Unravelling the Biochemical and Molecular Priming Effect of a New Yeast-Derived Product: New Perspectives towards Disease Management

Abstract

Plants constantly face the environment that surrounds them and fight for survival against biotic and abiotic stress factors. To deal with harmful conditions, plants have developed a multilayer defence system, making them capable of recognising threats and promptly recovering from them. This phenomenon, which takes advantage of the “memory effect”, is referred to as bio-priming and represents a new frontier in terms of crop protection. Here, we investigated the “indirect” protective mechanisms of a new yeast extract formulate in Vitis vinifera cv. Sangiovese plants at both the biochemical and genic levels. The formulate was applied once a week for three consecutive weeks, and grapevine leaves were sampled from the first to the fifth day after treatment (dat) at every week of the experiment. Increased levels of jasmonic acid (every week at 2 dat; +70% as average) and abscisic acid (at 1 dat of the first week, more than 1.7-fold higher than the control) and the underproduction of salicylic acid (from 2 dat; −18%) confirmed that these signalling molecules/”specialised compounds” are actively involved in the early activation of defence pathways in treated vines. In addition, pr2 and chit1b, two genes involved in regulating hormonal crosstalk, were significantly up-regulated (both in the first and second week of the trial) and were also found to underlie upstream molecular activation. The results obtained by this investigation confirm the use of this new product to prime and protect grapevines from a wide range of fungal and fungal-like plant pathogens through the induction of defence responses.

1. Introduction

Existing approaches currently adopted in plant disease management are overwhelmingly based on chemical applications which have an adverse impact on the environment/human health and are limited by resource availability [1]. In 2009, the European Directive 2009/128/EC invited Member States to restrict the use of pesticides in agriculture because of the significant increase in environmental issues along with the generation of resistant pathogens [2]. In 2020, through the Farm to Fork strategy, the European Commission adopted a series of measures to improve European food systems, making them fairer, healthier, and more eco-friendly. New approaches to counteract the incidence/severity of plant diseases might act directly, by suppressing pathogens [3], or indirectly, by improving the plant immune system [4] or developing a process that prepares plants for a strong/fast defence activation, in the face of unfavourable growth conditions (i.e., bio-priming) [5].

Various abiotic and biotic stressors (such as plants, microbes, wounding, treatment with natural/synthetic compounds, beneficial microbes, and pathogen-derived effectors) constantly pose challenges to agricultural production; to cope with these stressors, plants have developed sophisticated defence mechanisms (at the physicochemical and molecular levels), thus promoting their own health [6]. The innate capability of plant species to produce an adequate adaptive response to stimuli/stress is known as basal tolerance, but this mechanism is often not sufficient. Consequently, plants may exhibit the so called “acquired resistance” by memorising previous stimuli/stress factors (in the form of functional and genic adjustments) and activating a faster and more robust response to efficiently cope with potential subsequent exposure [7]. This phenomenon is referred to as priming-induced acquired tolerance, and it stimulates cells to counteract low doses of primed distress/eustress in a manner than they would not if non-primed [8]. Defence priming is correlated with the development of various types of immunity (e.g., local and systemic), including systemic acquired resistance (SAR), induced systemic resistance (ISR), wound-induced resistance, and resistance provided by beneficial microorganisms [9]. At the functional level, priming often induces the accumulation of elicitor molecules/components, such as reactive oxygen species (ROS), phytohormones, secondary metabolites, lipid messengers, and Ca²⁺ flux, and the activation of antioxidant enzymes [10,11]. Plant responses to pathogens/pests are coordinated through signalling pathways that involve ethylene (Et), salicylic acid (SA), and jasmonic acid (JA) [12]. Generally, the Et/JA pathway is activated in defence against necrotrophic pathogens and results in ISR [13]. Conversely, the SA pathway is activated following colonisation by biotrophic pathogens, resulting in SAR. An increase in the SA content (and generally in that of secondary metabolites) is correlated with the production of ROS, which can lead to cell wall rearrangement by preventing the systemic diffusion of the pathogen [14]. At the genic level, the priming alters the kinetics of gene expression by following several trajectories: (i) supported expression of priming-induced genes through recovery; or (ii) reverting to the constitutive level during recovery followed by overproduction in response to the stimulus/stress [15]. In particular, the simultaneous activation of different genes encoding enzymes involved in hormone metabolism in the presence of both bio- and necrotrophic pathogens represents the core of the plant defence arsenal [16]. The extent, intensity, and recurrence of priming influence the duration of stimulus/stress memory, which may last from a few hours to days (short- or long-term, respectively). Consequently, it would be significant to clarify whether single or combined mechanisms underlie priming-induced tolerance/defence.

Although priming has been studied for decades, little is known about the molecular basis of this phenomenon. A complex network of events in transcriptional regulation involving resistance-related genes and/or genes related to specific plant processes is responsible for plant defence and/or adaptation to unfavourable growth conditions [17]. The genic mechanisms triggering priming include the induction of oxidative bursts, secondary metabolites, signalling molecules, phytohormones, and raised levels of pattern recognition receptors and antimicrobial compounds [18]. The induction of the primed state may trigger an enhanced accumulation of these regulatory proteins, which remain inactive until plants are exposed to a specific stress-derived signal. Consequently, a strong and fast stress-induced signal transduction develops in the primed plant cell, resulting in a stronger and faster activation of stress-specific and appropriate defence-related genes [19]. Upon exposure to another type of stress factor, a different set of signalling proteins becomes activated, leading to a stimulation of another defence response. Although the way in which plants arrange their defensive priorities is incompletely understood, there has already been an appreciable translation of knowledge obtained from model plants in the laboratory to the field [20].

Grapevine (Vitis vinifera L.) is considered a model plant for investigating disease resistance. In the last few years, a range of experimental evidence of the interaction among grapevine, pathogens, and resistance inducers has been obtained, indicating which signalling pathways/molecules are reasonable targets to be manipulated for increasing the defensive capacity and disease resistance of grapevine [18,21]. Several defence related genes in grapevine have been reported to be involved in signalling pathways connected with the response to pathogens or elicitor, including those encoding for the putative hypersensitive response marker HSR1, and for pathogenesis-related (PR) proteins, such as pr1, pr2 or pr5 [22,23]. Other genes, such as pal1 or chi1b, encoding for phenylalanine-ammonia lyase and chitinase 1b, respectively, have been reported as being rapidly activated in response to biostimulants [24,25].

This species is the most frequently treated cropping system due to the great number of diseases that may affect it during the vegetative and reproductive phases [26]. Consequently, there is an urgent need to understand/elucidate the capacity of the plant immune system to achieve better protection of grapevine.

The aim of the present study is to characterise, at the biochemical and genic level, the ‘indirect’ protective mechanism(s) induced by the application of a new yeast extract formulate (YE) in V. vinifera plants. We postulate a ‘bio-priming’ effect of YE through the activation of specific phytohormones/“specialised compounds” and appropriate defence-related genes in grapevine.

2. Materials and Methods

2.1. Plant Material and Experimental Design

In August 2021, two hundred V. vinifera cv. Sangiovese plants were purchased from a local nursery and placed at the field station of San Piero a Grado (Pisa, Italy), at the Department of Agriculture, Food and Environment, University of Pisa. For 1 month, plants were kept in semi-controlled conditions until the beginning of the experimental activities. Uniform-potted plants (with the same size) were selected and divided as follows: 80 plants treated with YE (ABE-IT 56, Saccharomyces cerevisiae Stamm DDSF623, Kwizda Agro GmbH, Vienna, Austria), and 80 plants sprayed with water (untreated control). Treatments with YE were performed at 9:00 am by foliar application followed by one treatment once a week for three consecutive weeks at the dose (1.25 L ha⁻¹, in 500 L of water) suggested by the owner company, until runoff (0.25 mL of YE for m⁻² of leaf surface) was achieved. Three leaves from five individual vines in each condition (untreated and YE-treated plants), were collected each day, i.e., from the 1st to the 5th day after treatment (dat) for three consecutive weeks (Figure S1).

2.2. Biochemical Analyses

Ethylene emission was determined according to the method described by Marchica et al. [27]. Leaves were excised, closed in 15 mL glass vials, and incubated for 2 h at 18 °C. One millilitre of gas, taken from the headspace of each sample, was injected in a gas chromatograph (an Agilent 8890B; Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a capillary column (Agilent HP-PLOT/Q+PT: coating thickness 0.20 μm; 0.32 mm × 30 m, Agilent Technologies Inc., Santa Clara, CA, USA), and single quadrupole mass detector (Agilent 5977B, Agilent Technologies Inc., Santa Clara, CA, USA).

The total SA (conjugated and free forms) content was determined following the method described by Modesti et al. [21]. Leaves were added to methanol (90% v/v), sonicated and centrifuged (13,000× g at 18 °C for 15 min). The pellet was re-suspended in 100% methanol, while the supernatant was kept before undergoing the same procedure as reported above. Both supernatants were then mixed and left to evaporate at 35 °C under a vacuum (RVC 2-25 CDplus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). The residue was resuspended in 5% (w/v) trichloroacetic acid and partitioned by using ethyl acetate/cyclohexane (v/v). The upper phases were concentrated under a vacuum at 35 °C, and the lower aqueous phases were hydrolysed by adding HCl (8 N) and incubating for 1 h at 80 °C. The SA collected from both the phases, after it was combined and dissolved in a mixture containing sodium acetate buffer (0.2 M, pH 5.5), water (90%) and MeOH (10%), was separated at 40 °C by an ultra-high pressure liquid chromatography system (UHPLC; Dionex UltiMate 3000, Thermo Scientific, Waltham, MA, USA) equipped with an C18 column (Acclaim 120, 150 mm length × 4.6 mm internal diameter 5 μm particle size, Thermo Scientific, Waltham, MA, USA), and a Fluorescence Detector (UltiMate™ 3000, Thermo Scientific, Waltham, MA, USA) with excitation and emission at 305 and 407 nm, respectively.

Jasmonic acid was quantified according to the method proposed by Modesti et al. [21]. Leaves were added to 100% methanol, sonicated and centrifuged (3000× g at 18 °C for 30 min). The filtered supernatant was then evaporated under a vacuum at 35 °C. Next, the residue was re-suspended with ethyl acetate and injected into a GC-MS (previously described) equipped with a capillary column (Agilent DB-5MS (UI) 30 m × 0.25 mm; coating thickness 0.25 μm, Agilent Technologies Inc., Santa Clara, CA, USA).

Abscisic acid (ABA) was quantified using the Phytodetek^® Immunoassay Kit (Agdia, Elkhart, IN, USA), according to Marchica et al. [27]. Leaves were added to distilled water, sonicated and centrifuged (13,000× g for 30 min at 18 °C). The determination of ABA was performed at 415 nm by using a Victor3 1420 Multilabel Couter microplate reader (Perkin Elmer Inc., Waltham, MA, USA).

The hydrogen peroxide (H₂O₂) content was quantified using the AmplexTM Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes, Life Technologies Corp., Carlsbad, CA, USA), according to Pisuttu et al. [28]. Leaves were added to potassium phosphate buffer (20 mM and pH 6.5), centrifuged (12,000× g for 15 min at 4 °C), and kept in the dark at 25 °C for 30 min. The samples then underwent 530 nm (excitation) and 590 nm (emission) of resorufin fluorescence with the fluorescence/absorbance microplate reader described above.

Superoxide radical (^●O₂⁻) levels were determined according to the method proposed by Tonelli et al. [29]. Leaves were added to potassium phosphate buffer (50 mM and pH 7.5), centrifuged (12,000× g for 15 min at 4 °C), and incubated in the dark at 25 °C for 30 min. The samples then underwent 470 nm excitation with the same fluorescence/absorbance microplate reader mentioned above. The antioxidant activity of the frozen foliage samples was evaluated by determining their hydroxyl radical averting capacity (HORAC) and oxygen radical absorption capacity (ORAC), according to the method proposed by Marchica et al. [30]. Leaves were added to 100% of methanol, sonicated and centrifuged (13,000× g at room temperature for 15 min). The supernatants were mixed with fluorescein, which was used as a fluorescent probe, and incubated at 37 °C for 30 min. Both assays were quantified at 480 nm (excitation) and 530 nm (emission) with the same fluorescence/absorbance microplate reader described above. The results were expressed with antioxidant standard curves in gallic acid equivalents for HORAC and Trolox equivalents for ORAC.

2.3. Gene Expression Analyses

The relative expression of the plant defence-related genes was evaluated by quantitative Real-Time PCR by using Rotor-Gene Q cycler (Qiagen, Milan, Italy). Four genes connected with the SA-depending signalling, i.e., pr1, pr2 (β-1,3-glucanase), pr5 (thaumatin-like protein), and enhanced disease susceptibility 1 (eds1), two genes involved in JA/Et-depending signalling, i.e., chit1b, hypersensitive response marker 1, (hsr1), and a shared genetic element of both involved in basal resistance (pal1) were included in the analyses.

RNA was extracted from leaf material according to the method proposed by Fontana et al. [31]. RNA samples were treated with DNase I (AMPD1 DNase I Amplification Grade, Sigma, Milan, Italy), according to the manufacturer’s instructions, and 400 ng of DNase I-treated RNA were used for cDNA synthesis using the Maxima First Strand cDNA synthesis kit (K1642, Applied Biosystems, Monza, Italy).

PCR reactions were set up with cDNA, QuantiNova SYBR^® Green PCR Master Mix 2X (Qiagen, Milan, Italy), each primer and Nuclease-Free water. All PCR reactions were performed as follows: initial activation, 95 °C for 2 min; 40 cycles of denaturation, 95 °C for 5 sec, and combined annealing/extension, 60 °C for 10 s. Threshold cycles (CTs) were calculated with Rotor-Gene Q Series Software v2.3.1 (Qiagen, Milan, Italy), using the pyruvate-decarboxylase 1 (pcd1) gene as endogenous control [32]. Data were expressed as 2^−ΔΔCt [33]. Primers for each gene were designed using Geneious 10.0.9 (www.geneious.com, accessed on 1 September 2021), and in silico analysed using the NetPrimer platform available at www.premierbiosoft.com/NetPrimer/AnalyzePrimer.jsp (accessed on 1 September 2021). The primers used for gene expression analysis were checked for efficiency and dimer formation and are listed in Table S1.

2.4. Statistical Analysis

The Shapiro-Wilk’s and Levene’s tests were used to assess the normal distribution of data and the homogeneity of variance. The results from biochemical analyses were submitted to a one-way analysis of variance (ANOVA), to evaluate the effect of “time”, and to a Student’s t-test, to analyse the effect of “treatment”. The results from molecular analyses were submitted to a one-way ANOVA to evaluate the effect of “treatment”. Comparisons among means were determined by the Tukey’s HSD post hoc test by using JMP Pro 14.0 software (SAS Institute Inc., Cary, NC, USA). For all the analyses, statistically significant effects were considered for p ≤ 0.05.

3. Results

3.1. Biochemical Response

According to the one-way ANOVA test, the effect of time was significant for all the examined phytohormones/signalling molecules (Figure 1). In YE-treated plants, Et emission did not show any change when compared to 0 dat, except during the second (at 2, 3 and 5 dat; −31, −29 and −35%, respectively) and the third week of application (−36% as average), where it significantly decreased (Figure 1A). The concentration of total SA significantly decreased already at 2 dat during the first week of YE application (−23%, in comparison to 0 dat), and even more at all the other days of analysis (−37, −44 and −20% at 3, 4 and 5 dat, during the first week; −30, −39, −33, −59 and −44% during all the second week examined times, respectively; −26, −49 and −25% at 1, 4 and 5 dat of the third week), reaching a minimum at 4 dat during the second week (7.3 ± 0.7 µg g⁻¹ FW; Figure 1B). Conversely, an evident increase of JA content was observed at each 2 dat of the three weeks (as average +57%; Figure 1C). No significant differences were observed during the other days of analysis. Finally, the content of ABA significantly increased at 1 dat during each of the three weeks (more than 3-fold higher than 0 dat, on average; Figure 1D). No statistical differences were observed in ABA content during the other days of analysis, except during the second (3-fold higher than 0 dat at 3 dat) and the third week of application (2-fold higher than 0 dat at 4 dat, respectively).

According to the one-way ANOVA test, the effect of time was significant for all the examined ROS (Figure 2). During the first week of YE application, the H₂O₂ concentration significantly increased at 4 dat (+61%; Figure 2A). Similarly, a marked production of H₂O₂ was observed during the second and the third week of application (+37, +65 and + 35% at 2, 4 and 5 dat; +95 and +38% at 2 and 3 dat, respectively). Any significant difference occurred between 0 dat and the other days of analysis. In YE-treated plants, ^●O₂⁻ did not change when compared to 0 dat throughout the experiment, except at 3 dat during the first week of application (+85%) and at 4 dat of the second week of trial (+64%; Figure 2B).

According to the one-way ANOVA test, the effect of time was significant for the antioxidant activity (expressed as the HORAC and ORAC values; Figure 3). In YE-treated plants, HORAC values increased at 4 dat of the first week (+10%), at 3 and 4 dat during the second week (+10 and +11%), and at 3 dat of the third week of application (+10% in comparison to 0 dat; Figure 3A), respectively. No significant differences were observed when compared to 0 dat in the remaining days of analysis. Similarly, ORAC values were unchanged, except during the first (+21 and +28% at 3 and 5 dat, respectively) and the third (+22% at 3 dat) week of application (Figure 3B).

The Student’s t-test revealed that the Et emission was lower in YE-treated plants compared to control during the first and the third week of application (at 3 and 4 dat, respectively), when it significantly decreased (−29 and −28%;

Table 1. Variation in the ethylene (Et), total salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) contents in Vitis vinifera cv. Sangiovese leaves treated with yeast extract (YE) or with water (C). Applications were performed for three consecutive weeks (W1, W2 and W3), and each treatment once a week. Data are shown as mean ± standard deviation (n = 5). p-values of Student’s t test are shown for each parameter of C and YE (*** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, ns p > 0.05). Abbreviations: dat, days after treatment.

		Et (nl g−1 FW h−1)			SA (µg g−1 FW)			JA (µg g−1 FW)			ABA (ng g−1 FW)
	dat	C	YE	p	C	YE	p	C	YE	p	C	YE	p
W1	0	1.44 ± 0.14	1.44 ± 0.14	ns	16.11 ± 1.12	17.75 ± 0.80	ns	1.25 ± 0.09	1.21 ± 0.17	ns	30.65 ± 2.20	28.43 ± 2.24	ns
	1	1.25 ± 0.13	1.16 ± 0.04	ns	16.82 ± 0.82	17.92 ± 0.79	ns	1.01 ± 0.25	1.13 ± 0.07	ns	47.96 ± 0.42	103.89 ± 10.27	***
	2	1.23 ± 0.10	1.28 ± 0.11	ns	16.54 ± 1.01	13.65 ± 1.08	**	1.10 ± 0.16	1.74 ± 0.04	***	43.31 ± 10.77	45.33 ± 7.01	ns
	3	1.56 ± 0.11	1.11 ± 0.04	**	17.51 ± 0.98	11.12 ± 0.86	***	1.30 ± 0.05	1.13 ± 0.10	ns	35.16 ± 0.80	36.63 ± 4.63	ns
	4	1.06 ± 0.10	1.17 ± 0.14	ns	16.22 ± 0.83	10.01 ± 0.92	***	0.99 ± 0.09	1.08 ± 0.13	ns	37.13 ± 2.11	32.53 ± 0.64	ns
	5	1.14 ± 0.14	1.29 ± 0.08	ns	19.29 ± 0.52	14.13 ± 0.89	***	1.05 ± 0.16	1.09 ± 0.07	ns	35.82 ± 4.82	37.47 ± 6.16	ns
W2	1	1.21 ± 0.13	1.22 ± 0.12	ns	14.35 ± 1.37	12.48 ± 0.79	*	1.03 ± 0.05	0.99 ± 0.14	ns	55.89 ± 5.31	68.14 ± 4.08	*
	2	0.95 ± 0.06	1.00 ± 0.06	ns	15.66 ± 0.46	10.81 ± 1.27	***	1.09 ± 0.18	1.84 ± 0.05	***	42.89 ± 4.54	31.23 ± 6.41	ns
	3	1.08 ± 0.18	1.03 ± 0.09	ns	14.33 ± 0.65	11.91 ± 0.51	***	1.03 ± 0.05	1.09 ± 0.16	ns	45.27 ± 5.08	85.23 ± 6.50	***
	4	1.13 ± 0.10	1.21 ± 0.09	ns	11.06 ± 0.85	7.32 ± 0.73	***	1.12 ± 0.12	0.98 ± 0.11	ns	34.72 ± 1.76	44.96 ± 3.68	*
	5	0.96 ± 0.05	0.94 ± 0.03	ns	12.75 ± 1.19	9.86 ± 0.79	**	1.03 ± 0.14	1.30 ± 0.12	ns	31.64 ± 3.33	28.28 ± 5.26	ns
W3	1	0.97 ± 0.12	0.88 ± 0.03	ns	15.36 ± 0.80	13.22 ± 1.17	ns	1.25 ± 0.20	1.11 ± 0.10	ns	26.35 ± 4.35	44.56 ± 4.06	***
	2	0.99 ± 0.04	0.95 ± 0.06	ns	16.01 ± 1.00	16.08 ± 0.95	ns	1.14 ± 0.07	2.10 ± 0.09	***	18.20 ± 1.46	18.91 ± 2.47	ns
	3	1.01 ± 0.03	0.97 ± 0.06	ns	13.29 ± 1.12	15.44 ± 0.93	ns	1.06 ± 0.05	1.10 ± 0.07	ns	42.84 ± 1.34	34.70 ± 5.22	ns
	4	1.14 ± 0.11	0.81 ± 0.08	*	10.64 ± 1.07	9.08 ± 0.34	ns	1.20 ± 0.24	1.25 ± 0.04	ns	28.35 ± 6.22	58.93 ± 9.37	**
	5	1.04 ± 0.05	1.00 ± 0.04	ns	11.53 ± 1.18	13.23 ± 0.74	ns	1.11 ± 0.16	1.12 ± 0.08	ns	51.92 ± 1.97	48.14 ± 2.30	ns

). No other significant differences were recorded throughout the experiment. The total SA content in YE-treated plants was lower than the control through the experimental period (−30% as average), except at 0 and 1 dat of the first week and during all days of analysis of the third week of application, where no significant differences were observed. By contrast, the JA content evidently increased in YE-treated plants at 2 dat during each of the three weeks (+58, +69 and +84% in comparison to control, respectively). Finally, in YE-treated plants, ABA significantly increased at 1 dat during each of the three weeks (more than 1.7-fold higher, on average), and at 3 and 4 dat of the second week (+88 and +29%, respectively), and at 4 dat of the third week of application (2-fold higher than the control), when compared to control. No significant differences were observed in the ABA content during the other days of analysis.

The Student’s t-test revealed that the H₂O₂ levels were higher in YE-treated plants when compared to the control at 4 dat during the first week of YE application (+41%), at 4 and 5 dat of the second week (+53 and +30%), and at 2 and 3 dat of the third week of experiment (+73 and +17%;

Table 2. Variation in hydrogen peroxide (H₂O₂) and superoxide radical (^●O₂⁻) in Vitis vinifera cv. Sangiovese leaves treated with yeast extract (YE) or with water (C). Applications were performed for three consecutive weeks (W1, W2 and W3), and each treatment once a week. Data are shown as mean ± standard deviation (n = 5). p-values of Student’s t test are shown for each parameter of C and YE (*** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, ns p > 0.05). Abbreviations: dat, days after treatment.

		H2O2 (mmol g−1 FW)			●O2− (mmol g−1 FW)
	dat	C	YE	p	C	YE	p
W1	0	0.20 ± 0.02	0.19 ± 0.01	ns	0.006 ± 0.000	0.006 ± 0.001	ns
	1	0.21 ± 0.03	0.24 ± 0.01	ns	0.006 ± 0.000	0.007 ± 0.001	ns
	2	0.23 ± 0.02	0.22 ± 0.03	ns	0.006 ± 0.001	0.006 ± 0.000	ns
	3	0.21 ± 0.03	0.22 ± 0.04	ns	0.007 ± 0.001	0.012 ± 0.001	***
	4	0.22 ± 0.01	0.31 ± 0.03	**	0.007 ± 0.000	0.009 ± 0.001	*
	5	0.21 ± 0.02	0.24 ± 0.02	ns	0.006 ± 0.000	0.006 ± 0.001	ns
W2	1	0.22 ± 0.01	0.22 ± 0.00	ns	0.009 ± 0.001	0.010 ± 0.000	ns
	2	0.24 ± 0.02	0.27 ± 0.02	ns	0.009 ± 0.001	0.009 ± 0.000	ns
	3	0.24 ± 0.00	0.23 ± 0.02	ns	0.011 ± 0.000	0.010 ± 0.001	ns
	4	0.21 ± 0.01	0.32 ± 0.00	***	0.011 ± 0.001	0.011 ± 0.001	ns
	5	0.20 ± 0.02	0.26 ± 0.01	**	0.010 ± 0.000	0.011 ± 0.000	ns
W3	1	0.22 ± 0.01	0.24 ± 0.01	ns	0.006 ± 0.000	0.007 ± 0.001	ns
	2	0.22 ± 0.02	0.38 ± 0.03	***	0.006 ± 0.001	0.008 ± 0.000	ns
	3	0.23 ± 0.01	0.27 ± 0.01	**	0.007 ± 0.000	0.006 ± 0.001	ns
	4	0.19 ± 0.01	0.21 ± 0.03	ns	0.007 ± 0.000	0.007 ± 0.000	ns
	5	0.18 ± 0.01	0.18 ± 0.02	ns	0.006 ± 0.001	0.007 ± 0.000	ns

). The superoxide radical levels increased in YE-treated plants at 3 and 4 dat of the first week of trial (+71 and +29%, respectively). No significant differences were observed in the remaining days of analysis.

The Student’s t-test showed that the HORAC levels in YE-treated plants did not change when compared to the control throughout the experiment, except at 4 and 3 dat of the first and third week of application, respectively (+9 and +13%;

Table 3. Variation in hydroxyl radical averting capacity (HORAC) and oxygen radical absorption capacity (ORAC) in Vitis vinifera cv. Sangiovese leaves treated with yeast extract (YE) or with water (C). Applications were performed for three consecutive weeks (W1, W2 and W3), and each treatment once a week. Data are shown as mean ± standard deviation (n = 5). p-values of Student’s t test are shown for each parameter of C and YE (*** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, ns p > 0.05). Abbreviations: dat, days after treatment; GAE, gallic acid equivalents; TE, Trolox equivalents.

		HORAC (mmol GAE g−1 FW)			ORAC (mmol TE g−1 FW)
	dat	C	YE	p	C	YE	p
W1	0	128.52 ± 4.38	130.25 ± 2.66	ns	125.24 ± 1.00	124.24 ± 2.49	ns
	1	128.73 ± 3.33	128.71 ± 5.24	ns	126.09 ± 4.43	124.85 ± 7.95	ns
	2	128.66 ± 2.09	128.54 ± 5.17	ns	125.00 ± 3.76	125.76 ± 5.74	ns
	3	129.91 ± 2.48	135.40 ± 3.76	ns	131.98 ± 3.04	150.46 ± 4.84	***
	4	131.64 ± 2.01	143.59 ± 2.63	*	127.56 ± 4.30	139.12 ± 2.53	**
	5	132.34 ± 3.32	137.68 ± 1.05	ns	125.93 ± 0.12	159.51 ± 3.62	***
W2	1	128.28 ± 0.92	132.63 ± 2.71	ns	126.69 ± 4.91	122.58 ± 7.01	ns
	2	132.09 ± 4.19	136.83 ± 3.26	ns	124.98 ± 5.48	131.38 ± 1.50	ns
	3	144.68 ± 1.48	143.09 ± 4.83	ns	127.65 ± 7.32	126.86 ± 5.88	ns
	4	137.11 ± 2.57	144.66 ± 1.51	ns	126.59 ± 7.24	119.51 ± 7.13	ns
	5	132.29 ± 2.75	131.60 ± 1.97	ns	120.83 ± 3.53	121.80 ± 6.00	ns
W3	1	127.58 ± 2.43	127.72 ± 1.42	ns	129.61 ± 3.26	133.44 ± 3.04	ns
	2	127.38 ± 2.49	127.74 ± 1.83	ns	123.65 ± 3.54	130.22 ± 5.40	ns
	3	126.95 ± 3.60	143.12 ± 2.84	***	130.81 ± 3.33	151.62 ± 6.73	***
	4	128.45 ± 4.34	124.34 ± 4.50	ns	133.16 ± 1.78	132.18 ± 4.83	ns
	5	126.97 ± 2.89	129.32 ± 1.72	ns	131.27 ± 7.25	134.58 ± 4.64	ns

). Similarly, the ORAC values did not show significant differences during the experiment, except for increased levels at 3, 4 and 5 dat during the first week (+14, +9 and +27%, respectively) and at 3 dat of the third week of trial (+16%).

3.2. Transcriptional Responses

The application of YE resulted in changes in gene expression in treated leaves compared to the untreated ones; genes with a fold-change threshold of at least +2/−2 were considered differentially expressed. During the first week of YE application, the relative expression of chit1b gene slightly decreased at 2 dat (0.48-fold), and significantly up-regulated at 3 and 4 dat (3.87 and 3.65-fold, respectively; Figure 4A). Similar behaviour was observed during the second week of application with a fold-change higher than 2 at all the samplings (except at 4 days). No significant differences between untreated and YE-treated plants were documented during the other days of analysis. The relative expression of eds1, pr1 and pal1 did not ever significantly overcome the fixed up-regulation threshold (+2 ≤ fold change ≤ 0.5; Figure 4B,C,F). In YE-treated plants, relative expression of pr2 significantly increased during the first (2.05-fold at 1 dat), the second (2.08, 2.44 and 2.84-fold at 2, 3 and 4 dat, respectively), and the third week (2.46-fold at 1 dat) of application (Figure 4D). The relative expression of pr5 did not show changes in YE-treated plants when compared to the control throughout the experiment, except during the first (2.75-fold at 3 dat) and the third (2.60-fold at 2 dat) week of application, when it was found to be up-regulated (Figure 4E). In YE-treated leaves, the relative expression of hsr1 slightly decreased during the first (at 2 and 5 dat; 0.45 and 0.40-fold, respectively) and the third (at 2 dat; 0.33-fold) week of application (Figure 4G). Conversely, the expression of hsr1 was found to be up-regulated during the second week of application with a fold-change higher than 2 at all the samplings (except at 4 days). No significant differences between untreated and YE-treated plants were observed on the other days of analysis.

4. Discussion

In the present study, the application of YE triggered a marked increase in JA levels at 2 dat during the three weeks of the experiment, suggesting that this phytohormone is engaged in YE-priming-induced responses. The subsequent or concomitant (but transient) accumulation of H₂O₂ and ^●O₂⁻ confirmed that JA represents a self-amplifying system and that it stimulates ROS production [34]. In addition, during YE application, it might stimulate an ABA-related priming, as documented by the significant increase in the levels of this hormone (observed at 1 dat), during the three weeks of the experiment. The absence of any enhancement of SA throughout the experiment suggests that this secondary metabolite did not play a pivotal role in the modulation of YE-priming-induced and/or signalling responses [35]. In particular, the reduction of SA (observed already at 2 dat of the first and the second week of YE application, and even more during all the other days of analysis) may be related to the consumption of this compound promoted by the cells to counteract ROS accumulation [36]. This result indicates that SA was employed as a mediator of cell survival by providing defence reactions (e.g., phenylpropanoid pathway), rather than as part of the signalling pathways [34], and by regenerating active reduced forms (as documented by the rise of the total antioxidant capacity concomitant with the peaks of ROS) [35]. This is also confirmed by the increased expression profiles of pal1 (gene encoding enzyme in the early part of the phenylpropanoid biosynthesis pathways) [37] throughout the experiment (except at 2 dat during the first week), although they did not ever significantly overcome the fixed up-regulation threshold. Consequently, it is possible to speculate that the intensity, extent and recurrence of YE-priming guaranteed the maintenance of ROS-antioxidant homeostasis [38]. In addition, the absence of any alteration in Et production (except at 4 dat during the third week) confirmed a crosstalk between JA- and ABA-related signalling/adaptive responses due to YE application. Finally, it is possible to conclude that these molecules/hormones play a crucial role in modulating the YE-induced priming.

At the genic level, priming alters the protein and transcript abundances and/or functionality. These variations include a multifaceted network of events in transcriptional regulation which involves phytohormone-responsive marker genes and/or genes related to specific plant processes [17]. Here, the most marked changes in plant defence-related gene expression were observed for chit1b and hsr1 (especially during the first and second weeks of YE application) by indicating their involvement in an early, quick and local defence response. In addition, the concomitant increase in the JA content confirmed that these genes are activated by the accumulation of this hormone, being highly responsive to JA/Et [39]. Like plant chitinases, the genes involved in the hypersensitive response (HR) are associated with the plant’s effector-triggered immunity, the activation of which often initiates after an early ROS-burst [40]. Thus, YE application seems to stimulate JA-mediated basal resistance in treated V. vinifera plants through ROS accumulation by triggering the activation of a second and more specific defence layer involving the induction of genes coding for PR (e.g., chit1b) [41] or HR-connected proteins (e.g., hsr1) [42]. These genes play a pivotal role in the systemic response, and their activity lasts longer (as confirmed by their up-regulation observed during the second and the third week of application), indicating that all these mechanisms might be able to gradually shift the local defence response to a systemic one [40]. It is worth noting that the effective induction of PR proteins is usually accomplished by low SA concentrations [43], which is in line with the absence of any enhancement of SA throughout the experiment and the minor variations on the expression of SA-responsive genes (pr1, pr2, pr5 and eds1) observed here. Therefore, it can be proposed that YE-induced priming in treated V. vinifera plants involves JA/ABA-mediated defence responses, including the activation of plant basal resistance and genes connected with the biosynthesis of specific resistance proteins.

5. Conclusions

In conclusion, our study demonstrated the activation of specific defence signals, at both the biochemical and molecular levels, induced by the application of a YE based product onto the grapevine canopy. In particular, the JA/ABA-mediated pathway here triggered, together with the up-regulation of specific genes (as chit1b, pr2 and hsr1), which lasted throughout all the experiment, support our speculation that this new product can boost the natural defence of grapevine against a wide range of biotic stresses. Further research should investigate the performance of YE in relation to the major V. vinifera patho-systems to fully harness its potential as a resistance inducer. Finally, to respond to consumer demand for residue-free products, this YE formulation should receive approval from the relevant authorities for widespread use.