Biochemical Changes in Leaves of Vitis vinifera cv. Sangiovese Infected by Bois Noir Phytoplasma.

Bois noir is a disease associated with the presence of phytoplasma ‘Candidatus Phytoplasma solani’ belonging to the Stolbur group (subgroup 16SrXII-A), which has a heavy economic impact on grapevines. This study focused on the changes induced by phytoplasma in terms of the profile and amount of secondary metabolites synthesized in the phenylpropanoid pathway in leaves of Vitis vinifera L. red-berried cultivar Sangiovese. Metabolic alterations were assessed according to the disease progression through measurements of soluble sugars, chlorophyll, and phenolic compounds produced by plant hosts, in response to disease on symptomatic and asymptomatic Bois noir-positive plants. Significant differences were revealed in the amount of soluble sugars, chlorophyll, and accumulation/reduction of some compounds synthesized in the phenylpropanoid pathway of Bois noir-positive and negative grapevine leaves. Our results showed a marked increase in phenolic and flavonoid production and a parallel decrease in lignin content in Bois noir-positive compared to negative leaves. Interestingly, some parameters (chlorophyll a, soluble sugars, total phenolic or flavonoids content, proanthocyanidins, quercetin) differed between Bois noir-positive and negative leaves regardless of symptoms, indicating measurable biochemical changes in asymptomatic leaves. Our grapevine cultivar Sangiovese results highlighted an extensive modulation of the phenylpropanoid biosynthetic pathway as a defense mechanism activated by the host plant in response to Bois noir disease.


Introduction
Phytoplasmas are obligate plant-pathogenic organisms of the Mollicutes class, which infect many crops and hundreds of plant genera cultivated worldwide; thus, causing heavy economic losses [1,2]. Phytoplasmas have become an increasingly serious threat to many perennial plants, such as fruits, shrubs, woods, ornamental trees and, in particular, grapevine in Europe. In Italy, which is the second-largest wine producer in Europe, phytoplasmas are a serious limiting factor for grape production, particularly in traditional grapevine-growing regions [3].
One of the most important diseases in the main viticultural areas of Italy is "Bois noir" (BN), caused by the phytoplasma 'Candidatus Phytoplasma solani' of the Stolbur group (subgroup 16SrXII-A) [4].  Figure 1C), while in September, the same plants were classified as belonging to severity class 3 (= more than three shoots with symptoms of reddening leaf, Figure 1D). Thus, BN-positive samples collected in July were considered as asymptomatic, while those collected in September were considered as symptomatic.

Pigments and Soluble Sugars
The amount of chlorophyll a measured in BN-negative leaves was higher than in BN-positive leaves in two different key-stages. This, thus, showed the better photosynthesis activity of BNnegative leaves and a reduction in its amount in the BN-positive leaves from −15% to −44% from July to September (Table 1). On the other hand, the measured quantity of chlorophyll b, total chlorophylls (a+b), and carotenoids (Cars), despite being lower in the BN-positive leaves compared to BN-negative leaves in July, was considerably less in September, when the symptoms of phytoplasma disease were clearly visible on the leaves. The Chl a/b ratio decreased in BN-positive leaves, from −14% in July to −19% in September (Table 1), while the Car/Chls ratio was unaffected and no differences between BN-negative and BN-positive leaves were recorded ( Table 1).
The soluble sugars detected in BN-positive leaves were higher than the BN-negative leaves in two different key-stages. In particular, in the BN-positive leaves, the soluble sugar content was over +68% in BN-positive leaves sampled in July, and + 31% in BN-positive leaves sampled in September, as reported in Table 1. Table 1. Differences in the content (mg·g −1 dry weight (DW)) of chlorophyll (Chls), carotenoids (Cars), their ratio and soluble sugars in BN-positive and BN-negative leaves collected in July and September.

Pigments and Soluble Sugars
The amount of chlorophyll a measured in BN-negative leaves was higher than in BN-positive leaves in two different key-stages. This, thus, showed the better photosynthesis activity of BN-negative leaves and a reduction in its amount in the BN-positive leaves from −15% to −44% from July to September (Table 1). On the other hand, the measured quantity of chlorophyll b, total chlorophylls (a+b), and carotenoids (Cars), despite being lower in the BN-positive leaves compared to BN-negative leaves in July, was considerably less in September, when the symptoms of phytoplasma disease were clearly visible on the leaves. The Chl a/b ratio decreased in BN-positive leaves, from −14% in July to −19% in September (Table 1), while the Car/Chls ratio was unaffected and no differences between BN-negative and BN-positive leaves were recorded (Table 1). The soluble sugars detected in BN-positive leaves were higher than the BN-negative leaves in two different key-stages. In particular, in the BN-positive leaves, the soluble sugar content was over +68% in BN-positive leaves sampled in July, and + 31% in BN-positive leaves sampled in September, as reported in Table 1. 2.3. Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and Proanthocyanidins (PAs) Statistically significant differences in TPC and TFC leaf extracts were observed between BN-positive and BN-negative leaves at the key stages analyzed (Figure 2). TPCs measured in BN-positive samples collected in July and September were +38% and +29% higher compared to BN-negative leaves, respectively. Similarly, TFCs measured in two key-stages were +55% and +65% higher in BN-positive leaves compared to BN-negative leaves. These results indicated higher amounts of both total phenolics and flavonoids in BN-positive leaves regardless of the presence of disease symptoms on leaves.   Table 2 shows the phenolic compounds and anthocyanins extracted from the BN-positive and BN-negative leaves of grapevine cv. Sangiovese and identified by negative and positive ionization mode, using High-Performance Liquid Chromatography coupled to Electrospray Ionization Time-  On the other hand, the differences between the BN-negative and BN-positive leaves in PA amount was statistically significant only in July with higher values in the BN-positive leaves (+26%) despite the samples not clearly showing symptoms of BN disease. The BN-positive leaves sampled in September, with clear symptoms of phytoplasma disease, did not show differences in the amount of PAs compared to the BN-negative leaves sampled at the same stage ( Figure 2). Table 2 shows the phenolic compounds and anthocyanins extracted from the BN-positive and BN-negative leaves of grapevine cv. Sangiovese and identified by negative and positive ionization mode, using High-Performance Liquid Chromatography coupled to Electrospray Ionization Time-of-Flight Mass Spectrometry (HPLC ESI/MS-TOF). The qualitative analysis of all the extracts identified 42 compounds, which are listed in Table 2 with the respective retention times, experimental and calculated m/z, molecular formula, errors, score, and literature references.  The qualitative analysis of the cv. Sangiovese BN-positive and BN-negative leaf extracts did not show significant differences, indicating that there was no evident qualitative variation between the phenolic and anthocyanin profiles identified in our study. In the extracts, 42 compounds were detected, 36 of which were annotated or identified. However, only compounds that have differences lower than 5 ppm are considered properly annotated ( Table 2).
As shown in Figure 3, all three anthocyanins were detected only in traces in the BN-negative leaves sampled both in July and in September. On the other hand, in BN-positive leaves, in July the anthocyanins were present only in traces, while in September, there was a considerable accumulation only of cyanidin 3-glucoside (n. 41). In fact, when in September, on the BN-positive leaves the symptoms of disease were clearly visible (Figure 1), the amount of cyanidin 3-glucoside (n. 41) was more than 10-fold higher than the BN-negative leaves, where no chlorotic spots and reddish-purple Pathogens 2020, 9, 269 7 of 17 coloration of leaves were observed. In September, however, the leaves collected from the same plants did not show an accumulation or statistically positive variation in delphinidin 3-glucoside (n. 40) and peonidin 3-glucoside (n. 42), the other two anthocyanins quantified, compared to BN-negative leaves and/or compared to the previous sampling performed in July ( Figure 3). (n. 38) and syringetin-3-O-galactoside (n. 39); sugars: hexose derivate isomer 1-3 (n. 17, 18, 20); stilbenoids, only 5.56% of total compounds detected (viniferin (n. 12) and resveratrol glucoside (n. 36)) and anthocyanidins: delphinidin 3-glucoside (n. 40), cyanidin 3-glucoside (n. 41), and peonidin 3-glucoside (n. 42).
Quantitative analyses were performed on the most representative secondary metabolites commonly associated with phytoplasma disease in grapevines.
Anthocyanins. The three anthocyanins, delphinidin 3-glucoside (n. 40), cyanidin 3-glucoside (n. 41), and peonidin 3-glucoside (n. 42), identified in the leaf extracts (Table 2), were quantified and the respective amounts are shown in Figure 3. As shown in Figure 3, all three anthocyanins were detected only in traces in the BN-negative leaves sampled both in July and in September. On the other hand, in BN-positive leaves, in July the anthocyanins were present only in traces, while in September, there was a considerable accumulation only of cyanidin 3-glucoside (n. 41). In fact, when in September, on the BN-positive leaves the symptoms of disease were clearly visible (Figure 1), the amount of cyanidin 3-glucoside (n. 41) was more than 10-fold higher than the BN-negative leaves, where no chlorotic spots and reddish-purple coloration of leaves were observed. In September, however, the leaves collected from the same plants did not show an accumulation or statistically positive variation in delphinidin 3-glucoside (n. 40) and peonidin 3-glucoside (n. 42), the other two anthocyanins quantified, compared to BN-negative leaves and/or compared to the previous sampling performed in July (Figure 3).
Flavan-3-ols, flavonols and stilbenoid. Figure 4 reports the amounts of the other secondary metabolites quantified in leaves of grapevine cv. Sangiovese sampled in the two key-stages (July and September). Flavan-3-ols, flavonols and stilbenoid. Figure 4 reports the amounts of the other secondary metabolites quantified in leaves of grapevine cv. Sangiovese sampled in the two key-stages (July and September).
As shown in Figure 4, significant differences in the amounts of flavan-3-ols were observed between BN-positive and BN-negative leaves sampled in September. In fact, the amounts of catechin (n. 10) and epicatechin (n. 15) measured in July in BN-positive and BN-negative leaves were almost the same and no significant difference was observed. In September the amounts of catechin (n. 10) and epicatechin (n. 15) increased significantly only in BN-positive leaves, which were 1.5 and 4-fold higher than BN-negative leaves, respectively. Flavonols were the prevalent group detected in grapevine leaf extracts ( Table 2) and four different quercetin-glycosides were quantified (quercetin-glucoside (n. 26, 28), quercetin 3-glucuronide (n. 27), quercetin 3-O-rhamnoside (n. 33), and quercetin (n. 38)). Of all the flavonols quantified, only quercetin had a significantly higher concentration (about 2-fold higher) in BN-negative leaves sampled in July than BN-positive leaves which still showed no symptoms attributable to phytoplasma disease. In September, an alteration in the amount of flavonols in the BN-positive symptomatic leaves was not observed although the symptoms of disease on the leaves were clearly visible ( Figure 4). Furthermore, the content of resveratrol glucoside (n. 36) was quantified in the BN-positive and BN-negative samples in the two key-stages of July and September, and in the presence and absence of disease symptoms on the leaves, respectively ( Figure 1).
As shown in Figure 4, the amount of resveratrol glucoside (n. 36) in BN-positive leaves was noticeably higher in September and statistically greater than in BN-negative leaves. Specifically, in September the amount of resveratrol glucoside (n. 36) in BN-positive leaves was more than twofold higher than the BN-negative leaves (Figure 4).  As shown in Figure 4, significant differences in the amounts of flavan-3-ols were observed between BN-positive and BN-negative leaves sampled in September. In fact, the amounts of catechin (n. 10) and epicatechin (n. 15) measured in July in BN-positive and BN-negative leaves were almost the same and no significant difference was observed. In September the amounts of catechin (n. 10) and epicatechin (n. 15) increased significantly only in BN-positive leaves, which were 1.5 and 4-fold higher than BN-negative leaves, respectively. Flavonols were the prevalent group detected in grapevine leaf extracts ( Table 2) and four different quercetin-glycosides were quantified (quercetin-

Lignin Distribution in BN-Positive and BN-Negative Sangiovese Leaves
Histological observations of cv. Sangiovese leaves showed that the impact of the phytoplasma disease on lignin distribution differed between the July and September sampling periods ( Figure 5). A statistically significant decrease in lignin (−31%) was reported only in BN-positive leaves sampled in September, while no significant difference was found in lignin distribution between BN-positive and BN-negative leaves sampled in July ( Figure 5).

Discussion
Some pathogens, including phytoplasma of grapevine, can affect the carbohydrate metabolism of host plants and, consequently, induce other important metabolic alterations [12,38]. These include modifications in photosynthesis and secondary metabolism, especially a modification in the phenylpropanoid biosynthesis with a reduction and/or accumulation of some plant secondary metabolites, such as anthocyanin, phenolic compounds, and lignin [15,17,18]. The most common symptom of grapevine infected by phytoplasma is leaf yellowing (in white-berried grapes) or reddening (in red-berried grapes) caused by extensive modification in the synthesis and transport of soluble carbohydrates and starch. In fact, there is evidence of the anomalous accumulation of carbohydrates, the decrease in photosynthetic activity, the breakdown of chlorophyll, carotenoids and their biosynthesis inhibition in many phytoplasma-infected plants [8,[10][11][12][13].

Discussion
Some pathogens, including phytoplasma of grapevine, can affect the carbohydrate metabolism of host plants and, consequently, induce other important metabolic alterations [12,38]. These include modifications in photosynthesis and secondary metabolism, especially a modification in the phenylpropanoid biosynthesis with a reduction and/or accumulation of some plant secondary metabolites, such as anthocyanin, phenolic compounds, and lignin [15,17,18]. The most common symptom of grapevine infected by phytoplasma is leaf yellowing (in white-berried grapes) or reddening (in red-berried grapes) caused by extensive modification in the synthesis and transport of soluble carbohydrates and starch. In fact, there is evidence of the anomalous accumulation of carbohydrates, the decrease in photosynthetic activity, the breakdown of chlorophyll, carotenoids and their biosynthesis inhibition in many phytoplasma-infected plants [8,[10][11][12][13].
Our findings on the leaves of red-berried grape cv. Sangiovese confirmed an increase in soluble sugar content and a reduction in the chlorophyll content of the leaves positive to the pathogen, in accordance with data in the literature on white-berried grape cv. Chardonnay [11,39,40]. In particular, an increase in the soluble sugar content was observed in the BN-positive leaves sampled in two different key stages. On the other hand, a significant reduction in chlorophyll a content was observed even when the characteristic symptoms of phytoplasma disease were not yet visible on the leaves. However a drastic and significant reduction in the total content of chlorophylls and carotenoids was observed in September, when the plants infected by the pathogen show leaves with obvious symptoms (Table 1).
Our results would seem to indicate that the accumulation of soluble sugars and the reduction in chlorophyll content in the BN-positive leaves interfere and change the secondary metabolism. In fact, the source-sink transition in relation to pathogen infection is commonly linked to coordinated defense responses and changes in secondary metabolite production [11,[13][14][15]18,19,41,42].
In previously untested red-berried cv. Sangiovese, we also analyzed the qualitative profiles and respective amounts of phenolics, flavonoids, stilbenoids, and lignins produced by plant hosts in response to pathogen infection. The qualitative profiles of methanolic extracts of grapevine leaves showed no differences in either BN-positive or BN-negative samples, and the 36 compounds annotated and/or identified by HPLC ESI/MS-TOF have previously been found in grapevine leaf extracts, as reported in Table 2.
On the other hand, statistically significant differences were revealed in the amount of some chemical classes and their single compounds grouped as phenolics and flavonoids, according to the health status and/or period of sampling. In fact, our results show a markedly increased phenolic and flavonoid production in asymptomatic or symptomatic BN-positive leaves compared to BN-negative leaves collected in the same period. However, the amount of proanthocyanidins increased only in asymptomatic BN-positive leaves sampled in July, as reported in Figure 2.
Among the identified phenolics and flavonoids in the leaves of grapevine cv. Sangiovese (Table 2), the main secondary metabolites known as antioxidant compounds involved in defense reactions, have been quantified [13,18,40,43,44]. The most important alterations induced by BN disease in phenylpropanoid pathway products were observed in the amount of anthocyanin (cyanidin 3-glucoside (n. 41)), flavan-3-ols (catechin (n. 10) and epicatechin (n. 11)), stilbenoid (resveratrol glucoside (n. 36)), and lignin (Figures 3-5). The amount of cyanidin 3-glucoside (n. 41) quantified in the BN-positive leaves with clear symptoms of disease sampled in September was considerably higher (over 10-fold higher) compared to the BN-negative leaves sampled in the same phase. However, no statistical difference was observed in July between BN-positive compared to BN-negative leaves (Figure 3).
Although an increase in total anthocyanins in leaves of Barbera red-berried grapevine infected by Flavescence dorée phytoplasma has already been observed [13], our results show for the first time changes in the anthocyanin profile and in particular, that only cyanidin 3-glucoside (n. 41) accumulates in Sangiovese grapevine BN-positive leaves. Increases in the amounts of catechin (n. 10), epicatechin (n. 15) and resveratrol glucoside (n. 36) were also observed in BN-positive leaves with clear symptoms of disease (Figure 3), in accordance with data reported by [17,19] on Chardonnay leaves and 1-year-old canes, and by [13] on Barbera leaves. However, higher amounts of quercetin (n. 38) (Figure 4) were quantified in BN-negative leaves sampled in July.
It has been reported [45] that constitutive higher amounts of flavonols, and in particular quercetin (n. 38), could limit the diffusion of specific pathogens in grape leaves, and further investigations are necessary to understand whether the reduction in this compound could become an early indicator of the health status of grapevine, well before the appearance of symptoms. In fact, several global transcription profiles in Chardonnay and Manzoni Bianco grapevine infected with BN phytoplasma have revealed that the genes involved in the phenylpropanoid biosynthetic pathway are up-regulated in phytoplasma-infected leaves [11,16].
Further evidence of an alteration in the biosynthetic phenylpropanoid pathway products in grapevine cv. Sangiovese infected by BN, was a statistically significant difference in the content of lignin. In fact, as shown in Figure 5, in September the lignin content in BN-positive leaves was significantly lower than in BN-negative leaves. This suggests that in response to phytoplasma invasion, the synthesis of phenolic compounds, flavonols, and lignin uses the same precursor pool (hydroxycinnamic acid) and, thus, their excessive use can affect the response of other pathways, as observed in Vitis spp. and other plant species in relation to pathogen disease (see Figure 6) [19,46]. Pathogens 2020, 9, x FOR PEER REVIEW 13 of 19 lignin. In fact, as shown in Figure 5, in September the lignin content in BN-positive leaves was significantly lower than in BN-negative leaves. This suggests that in response to phytoplasma invasion, the synthesis of phenolic compounds, flavonols, and lignin uses the same precursor pool (hydroxycinnamic acid) and, thus, their excessive use can affect the response of other pathways, as observed in Vitis spp. and other plant species in relation to pathogen disease (see Figure 6) [19,46]. Although all these secondary metabolites have long been recognized as molecules implicated in the defense mechanisms activated by the host plant, they do not seem to be beneficial to the cv. Sangiovese infected by BN phytoplasma despite their biosynthesis being either activated or inhibited in BN-positive compared with BN-negative leaves. Further studies are therefore needed to understand whether or not these bio-compound accumulations have any direct protective role toward the establishment of phytoplasma infection.

Plant Samples and Phytoplasma Detection
The study was conducted during 2018 within an experimental area selected as representative of a vineyard located in Tuscany (central Italy), where BN-positive and BN-negative plants were detected through multi-year monitoring (see following Section 4.2 for details on tests). Sampling was carried out at two different key-stages according to symptom appearance on leaves (July and September, respectively with asymptomatic or symptomatic leaves). Sampling was performed on the same plants all the time, collecting 10-15 leaves from five BN-positive plants and five BN-negative plants. In both sampling periods, the severity of symptoms was classified according to a grapevine yellows symptomatic scale from 0 to 3, as reported in [47]: (i) symptom severity class 0 = plants with Although all these secondary metabolites have long been recognized as molecules implicated in the defense mechanisms activated by the host plant, they do not seem to be beneficial to the cv. Sangiovese infected by BN phytoplasma despite their biosynthesis being either activated or inhibited in BN-positive compared with BN-negative leaves. Further studies are therefore needed to understand whether or not these bio-compound accumulations have any direct protective role toward the establishment of phytoplasma infection.

Plant Samples and Phytoplasma Detection
The study was conducted during 2018 within an experimental area selected as representative of a vineyard located in Tuscany (central Italy), where BN-positive and BN-negative plants were detected through multi-year monitoring (see following Section 4.2 for details on tests). Sampling was carried out at two different key-stages according to symptom appearance on leaves (July and September, respectively with asymptomatic or symptomatic leaves). Sampling was performed on the same plants all the time, collecting 10-15 leaves from five BN-positive plants and five BN-negative plants. In both sampling periods, the severity of symptoms was classified according to a grapevine yellows symptomatic scale from 0 to 3, as reported in [47]: (i) symptom severity class 0 = plants with no symptoms, (ii) symptom severity class 1 = one shoot with mild leaf symptoms, (iii) symptom severity class 2 = two to three shoots with leaf symptoms, and (iv) symptom severity class 3 = more than three shoots with symptoms of reddening leaf and berry shrivel.
The leaves collected were stored at −20 • C until DNA extraction for phytoplasma detection or lyophilized (Christ alpha 2-4 LSC plus, Osterode am Harz, Germany) for biochemical analysis.
The DNA was extracted according to [48] with some modifications reported in [49]. Specific detection of BN phytoplasma was carried out by amplification of 16S ribosomal DNA through TaqMan assay following reaction conditions, as described in [50]. The grapevine chloroplast chaperonin 21 gene and DNA extracted from BN-negative and BN-positive plants were used as endogenous, negative and positive controls, respectively. A threshold cycle of <37 was associated with the presence of BN phytoplasmas [50]. Both

Analysis of Pigments and Soluble Sugars
Chlorophylls and carotenoids were extracted from lyophilized leaves and homogenized using liquid nitrogen with 80% acetone at a ratio of 1:100 (w/v) under stirring for 30 min. The amounts of chlorophylls and carotenoids were calculated by applying the formula in [54] and the absorbance was read with a JASCO V-550 UV/VIS spectrophotometer (JASCO Corporation 2967-5, Ishikawa-machi, Hachioji-shi Tokyo, Japan). The results were expressed in mg·g −1 dry weight (DW), and all measurements were performed in triplicate for each analyzed sample (n = 5, BN-positive and BN-negative plants, respectively).
The content of soluble sugars, expressed as mg·g −1 DW, was calculated using a commercial enzymatic kit from Megazyme (Megazyme International Ltd., Ireland, cat. no. K-SUFRG 06/14), according to the manufacturer's protocol.

Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and Proanthocyanidins (PAs)
The collected leaves were lyophilized and ground with a mortar and pestle in liquid nitrogen to which the extraction buffer (methanol:water:formic acid, 60:39.9:0.1) at a ratio of 1:10 was added, and left to stir in the dark for 2 h. After centrifugation at a maximum speed (5000× g), the resulting solutions were filtered into glass vials using a 0.2 µm polytetrafluoroethylene (PFTE) membrane and analyzed as described below. Three replicates for each harvested sample (n = 5, BN-positive, and BN-negative plants, respectively) were carried out.
The total phenolic content (TPC) was determined using the spectrophotometric Folin-Ciocalteau method [55] and the data were expressed as mg of caffeic acid equivalent (CAE)·g −1 DW.
The total flavonoid content (TFC) and amount of proanthocyanidins (PAs) were evaluated as reported by [56] and the absorbance was read with a JASCO V-550 UV/VIS spectrophotometer. The TFC amount was calculated by determining the absorbance at 280 and 540 nm and reported as mg of catechin equivalent (CE)·g −1 DW. The proanthocyanidin (PA) quantification was measured before and after hydrolysis into cyanidins (HCl 12 N with 300 mg·L −1 of FeSO 4 x7H 2 O for 50 min in a thermostatic bath at 100 • C at reflux). The results were expressed as mg·g −1 DW.

HPLC ESI/MS-TOF Analysis
The phenolic characterization and quantification on leaf extracts (see Section 2.3) were performed using an Agilent 1200 High Performance Liquid Chromatography (HPLC) System (Agilent Technologies, Palo Alto, CA, USA) equipped with a standard autosampler and an Agilent Zorbax Extend-C18 analytical column (5 × 2.1 cm, 1.8 µm), as reported by [28,36]. The HPLC system was coupled to an Agilent diode-array detector (wavelength 280 nm) and an Agilent 6320 TOF mass spectrometer equipped with a dual ESI interface (Agilent Technologies) operating in negative ion mode. Detection was carried out within a mass range of 50-1700 m/z. Accurate mass measurements of each peak from the total ion chromatograms (TICs) were obtained by means of an ISO Pump (Agilent G1310B) using a dual nebulizer ESI source that introduces a low flow (20 µL min −1 ) of a calibration solution containing the internal reference masses at m/z 112.9856, 301.9981, 601.9790, and 1033.9881, in negative ion mode. The anthocyanins were identified with the same method, but with positive ionization (detection wavelength 280 and 520 nm), using the internal reference masses at m/z 121.050873, 149.02332, 322.048121, and 922.009798, as reported by [57].

Histology: Lignin Distribution in Leaves
Free-hand cross sections of BN-positive and BN-negative fresh leaves were mounted onto microscope slides for observation under a microscope. Lignin auto-florescence was detected using UV-excitation under DAPI-filter. Images were taken on a confocal laser-scanning microscope (Carl Zeiss LSM 700 laser scanning microscope, Jena, Germany). For each section, several images were captured in order to obtain a representative distribution of lignin throughout the leaf layer (including signals captured from leaf veins); all images were acquired with the same microscope settings and analyzed with the Zeiss LSM image examiner software v. 4.2.0.121. The intensity of autofluorescence signal was quantified using Image J software.

Statistical Analysis
All data were reported as the mean ± SD in triplicate for each analyzed sample (n = 5, BN-positive and BN-negative plants, respectively). The statistical analysis was performed using multiple t-tests (FDR = 5%) to highlight the differences between BN-positive and BN-negative leaves for each physiological parameter analyzed. Statistical analyses were performed using GraphPad v. 6.01.

Conclusions
We believe that this is the first biochemical description of leaves of grapevine cv. Sangiovese infected by BN phytoplasma. Our study highlighted the modulation of the phenylpropanoid biosynthetic pathway through the accumulation and/or reduction of some end-products as a defense response status. Our findings represent a starting point for future insights regarding the role of these metabolites in phytoplasma pathogenesis and the response of grapevine cv. Sangiovese. Further studies are needed to clarify the possible benefits of the accumulation or reduction of the main metabolites in relation to phytoplasma survival and the same host plant.