curative solutions and the limited efficacy of currently available preventive measures, there is a pressing need for sustainable, field-ready strategies capable of mitigating disease impact. In this study, we assessed the performance of AF5, a novel foliar formulation composed of potassium acetate, α-tocopherol, and propylenic glycol, applied throughout the growing season. Field trials were conducted over three consecutive years in three vineyards located in distinct pedoclimatic areas of northern Italy. Across all sites and seasons, AF5 consistently reduced esca incidence and severity, with decreases ranging from 26 to 40% and 35% to 50%, respectively, compared with the conventional management protocol adopted by each winery. To investigate the physiological basis of this mitigation, we measured leaf-level gas exchange in symptomatic and asymptomatic vines. AF5-treated symptomatic plants displayed higher stomatal conductance and transpiration than untreated symptomatic vines, indicating a partial alleviation of the hydraulic limitations typically associated with esca-induced xylem dysfunction. To complement these observations, RNA sequencing of leaf tissues revealed a clear transcriptional activation of defence-related pathways in AF5-treated symptomatic vines. Genes involved in pathogen perception (e.g., receptor-like kinases), ethylene and jasmonate signalling cascades, and immune-regulatory transcription factors such as WRKYs and FRK1 were strongly induced, suggesting that AF5 enhances host defensive preparedness. Overall, this study provides the first multi-year, multi-site evidence that AF5 can reduce disease expression while supporting physiological performance and activating key immune pathways. These results position AF5 as a promising, easily implementable tool for integrated esca management in commercial vineyards.

The online version contains supplementary material available at 10.1186/s12870-026-08204-7.

Figures

Disease incidence: Disease incidence trends in 2021, 2022 and 2023 along the three vineyards. a-d-g Represent the vineyard located in Cozzuolo, b-e–h represent the vineyard located in Lonigo and c-f-i represent the vineyard located in Fossalta. Along the experimental trial, the AF5 treated plants across all the considered locations, and during the three years, displayed a significant reduction of esca incidence. Specifically, the Cozzuolo vineyard displayed a disease incidence of significantly lower in AF5 treated plants across 2021, 2022 and 2023. Similar data were observed for the Lonigo and

Disease severity: Disease severity trends in 2021, 2022 and 2023 along the three vineyards. a-d-g Represent the vineyard located in Cozzuolo, b-e–h represent the vineyard located in Lonigo and c-f-i represent the vineyard located in Fossalta. Along the experimental trial, the AF5 treated plants across all the considered locations, and during the three years, displayed a significant reduction of esca severity. Specifically, the Cozzuolo vineyard displayed a significantly lower disease incidence in AF5 treated plants across 2021, 2022 and 2023. Similar data were observed for the Lonigo and Fossa

Ecophysiological measurements: Leaf ecophysiological measurements were collected in the Lonigo field during the year 2023. Net photosynthesis (Pn), substomatal CO_2_ concentration (C_i_), stomatal conductance (g_s_), transpiration (E), intrinsic water use efficiency (iWUE) and instantaneous carboxylation efficiency (iCE) have been collected for STD and AF5, symptomatic (SYN) or asymptomatic (AS) plants. a Pn did not show any significant difference along conditions and treatments. b Ci did not show any significant difference along conditions and treatments. c g_s_ was significantly lower only i

RNAseq analysis: The same leaf used for the ecophysiological measurements have been used for RNA extraction and subsequent RNA sequencing. a Differentially expressed genes (DEGs) were analysed to identify the main co-expression pathway enriched in the AF5-treated and symptomatic leaves. As reported, most of gene identified with this analysis belong to responses against stressful factors (e.g., fungi, chitin, other organisms, biotic stresses, etc.…). b Hierarchical clustering and heatmap representation of the key defence-related genes in AF5-treated and symptomatic leaves. Several genes belongi

Full text

Introduction

Cultivated across more than 7.2 million hectares globally, the grapevine (Vitis vinifera L.) stands as the most economically significant perennial fruit crop, holding substantial commercial value for the production of wine, juice, fresh table grapes, and dried fruit. Grapevines are also particularly susceptible to a diverse array of pathogens, especially oomycete and fungal pathogens, which cause devastating diseases leading to yield reduction and/or vine mortality [24, 25, 36, 46]. While some fungal diseases primarily impact fruit yield and quality within the ongoing season, they seldom have long-term effects or result in vine death, thereby not posing a significant threat to the long-term exploitation of vineyards. In contrast, grapevine trunk diseases (GTDs), caused by wood colonizing fungi, are more severe, leading to vine mortality and contributing to vineyard decline over time [22, 23, 30]. The causal agents of GTDs comprise a diverse array of both ascomycetes and basidiomycetes, which predominantly colonize the vascular tissues of grapevines. This colonization disrupts plant physiology, alters microbial ecology, and triggers plant defence mechanisms [7, 13, 43]. Among these diseases, the esca complex is recognized as one of the most prevalent and devastating wood diseases affecting European grapevines [26, 31]. The incidence and severity of esca are currently escalating, particularly in warmer viticultural regions [20]. This trend suggests that climate change may be influencing both the progression of esca and the grapevine's response to these pathogens.

Typical symptoms of esca include inner wood necrosis, manifesting as either brown-black streaks attributed to ascomycetes [42] or as white rot, characterized by a yellowish-white discoloration of the wood, which becomes soft, spongy, and brittle, caused by basidiomycetes [11]. Additionally, in the leaves, peripheral and interveinal chlorosis happens, leading to necrosis and the appearance of the so called "tiger striped leaves". The latter is commonly observed during mid to late summer resulting from a hydraulic impairment caused by a compromised xylem system [10] and the presence of phytotoxic metabolites of fungal origins [3].

Historically, the management of GTDs, including esca, has been achieved with the use of sodium arsenite [17], a fungicide banned more than 20 years ago due to its high toxicity to human health [49]. Only recently a dual mechanism for sodium arsenite efficacy has been reported, suggesting that it acts directly as fungicide but it also exert a stimulating effect by improving plant defence response [51]. To find any alternative, the effectiveness of other antifungal molecules belonging to the triazoles [29], organophosphorus compounds [18], as well as many other organic compounds [41] have been evaluated so far showing unsatisfactory results. Additionally, innovative approaches exploiting nanocarrier [19], hydroxyapatite [4] and plant extract (i.e., Allium extracts, lignin sulfate, lemon peel extract, green coffee) [41] have been evaluated resulting in low efficacy or too complex application method for the exploitation in field conditions. For this reason, presently, the main strategy for esca and GTDs management relies on preventive practices (mostly relying on the application of pruning-wounds protectants, both of chemical (e.g., Fluopyram, Myclobutanil, polymer of cyclohexane) or biological (i.e., Trichoderma spp.) origins [12],del Pilar [47] or on trunk surgery to remove as most as possible of the necrotic tissue [38, 45]. However, the latter two strategies are insufficient to control the GTDs development, leading to an increase in symptomatic plants, also due to the ongoing climate change as previously reported [6].

Considering all the above-mentioned aspects, a more easily implementable, widely accessible and environmentally sustainable solution needs to be identified. The present work presents the data on the application of a novel and easy to use formulation composed of potassium acetate, α-tocopherol, and propylenic glycol, namely AF5, for the management of esca disease in field conditions. The selection of this formulation was made on recent observations on the effects of fertilizers for esca management [15] and considering that a formulation based on potassium and propylene glycol was never tested before. Three years of trials have been conducted across three different areas in the northwestern region of Italy, assessing the efficacy of the novel implemented protocol in terms of disease incidence and severity. Finally, to gain a deeper understanding of the observed disease reduction we also analysed ecophysiological performances and molecular responses of plants treated with the novel strategy compared to those treated with the conventional disease management protocol. Overall, this study provides for the first time the evidence for the possible implementation of a low-impact protocol for the management of esca disease.

Materials and methods

Experimental design, sampling and vineyard location

The experiment was repeated for three years (2021–2023), between April and July in open field condition, in three vineyards located in i) Fossalta di Portogruaro, Venice area, Veneto, Italy (GPS coordinates N45.764636, E12.900977), ii) Cozzuolo, UNESCO world heritage site “Le Colline del Prosecco di Conegliano e Valdobbiadene”, Veneto, Italy (GPS coordinates N45.952000, E12.280354) and iii) Lonigo, Vicenza area, Veneto, Italy (GPS coordinates N45.420255, E11.415359). The vineyards were cultivated with Glera (Lonigo and Cozzuolo) and Cabernet Sauvignon (Fossalta di Portogruaro) cultivars both grafted onto Kober 5BB rootstock. The plants in Lonigo were 15 years old at the beginning of the trial, those in Cozzuolo were 19 years old, and those in Fossalta di Portogruaro were 22 years old. The disease indexes in 2020 (year prior the beginning of the experiment) are summarized in Table 1.Table 1. Disease indexes in 2020 (year prior to the beginning of the experiment) along the three selected vineyard**Incidence (± SD)****Severity (± SD)**Lonigo13.2 ± 1.7%10.3 ± 1.2%Cozzuolo25.8 ± 2.3%14.8 ± 1.7%Fossalta di Portogruaro29.3 ± 2.6%15.1 ± 1.8%

The AF5 formulation provided by the Syneco SPA Company (San Giuliano Milanese, Italy) is composed by 71 to 81% propylenic glycol (1 to 4 carbon atoms), 21 to 27% of potassium acetate and 0.1 to 1% α-tocoferol. For each protocol 0.5 hectares (about 1,700 plants) have been treated. The “AF5” protocol consisted in the addition of 500 mL/ha of the AF5 foliar fertilizer to each treatment made by the beginning of the vegetative season until véraison (minimum of 10 applications). The AF5 fertilizer was mixed with the conventional active principles selected for the conventional protocol and applied at the same time. Timing of application was based on weather. The active principles used among the three fields were selected among the ones allowed by the regional phytosanitary agency (https://www.regione.veneto.it/web/fitosanitario/difesa-integrata). The complete list of products, dosage and schedule of treatments is reported in Supplementary Table 1.

In the experiment an alternative protocol “AF5” was compared to the standard treatment protocol adopted by the winery “STD”. An outline of the experimental design has been summarized in Supplementary Fig. 1. The experimental product being tested were applied according to the guidelines EPPO/OEPP PP 1/303(2)—Efficacy evaluation of products for the control of grapevine trunk diseases in vineyards.

Meteorological data

Meteorological data including precipitation, relative humidity, temperature and solar radiation were collected by the meteorological station located in each vineyard. The main environmental parameters along the three years are reported in Supplementary Table 2.

Evaluation of esca symptoms

Visual scouting of esca symptoms on leaves were conducted during the month of August of each year (BBCH [39] stages 83–85) to evaluate the treatments’ effectiveness. The disease indexes scoring (incidence and severity) was conducted using the Grape Assess Mobile App (University of Adelaide) [28, 54] using the trunk diseases custom assessment (for specification on app parameters refer to the documentation available at https://www.adelaide.edu.au/news/news100582.html). Incidence represents the ratio between symptomatic observations over the total number of observed plants, while severity represents the symptomatic canopy area of each plant as percentage. For each condition three biological independent group of observations were made. Each biological replicate was made by at least 150 observations (i.e., 150 plants): in total we collected at least 450 observations for each treatment and every year, meaning 900 observations for each vineyard and 2,700 across the three vineyards.

Yield and must evaluations

For the last year of experimentation (2023), at ripening (BBCH stage 89) [39], bunches were collected from single plants (3 biological replicates, p < 10 plants for each biological replicate), the total weight and number of bunches were recorded. Subsequently, ten bunches for each replicate and treatment were then randomly selected and squeezed manually for musts characteristics evaluation. Total soluble solids concentration (TSS) was determined with the aid of a refractometer while pH was measured using a pHmeter. Titratable acidity was measured by titration as described in OIV-MA-AS313-01 method and expressed as g L^−1^ of tartaric acid.

Ecophysiological measurements

Concomitantly to the esca scoring, ecophysiological parameters were collected for the field located in Lonigo during the three considered seasons (2021–2023) (BBCH stage 83–85) using an infra-red gas analyser (ADC-LCi T system; Analytical Development Company, BioScientific Ltd., UK) in a warm and sunny day (August 10th, 2023). The Lonigo field was selected for its logistical advantages and the ability to immediately store collected samples for subsequent molecular analyses. Measurements of net photosynthesis (Pn), transpiration rate (E), stomatal conductance (g_s_), intercellular CO_2_ concentration (Ci), water use efficiency (WUE, obtained as the ratio between Pn and E) and apparent carboxylation efficiency (iCE, obtained as the ratio between Pn and Ci [21]) were carried out on five randomly selected vines from each condition (AS and SYN plants) and treatment (STD and AF5). For each plant two fully developed non-senescent leaves at the same phenological age (4th to 5th leaf from the shoot apex) were measured using a portable infrared gas analyser in the hottest hours of the day as previously reported [5]. A total of 10 measures were collected for each condition and treatment. The asymptomatic plants we measured and sampled (both in STD and AF5 treatment) were healthy, meaning that no esca symptoms were ever scored in on those plants. During measurements, ambient parameters (i.e., vineyard conditions) were maintained: light intensity ranged from 1.500 to 1.700 µmol photons m^−2^ s^−1^, temperature ranged from 29 to 33 °C and the concentration of the CO_2_ in the air ranged from 420 to 440 ppm.

Samples processing and RNAseq analyses

Total RNA was isolated from leaf samples collected in 2023 in the same field where ecophysiological parameter were collected (Lonigo field). The same leaves used for ecophysiological measurements were collected (12 for each condition and treatment) and then subdivided in 3 biological replicates formed by 4 randomly selected leaves each. Samples were stored at −80 °C until nucleic acids extraction. To this aim the Spectrum Plant Total RNA Kit (Merck KGaA, Darmstadt, Germany) was used following the manufacturer’s instructions. The RNA quantity and purity was checked using a NanoDrop one spectrophotometer (Thermo Fisher Scientific) followed by a second quantification using a Qubit4 fluorometer (Thermo Fisher Scientific) and the integrity was confirmed on agarose gel. To proceed with RNA-seq analysis, at least 4 µg for each sample were sent to Macrogen Inc. (Europe) for cDNA library construction (TrueSeq total RNA sample kit, Illumina) and sequencing by Illumina Novaseq technology with an output of 40 M paired-end reads of 100 bp for each sample.

Raw RNAseq sequences were deposited in NCBI under the project PRJNA1247907. For all the bioinformatics analyses on RNAseq data three samples were used for each condition. To ensure that contamination from rRNA was below the selected threshold (less than 1%) the hidden Markov models (HMMs) [48] were run on the whole dataset. RNAseq data were subsequentially analysed using dedicated pipelines in Artificial Intelligence RNAseq Software AIR (accessible at https://transcriptomics.cloud). To determine gene expression of grapevine, reads were cleaned with Trimmomatic (V0.32) [9] and aligned against the grape reference coding sequence database [34, 52] (PN40024 CDS, http://www.grapegenomics.com/) using the built in DESeq2 [40] R package. Transcripts were annotated using annotation V1 of the grapevine genome [32]; Annotation, GO enrichment and differential expression comparison were done as previously described [43]. Briefly, transcripts were and grouped into functional gene classes according to the VitisNet GO. The BiNGO 3.0 plug-in tool in Cytoscape (v3.2, U.S. National Institute of General Medical Sciences (NIGMS), Bethesda, MD, USA) was used for GO enrichment analysis. Over-represented GO slim categories were identified using a hypergeometric test with a significance threshold of 0.05. Differentially expressed genes (DEGs) were identified in a pairwise comparisons using a p-value of 0.05% adjusted with the Benjamin-Hochberg method.

Statistical analysis

For each year, the significance of differences between STD and AF5 treatments for disease indexes was assessed using Student’s t test (p ≤ 0.05). While for leaf ecophysiology, quality features and production data comparison, a one-way analysis of variance (ANOVA) was applied using the SPSS statistical software package (version 23; SPSS Inc., Cary, NC, USA). Data normality and homogeneity of variance were assessed using the Shapiro–Wilk and Levene’s tests, respectively. When necessary, leaf gas exchange, disease incidence and severity data, were arc-sin-transformed to stabilize the variances and normalize their distribution. Multiple comparisons between means were conducted using Tukey’s honestly significant difference (HSD) post-hoc test (p ≤ 0.05). For each dataset, mean values ± standard deviations (SD) were calculated.

Results

Disease indexes

In the first year of experimentation (2021), the assessment of disease incidence and severity revealed statistically significant differences among the three vineyard locations under investigation (Fig. 1). In Cozzuolo, where the Glera variety was cultivated, disease incidence in plants managed under the standard (STD) protocol was recorded at 23.4 ± 1.9% (Fig. 1a). In contrast, plants treated with the innovative formulation (AF5) protocol exhibited a significantly lower incidence (−9.8%), with a recorded value of 14.6 ± 1.2% (Fig. 1a). A similar trend was observed in Lonigo, also featuring the Glera variety, where disease incidence was 14.5 ± 2.1% under the STD protocol, while the AF5-treated plants exhibited a significantly reduced (−5.8%) incidence of 8.7 ± 0.9% (Fig. 1b). In Fossalta di Portogruaro, where Cabernet Sauvignon was cultivated, the AF5 treatment again demonstrated a substantial reduction in disease incidence (−6.9%), with a value of 26.3 ± 1.4% compared to 33.2 ± 1.8% recorded in plants subjected to the STD protocol (Fig. 1c).Fig. 1. Disease incidence: Disease incidence trends in 2021, 2022 and 2023 along the three vineyards. a-d-g Represent the vineyard located in Cozzuolo, b-e–h represent the vineyard located in Lonigo and c-f-i represent the vineyard located in Fossalta. Along the experimental trial, the AF5 treated plants across all the considered locations, and during the three years, displayed a significant reduction of esca incidence. Specifically, the Cozzuolo vineyard displayed a disease incidence of significantly lower in AF5 treated plants across 2021, 2022 and 2023. Similar data were observed for the Lonigo and Fossalta vineyards. Data are presented as mean value of three independent replicates, each replicate was made of 150 observations. Asterisks above the bars represent significant differences as attested by the t-test

This trend continued over the following two years (2022 and 2023), with the AF5 treatment consistently showing a significantly lower incidence of symptomatic plants compared to the STD protocol (Fig. 1d-I). Specifically, in the Cozzuolo vineyard, disease incidence in STD-treated plants was 17.3 ± 2.1% in 2022 and increased to 24.3 ± 2.1% in 2023. In contrast, plants treated with the AF5 protocol displayed notably lower values, with incidences of 9.1 ± 1.4% (−8.2% with respect to STD) in 2022 and 9.4 ± 0.9% (−14.9% with respect to STD) in 2023. In the Lonigo vineyard, STD-treated plants exhibited a disease incidence of 10.2 ± 1.3% in 2022, which rose to 17.8 ± 1.7% in 2023, whereas AF5-treated plants showed significantly lower values of 6.1 ± 0.9% (−4.1% with respect to STD) and 6.3 ± 1.1% (−11.5% with respect to STD) in the respective years. Lastly, in the Fossalta di Portogruaro vineyard, disease incidence in STD-treated plants was recorded at 24.2 ± 2.2% in 2022 and increased to 28.7 ± 1.5% in 2023, while AF5-treated plants displayed significantly lower disease incidences of 15.8 ± 2.1% (−8.4% with respect to STD) and 18.7 ± 1.2% (−10% with respect to STD), respectively, in the same years.

In addition to disease incidence, also disease severity data showed significant differences between AF5- and STD-treated plants among the three vineyards (Fig. 2). From the outset of the study in 2021 (Fig. 2a), disease severity in the Cozzuolo vineyard was recorded at 12.7 ± 1.8% under the STD protocol, whereas AF5-treated plants exhibited a significantly lower severity of 7.8 ± 1.1% (−4.9%). A similar reduction was observed in Lonigo, where disease severity in STD-treated plants was 13.9 ± 1.9%, while plants receiving the AF5 treatment displayed a significantly lower severity of 8.2 ± 1.3% (−5.7%) (Fig. 2b). The Fossalta di Portogruaro vineyard followed the same trend, with disease severity reaching 16.4 ± 1.3% in STD-treated plants, whereas AF5-treated plants exhibited a substantially lower severity of 12.3 ± 0.8% (−4.1%) (Fig. 2c).Fig. 2. Disease severity: Disease severity trends in 2021, 2022 and 2023 along the three vineyards. a-d-g Represent the vineyard located in Cozzuolo, b-e–h represent the vineyard located in Lonigo and c-f-i represent the vineyard located in Fossalta. Along the experimental trial, the AF5 treated plants across all the considered locations, and during the three years, displayed a significant reduction of esca severity. Specifically, the Cozzuolo vineyard displayed a significantly lower disease incidence in AF5 treated plants across 2021, 2022 and 2023. Similar data were observed for the Lonigo and Fossalta vineyards. Data are presented as mean value of three independent replicates, each replicate was made of 150 observatios. Asterisks above the bars represent significant differences as attested by the t-test

The pattern of significant reductions in disease severity for AF5-treated plants was consistently observed in the subsequent two years of experimentation (2022 and 2023) across all vineyard locations (Fig. 2d-I). In the Cozzuolo vineyard, disease severity in STD-treated plants increased from 8.3 ± 1.7% in 2022 to 20.5 ± 2.1% in 2023. Conversely, plants subjected to the AF5 protocol exhibited significantly lower values of 4.7 ± 0.9% (−3.6% with respect to STD) in 2022 and 8.3 ± 0.9% (−12.2% with respect to STD) in 2023. In the Lonigo vineyard, the disease severity in STD-treated plants was 9.5 ± 1.9% in 2022, rising to 19.4 ± 1.7% in 2023, whereas AF5-treated plants demonstrated significantly reduced values of 4.9 ± 1.1% (−3.6% with respect to STD) and 7.7 ± 1.1% (−11.7% with respect to STD) in the respective years. Similarly, in the Fossalta di Portogruaro vineyard, the disease severity in STD-treated plants increased from 12.7 ± 1.3% in 2022 to 19.7 ± 1.5% in 2023, while AF5-treated plants maintained significantly lower values of 6.8 ± 1.1% (−5.9% with respect to STD) in 2022 and 9.7 ± 1.2% (−10% with respect to STD) in 2023.

Evaluation of leaf ecophysiological parameters and musts characteristics

To further deepen this subject, leaf ecophysiological parameters were measured to identify potential differences between treated and untreated plants. Over the three analysed seasons, leaf gas exchanges measurements showed the same trend (Fig. 3; Supplementary Figs. 2 and 3). In detail, during the last experimental season (2023), at the time of sampling, the first parameter analysed was net photosynthesis (Pn), which did not show any statistically significant differences among the treatments and conditions tested (Fig. 3a). However, a discernible trend was observed: AS plants, irrespective of whether they were subjected to the STD or AF5 treatments, displayed similar values. In contrast, although not statistically significant, SYN plants exhibited a difference between the treatments. Specifically, SYN-AF5 plants showed slightly higher Pn values compared to SYN-STD plants, albeit still lower than those of AS plants. Similarly, substomatal CO₂ concentration (C_i_) did not reveal any significant differences among treatments (Fig. 3b). Conversely, stomatal conductance (g_s_) was significantly reduced in SYN-STD plants (Fig. 3c) compared to all other conditions, including SYN-AF5, which exhibited values comparable to those of AS plants (both STD and AF). Similarly, transpiration rate (E) was significantly reduced exclusively in SYN-STD plants (Fig. 3d), whereas SYN-AF5 plants did not significantly differ from AS plants.Fig. 3. Ecophysiological measurements: Leaf ecophysiological measurements were collected in the Lonigo field during the year 2023. Net photosynthesis (Pn), substomatal CO_2_ concentration (C_i_), stomatal conductance (g_s_), transpiration (E), intrinsic water use efficiency (iWUE) and instantaneous carboxylation efficiency (iCE) have been collected for STD and AF5, symptomatic (SYN) or asymptomatic (AS) plants. a Pn did not show any significant difference along conditions and treatments. b Ci did not show any significant difference along conditions and treatments. c g_s_ was significantly lower only in the SYN-STD plants, while no differences between AF5 treated plants (both SYN or AS) and the AS-STD plants was observed. d E showed a behaviour similar to g_s_, with SYN-STD having significant lower values. e and f reporting iWUE and iCE did not show any significant difference along conditions and treatments. Data are presented as mean value of five randomly selected vines from each condition (AS and SYN plants) and treatment (STD and AF5). For each plant two fully developed non-senescent leaves at the same phenological age (4th to 5th leaf from the shoot apex) were measured. Different letters above the box denote significant differences as attested by the ANOVA analysis

Under our experimental conditions, iWUE did not exhibit significant differences among treatments, though a discernible trend was present (Fig. 3e). While not statistically significant, SYN-STD plants appeared to have higher iWUE values, whereas the other three conditions, including SYN-AF, displayed similar values.

The last parameter analysed, instantaneous carboxylation efficiency (iCE), was assessed through the ratios of Pn to Ci (Pn/Ci). As with previous parameters, no statistically significant differences were detected, but a trend was evident (Fig. 3f). Specifically, SYN plants exhibited consistently lower iCE values, regardless of treatment, while AS plants displayed higher values.

During the 2023 growing season, grape bunches were harvested, and basic must quality features were analysed to evaluate potential effects on must quality. As shown in Table 2, total soluble solids (TSS) were significantly higher and comparable between AS-STD and AS-AF treatments. The TSS value for SYN-AF was significantly lower than that of AS plants but remained significantly higher than that of SYN-STD, which exhibited the lowest TSS value. Acidity levels were significantly lower in AS plants, whereas SYN plants displayed significantly higher acidity. On the contrary, no significant differences were observed in pH across treatments and conditions. Finally, looking at the yield, expressed as Kg of bunches per plant, we observed that AS plants behaved similarly independently from the treatment. Conversely, SYN plants treated with AF5 exhibited a yield that was significantly lower than that of AS plants, but still significantly higher than SYN-STD plants. Notably, the lowest yield was recorded in the SYN-STD plants.Table 2. Production indexes analysis for the year 2023: Total soluble solids (TSS), Total acidity, pH and yield (Kg). Data are reported as average of three biological replicates ± standard deviation. Different letters correspond to treatments that differ for Tukey’s HSD test for p < 0.05**TSS (%)****Total acidity (%)pHYield (Kg/plant)**AS-STDL16.9 ± 0.09 c6.5 ± 0.10 a3.13 ± 0.065.2 ± 0.51 cAS-AF516.9 ± 0.08 c6.7 ± 0.12 a3.13 ± 0.045.3 ± 0.59 cSYN-STD15.1 ± 0.07 a7.5 ± 0.13 b3.11 ± 0.052.5 ± 0.30 aSYN-AF515.9 ± 0.08 b7.5 ± 0.12 b3.06 ± 0.043.5 ± 0.34 b

Transcriptomics analysis

To gain insights into the molecular responses triggered by the AF5 treatment, RNA sequencing analysis was performed on leaf samples collected from the same plants used for the ecophysiological measurements. Differentially expressed genes (DEGs) were identified by comparing AF5-treated plants to those subjected to the conventional treatment applied by the winery in both asymptomatic (AS) (Supplementary Table 3) and symptomatic (SYN) (Supplementary Table 4) conditions.

In SYN plants, the AF5 treatment induced a broad transcriptional reprogramming, with a significant enrichment of genes associated with both abiotic and biotic stress responses. Gene ontology (GO) enrichment analysis highlighted key biological processes related to defence responses, specifically in response to chitin, protein phosphorylation, ATP binding, and ribonucleotide binding, among others (Fig. 4a). Several DEGs were functionally linked to pathogen perception and defence signalling, including calmodulin-binding proteins, kinases involved in phosphorylation cascades, and transcription factors belonging to the WRKY family, known for their role in plant immune responses. Notably, genes associated with oxidative stress regulation, such as respiratory burst oxidase protein D (ATROBOHB), were upregulated in AF5-treated SYN plants. Further supporting this, the hierarchical clustering and heatmap representation (Fig. 4b) of upregulated genes (from the GO enrichment) in SYN-AF5 plants revealed the induction of several key regulators of plant defence. Among them, several WRKY genes, well-known markers of defence responses, and other genes involved in the plant defence (e.g., FRK1, DCL2, etc.…) were strongly upregulated.Fig. 4RNAseq analysis: The same leaf used for the ecophysiological measurements have been used for RNA extraction and subsequent RNA sequencing. a Differentially expressed genes (DEGs) were analysed to identify the main co-expression pathway enriched in the AF5-treated and symptomatic leaves. As reported, most of gene identified with this analysis belong to responses against stressful factors (e.g., fungi, chitin, other organisms, biotic stresses, etc.…). b Hierarchical clustering and heatmap representation of the key defence-related genes in AF5-treated and symptomatic leaves. Several genes belonging to the response against stressful factors are more expressed in the SYN samples (e.g., APK kinases, WRKY genes, PR-related proteins, etc.…)

In AS plants, the transcriptional impact of AF5 treatment was less pronounced, with a reduced number of DEGs compared to SYN plants. Nevertheless, GO term analysis highlighted an enrichment of genes involved in response to external biotic stimuli (e.g., VIT_09s0002g00230, VIT_04s0044g00270, VIT_04s0044g00220), nitrogen compound metabolic processes (e.g., VIT_11s0037g00590, VIT_00s2607g00010, VIT_00s2579g00010, VIT_02s0033g00800), and secondary metabolism regulation, specifically those related to hormones (e.g., VIT_15s0048g01350, VIT_00s0662g00030, VIT_00s0662g00040) (Supplementary Table 3).

Discussion

Esca disease remains one of the most destructive grapevine trunk diseases, causing significant economic losses in vineyards worldwide. Despite extensive research efforts, the management of this complex disease remains challenging, primarily due to the lack of curative treatments and the multifactorial nature of its aetiology, which involves a consortium of wood-colonizing fungi, environmental conditions, and host susceptibility [30, 50]. The present study aimed to evaluate the efficacy of an innovative treatment strategy (AF5) in reducing disease incidence and severity in symptomatic and asymptomatic grapevines, integrating physiological assessments, transcriptomic analyses, and field trials over three consecutive years.

AF5 treatment reduced disease incidence and severity while improving physiological performances in symptomatic plants

Field data demonstrated a significant reduction in both disease incidence and severity in vines treated with AF5 compared to those managed under the standard winery protocol (STD). This effect was observed consistently across different vineyard locations and two different grape varieties, suggesting that AF5 trigger a broad-spectrum protective effect independent of environmental or varietal differences. A notable reduction in symptomatic plants was particularly evident in vineyards with high esca pressure, reinforcing the hypothesis that AF5 contributes to limiting disease progression in affected vines. These findings align with previous studies reporting that innovative biostimulant-based or alternative treatments can enhance grapevine resilience against trunk diseases by modulating plant defences and reducing pathogen establishment [41, 45]. Indeed, one of the active substances widely used in the past and now banned, sodium arsenite, which was thought to act as fungicide, it has recently been demonstrated to act as an activator of defence responses in wood-infected grape plants [51]. This is in line to what we observed in our experimental conditions where the symptomatic treated plants displayed an enhancement in defence responses.

The ecophysiological measurements provided further insights into the impact of AF5 on grapevine health. While net photosynthesis (Pn) and substomatal CO₂ concentration (Ci) were not significantly affected, AF5-treated symptomatic plants exhibited improved stomatal conductance (g_s_) and transpiration rates (E) compared to SYN-STD plants. Since the photosynthetic apparatus remains unaffected, the significant reduction in g_s_ and E rates observed in SYN-STD plants compared to the other treatments and conditions highlights a hydraulic impairment, likely caused by a massive xylem colonization by pathogenic fungi, as previously reported [23]. Parallelly, the partial recovery observed in SYN-AF5 plants suggests that AF5 treatment helps maintaining the water balance and potentially delays the onset of leaf symptoms associated with hydraulic dysfunction. In fact, it has already been demonstrated that the onset of leaf symptoms negatively affect the sap flow with a clear correlation between symptoms severity and transpiration rate (E) [44]. Considering this data all together, in our experimental conditions, the AF5 symptomatic treated plants displayed an improved E, Pn and g_s_, suggesting that the observed reduction symptom severity can be associated to a recovery in the ecophysiological performances. This suggests that AF5 may mitigate esca-associated physiological constraints by improving water transport ability, a key factor influencing vine water relations and overall plant performance [10]. Indeed, the recovery in stomatal conductance and transpiration might result from the improved hydraulic functionality possibly linked to antioxidant and membrane-protective effects of α-tocopherol present in the AF5 formulation [16]. Though speculative, this observation aligns with the hypothesis that AF5 could counteract pathogen-induced xylem occlusion or oxidative damage, thereby supporting leaf-level gas exchange.

RNAseq demonstrates an activation of defence-related responses specifically in esca symptomatic plants

Furthermore, the analysis of must composition revealed that total soluble solids (TSS) were significantly lower in SYN-STD plants compared to SYN-AF5 and AS vines, indicating that esca can negatively impact fruit maturation [14]. AF5 treatment mitigated this effect, suggesting that it may contribute to partially restore both yield and quality in symptomatic plants.

RNA sequencing analysis highlighted a substantial transcriptional reprogramming in AF5-treated vines, particularly in symptomatic plants. GO enrichment analysis identified upregulated genes involved in stress responses, pathogen recognition, and hormone signalling. Among the upregulated genes, several encode components of signalling pathways related to ethylene (i.e., VIT_16s0050g02400, VIT_16s0100g00400, VIT_03s0063g00460, VIT_07s0031g00710 and VIT_07s0031g00720) and jasmonic acid (i.e., VIT_09s0002g00890, VIT_11s0016g00710, VIT_18s0001g12890, VIT_10s0003g03790 and VIT_10s0003g03800), suggesting a potential activation of these hormonal cascades. Notably, AF5 treatment induced the expression of FRK1 (FLG22-induced receptor-like kinase 1), WRKY transcription factors, and respiratory burst oxidase protein D (ATROBOHB), which play pivotal roles in plant immune responses and ROS-mediated defence mechanisms [13, 51]. The observed induction of DCL2 (DICER 2) suggests that AF5 treatment may also modulate RNA interference (RNAi)-mediated defence pathways, an emerging area of interest in plant-pathogen interactions with the recognition of small-RNAs (sRNAs) as a new class of effectors [53, 55].

Another key observation is the upregulation of genes associated with calmodulin-binding proteins and kinase activity, indicating that AF5 may enhance signal transduction cascades linked to systemic acquired resistance (SAR) [56]. The stronger transcriptomic response in symptomatic plants compared to asymptomatic ones suggests that AF5 primarily enhances pre-existing defence mechanisms rather than acting as a prophylactic agent. This is consistent with studies demonstrating that treatments with biostimulants effects often prime plant immunity, leading to a faster and stronger response upon pathogen challenge [1, 27]. In fact, while esca pathogens colonize the trunk, the systemic nature of defence signalling may enable leaves to act as priming sites, triggering SAR or other long-distance immune responses that restrict pathogen spread. Moreover, enhanced leaf defences may improve the plant’s overall physiological status [8], indirectly limiting the metabolic resources available for pathogen progression from the trunk.

The findings of this study provide compelling evidence for the efficacy of a novel strategy to mitigate esca disease progression. The reduction in disease incidence and severity, coupled with improved physiological performance and enhanced defence gene activation, supports the hypothesis that AF5 contributes to strengthening grapevine resilience against trunk diseases. The multifaceted action of AF5, which includes physiological stabilization, immune priming, and potential indirect interference with fungal colonization, offers a promising approach for integrating this treatment into sustainable vineyard management. Unlike conventional fungicides, which have limited efficacy against esca pathogens [41], AF5 appears to activate host-driven defence responses, providing a complementary strategy to current control measures such as pruning wound protection and trunk surgery [38]. Indeed, differently from all the measures available to date, this biostimulant works on plants already showing disease symptoms.

In contrast to the application of other foliar fertilizers AF5 provide a promising field-ready mitigation strategy for esca

Several recent studies have examined the use of foliar fertilization to mitigate symptoms of grapevine trunk diseases (GTDs), including esca, with varying outcomes. Calzarano et al. [15] demonstrated that repeated foliar applications of macro- and microelements could exacerbate esca foliar symptoms, potentially due to a decline in calcium content and a corresponding hypersensitive response. Conversely, Amarlu et al. [2] reported a significant reduction in symptom severity (from 56.6% to 19.95%) following foliar application of calcium, magnesium nitrate, and seaweed extract, which enhanced antioxidant enzyme activities (catalase and guaiacol peroxidase) and increased leaf area index. These contrasting findings are probably due to the underscored importance of formulation-specific effects and physiological contexts. Supporting this, Knoll et al. [37] found that foliar potassium applications in Vitis vinifera ‘Zweigelt’ decreased photosynthetic activity, indicating possible adverse effects depending on the nutrient composition. However, more recent findings highlight the potential of potassium, particularly in forms such as potassium sulfate and dihydrogen phosphate, to enhance grapevine physiological resilience by improving nutrient uptake, membrane stability, and stress tolerance [33, 35]. Within this framework, the AF5 formulation tested in the present work, which combines potassium acetate and α-tocoferol, appears to provide a balanced and effective nutritional stimulus. Indeed, unlike previous attempts that yielded inconsistent or detrimental effects, the AF5 formulation demonstrates a clear capacity to induce the activation of molecular defense pathways and contributes to the maintenance of a physiological equilibrium, suggesting it as a novel and effective tool for integrated esca disease management.

Future research should focus on elucidating the long-term effects of AF5 application, its interaction with vineyard microbiota, and its efficacy under varying climatic conditions. Given the increasing impact of climate change on trunk disease dynamics [20], strategies like the one here presented, that enhance grapevine physiological resilience, will be critical in developing integrated, low-impact disease management programs in the near future.

Conclusion

Overall, this study demonstrates that the newly developed treatment significantly reduces esca disease incidence and severity while promoting physiological stability and activating defence-related pathways in grapevines. The combination of field trials, ecophysiological assessments, and transcriptomic analyses provides strong support for its use as a viable support to be added to the already available preventive measures. Given the current challenges in controlling grapevine trunk diseases, it represents an innovative solution that could be widely and easily adopted in viticultural regions affected by esca.

Supplementary Information

Supplementary Material 1: Supplementary Figure 1. Experimental design: The experimental outline of the work has been summarized in the figure highlighting the field size (1 ha), the number of leaves evaluated for the physiological measurements and the subsequent molecular analysis. Supplementary Figure 2. Ecophysiological measurements: Leaf ecophysiological measurements were collected in the Lonigo field during the year 2021. a) Net photosynthesis (Pn); b) substomatal CO_2_ concentration (C_i_); c) stomatal conductance (g_s_); d) transpiration (E); e) intrinsic water use efficiency (iWUE), and f) instantaneous carboxylation efficiency (iCE) have been collected for STD and AF5, symptomatic (SYN) or asymptomatic (AS) plants. Data are presented as mean value of five randomly selected vines from each condition (AS and SYN plants) and treatment (STD and AF5). For each plant two fully developed non-senescent leaves at the same phenological age (4^th^ to 5^th^ leaf from the shoot apex) were measured. Supplementary Figure 3. Ecophysiological measurements: Leaf ecophysiological measurements were collected in the Lonigo field during the year 2022. a) Net photosynthesis (Pn); b) substomatal CO_2_ concentration (C_i_); c) stomatal conductance (g_s_); d) transpiration (E); e) intrinsic water use efficiency (iWUE), and f) instantaneous carboxylation efficiency (iCE) have been collected for STD and AF5, symptomatic (SYN) or asymptomatic (AS) plants. Data are presented as mean value of five randomly selected vines from each condition (AS and SYN plants) and treatment (STD and AF5). For each plant two fully developed non-senescent leaves at the same phenological age (4^th^ to 5^th^ leaf from the shoot apex) were measured. Supplementary Table 1. Commercial vineries protocols: List of dates, formulations, and dosage of agrochemicals are reported for each vineyard and year of experimentation. Supplementary Table 2. Meteorological data: meteorological data including precipitations, relative humidity, temperature and solar radiation were collected by the meteorological station located in the vineyard. Supplementary Table 3. Differentially expressed genes in asymptomatic plants: Differentially expressed genes (both over and under-expressed) comparing AF5 treated and STD asymptomatic plants have been identified and annotated. Supplementary Table 4. Differentially expressed genes in symptomatic plants: Differentially expressed genes (both over and under-expressed) comparing AF5 treated and STD symptomatic plants have been identified and annotated.