Grapevine Plants Management Using Natural Extracts and Phytosynthesized Silver Nanoparticles

Starting from the well-known antimicrobial properties of silver nanoparticles, the goal of this study is to evaluate the influence of two “green” recipes, namely an alcoholic extract of Dryopteris filix-mas (L.) Schott and a dispersion of silver nanoparticles phytosynthesized using the extract on grapevine pathogens. The influence of some grapevine parameters (pith/wood rapport, soluble sugars, starch, total sugars, total water content, length of young shoots, number of grapes) in field experiments was also studied. The study was conducted on four clones (Feteasca alba 97 St., Feteasca neagra 6 St., Feteasca regala 72 St., and Cabernet Sauvignon 131 St.) located in vegetation pots inside a greenhouse. For the phytosynthesis of the silver nanoparticles (AgNPs) we used a scaled-up technology, allowing us to obtain large quantities of nanoparticles-containing solution. The AgNPs analysis by X-ray diffraction and transmission electron microscopy confirmed the synthesis of spherical and quasi-spherical nanoparticles of 17 nm average diameter and 6.72 nm crystallite size. The field experiments registered different responses of the four clones to the treatment, using both the natural extracts and phytosynthesized nanoparticles solution. Both recipes exhibited a protective effect against the Uncinula necator pathogen. For the treatment using phytosynthesized nanoparticles, significant increases in the pith/wood ratio for white wine clones (Feteasca alba 97 St. and Feteasca regala 72 St.) were observed. The biochemical analyses revealed other significant increases of soluble sugars (red wine clones—Feteasca neagra and Cabernet Sauvignon/second year), starch (Feteasca alba and Cabernet Sauvignon in 2021 for both clones), total sugars (Feteasca alba and Feteasca neagra in 2021 for both clones), and of total water content (Feteasca alba and Feteasca neagra in 2021 for both clones), respectively. The applied treatments also led to an increase of young shoots length and grape numbers for all clones as compared to the control (chemical pesticide), which would suggest a potential biostimulant effect of the recipes.


Introduction
Viticulture plays an important global role, both from an economic and social point of view. Unfortunately, the vineyards are frequently attacked by numerous pathogens (fungus, viruses, viroids, phytoplasmas, bacteria) that, in a worst-case scenario, can cause serious

Collection and Treatment of Vegetal Material
The vegetal material was collected in 2018 from Valea Vâlsanului, Romania (45 • 20 41.1 N; 24 • 44 06.0 E, altitude of 735 m), being identified by Liliana Cristina Soare. A voucher specimen of the plant was preserved at the Argeş County Museum (voucher specimen no. 11330). The harvested plants were cleaned of impurities, washed with tap water, and dried at room temperature on paper sheets until a constant mass was achieved. The dried vegetal material was ground to a powder and preserved for further use [48].

Preparation and Characterization of Vegetal Extract (V1) and Silver Nanoparticles Solution (V2) 2.2.1. Preparation of Sample V1 (Vegetal Extract) and V2 (Photosynthesized Nanoparticles)
The vegetal extracts (further encoded as V1) were obtained using temperature extraction by scaling up a recipe, which has been proved effective at the laboratory scale, for the case of 50 L reactors (FSupplementary Material, Figure S1). The studied experimental conditions are as follows: 50% ethanol (Chimreactiv, Bucharest, Romania) as extraction solvent, ratio of vegetal material to solvent of 1:10 (w/v), extraction temperature of 50 • C, extraction time of 3 h, and continuous stirring. The water used for all experiments was distilled water, obtained in our laboratory.
After the extraction procedure, the extracts were allowed to cool down and filtered. The phytosynthesis procedure was performed by direct mixing of a part of the extract with an equal quantity of 1 mM AgNO 3 solution (Chimreactiv, Bucharest, Romania), thus obtaining the solution containing phytosynthesized silver nanoparticles, further encoded as V2. The procedure was performed under ambient conditions, in the presence of natural light, without stirring, the reaction being considered completed after 24 h.

Characterization of Samples V1 and V2A
A small quantity of the two types of materials was prelevated for controlling the extract composition and the success of the phytosynthesis procedure, respectively. Both types of materials (the vegetal extract and the phytosynthesized nanoparticles solution) were stored in clean recipients at 4 • C for further use (Supplementary Material, Figure S2).
The control of the vegetal material was performed by determining the total phenolics content of the vegetal extract (the method being previously presented [45], with a brief description in the Supplementary Material) for the natural extract (further encoded as V1). The phytosynthesized nanoparticles (NPs) solution (further encoded as V2) was characterized using X-ray diffraction and transmission electron microscopy (TEM) (to define the obtained nanoparticles). XRD analyses were performed using a 9 kW Rigaku SmartLab diffractometer (Rigaku Corp., Tokyo, Japan, operated at 45 kV and 200 mA, Cu Kα radiation-1.54059 Å), in scanning mode 2θ/θ, between 20 and 80 • (2θ). The components were identified by comparison with ICDD data. The crystallite size was determined using the Scherrer equation: where Dp = the average size of the crystallites, K = the Scherrer constant (K = 0.94), β = width at half-height of the diffraction maximum, θ = Bragg angle, λ = the wavelength (1.54059Å). Transmission electron microscopy analyses were performed using a Tecnai G2 F20 TWIN Cryo-TEM electron microscope (FEI Company, Hillsboro, OR, USA) at 300 kV acceleration voltage and 1Å resolution. For the analysis, a drop of nanoparticles-containing solution was directly placed on the copper grids, evaporated, and analyzed. The nanoparticle size distribution was examined via direct measurements of nanoparticles from TEM images (over 100 measurements) using ImageJ image analysis software (v. 1.53s, National Institutes of Health, Bethesda, MD, USA).
Other specific characteristics of the recipes constitute the subject of a pending patent application [49].

In Vitro Antimicrobial Studies
As a preliminary step, the two recipes were evaluated as antimicrobial agents using the agar well diffusion method [50,51] against Plasmopara viticola.
Briefly, diseased vine leaves were collected and washed with sterile water. The petiole was wrapped with absorbent cotton, and the small pieces of the abaxial surface of the leaves were placed in a Petri dish. Furtherly, they were cultured in a Laboshake shaker (Gerhardt GmbH & Co. KG, Königswinter, Germany) at 22 • C for 24-48 h, as previously described [7], and in concordance with literature data [52][53][54]. For the detached leaf assay, the sporangia were collected with a sterile brush. The suspension obtained was diluted into 10 5 sporangia/mL. Potato Dextrose Agar plates (abbreviated PDA) from Sigma-Aldrich (Sigma Aldrich, St. Louis, MO, USA; composition: agar, 15 g/L, dextrose, 20 g/L and potato extract, 4 g/L supplemented with 0.1 g/L chloramphenicol, used to avoid the bacterial contamination) were inoculated with 1 mL inoculum of the P. viticola.
Wells in the PDA plates were made using a sterile borer at the size of 6 mm diameter, and then 50 µL of tested sample were placed in them. The Petri plates were incubated at 22 • C for 48 h in a shaker. As a negative control, ethanol: water = 1:1 (v/v) was used, and Pergado F 45 WG (Syngenta AG, Basel, Switzerland) was selected as a positive control, at 0.25% concentration (as recommended by the producer). Each experiment was carried out in triplicate.  Figure S3). The plants were own-rooted and intended for obtaining the viticultural propagating material from higher biological categories (the initial propagating material). For this experiment 120 plants were used, divided into 10 plants/experimental group for each clone. They were separated from each other by a paravane in order to apply differentiated treatments.

Evaluation of Pathogen Symptoms
During the study, the appearance of the symptoms produced by the pathogens P. viticola and U. necator was monitored. During both years, only powdery mildew was formed, with the attack monitoring frequency (F), the intensity (I), and the attack degree (AD) being determined. These determinations were performed both for the control and for the experimental groups before and after the treatments' application. The frequency value was obtained by direct observations on the number of plants and leaves, and the collected data were computed using the following formula: where n is the number of plants or organs of the plant attacked by pathogens and N is the number of plants or organs observed. The intensity of the attack was determined by reporting the leaf attacked area to the total area observed, thus being calculated by the degree and extend of the attack. To reproduce the intensity of the powdery mildew and downy mildew attack, the following classification scale was used, which included the attacked surface (%) and the severity (note) of the attack intensity: for 1-3% = 1; 4-10% = 2; 11-25% = 3; 25-50% = 4; 26-50% = 4; 51-75% = 5; 76-100% = 6.
To determine the coverage attack degree, the following formula was used: where: I = the attacked note or surface (%); f = the number of cases with attack on each note; n = total number of cases with attack. The determination of the F, I, and AD has a special importance in assessing the damage caused, in establishing the rhythm of treatments application, and in establishing the effectiveness of various methods and means of crop protection against pathogens.
The relation linking the intensity of powdery mildew to the total number of plants at which the observations were made was used in order to determine the attack degree. Its value expression is given by the relation:

Biochemical Analyses and Grapevine Characteristics
At the same time, the evaluation of woody biological material of all considered clones was studied by determining the pith/wood (P/W) ratio, the total sugars (soluble sugars and starch), and the total water content. For the canes harvested from each studied grapevine plant, the dimensions of the pith and wood were measured at the base, middle, and top of the cane.
The assessment of the ratio between the pith and the wood was made considering the relationship presented in literature data [55]: P/W > 1− incompletely matured wood; P/W ≈ 1 -partially matured wood; P/W < 1 -wood well matured.
The canes' water content (%) was obtained by drying the samples at 60-62 • C in the oven until constant mass [56].
The extraction of soluble sugars was carried out with warm 80% ethylic alcohol. The starch was extracted with 52% perchloric acid from the sediment resulting from the extraction of soluble sugars. A dosing of sugar was done with 0.2% anthrone prepared in 95% sulfuric acid. The absorbance reading of the samples was performed using a spectrophotometer at a wavelength of 620 nm [57]. The soluble sugars and starch contents were calculated in a percentage related to the dry material.
In order to provide a full image of the treatment influence on the grapevine productivity, the length of the young shoots and the number of grapes per plant were determined over the year 2020. The measurement of the young shoots was performed using a roulette, with the results being expressed in cm. The number of grapes was determined by direct counting.
The statistical significance was analyzed with IBM SPSS Statistics version 25 for Windows, one-way ANOVA. Different superscript letters mean statistically differences between the results obtained for different experimental groups at p < 0.05. The results are presented as means ± SD of three experiments. For graphical representations, the OriginPro 2018 Data Analysis and Graphing Software (OriginLab Corporation, Northampton, MA, USA) was used.

Characterization of Extract and Extract Containing AgNPs
The control of the natural recipes proposed for treatment was performed, as presented in the previous section, using a phytochemical assay (for the natural extract). The evaluation of the nanoparticles crystalline structure was performed using X-ray diffraction. Their morphological properties were evaluated using transmission electron microscopy. The total phenolics content determined for sample V1 was 36 ± 1.25 mg/L GAE (gallic acid equivalents).
The silver in V2 was evaluated by XRD analysis. Approximately 10 mL of V2 were centrifuged and the obtained precipitate was deposited on specific glass support for analysis. Figure 1 presents the diffractogram of the analyzed sample.
productivity, the length of the young shoots and the number of grapes per plant were determined over the year 2020. The measurement of the young shoots was performed using a roulette, with the results being expressed in cm. The number of grapes was determined by direct counting.
The statistical significance was analyzed with IBM SPSS Statistics version 25 for Windows, one-way ANOVA. Different superscript letters mean statistically differences between the results obtained for different experimental groups at p < 0.05. The results are presented as means ± SD of three experiments. For graphical representations, the OriginPro 2018 Data Analysis and Graphing Software (OriginLab Corporation, Northampton, MA, USA) was used.

Characterization of Extract and Extract Containing AgNPs
The control of the natural recipes proposed for treatment was performed, as presented in the previous section, using a phytochemical assay (for the natural extract). The evaluation of the nanoparticles crystalline structure was performed using X-ray diffraction. Their morphological properties were evaluated using transmission electron microscopy. The total phenolics content determined for sample V1 was 36 ± 1.25 mg/L GAE (gallic acid equivalents).
The silver in V2 was evaluated by XRD analysis. Approximately 10 mL of V2 were centrifuged and the obtained precipitate was deposited on specific glass support for analysis. Figure 1 presents the diffractogram of the analyzed sample. The present phases identification was based on the comparison with ICDD entries. This resulted in confirmation of the silver nanoparticles synthesis (PDF card no. 01-087-0720) by the apparition of diffraction peaks corresponding to the (111), (200), (220), and (311) diffraction planes. Additionally, the presence of a secondary silver oxide phase was confirmed by the apparition of silver oxide diffraction peaks, corresponding to the diffraction planes (110), (111), (220), and (221) (PDF card no. 01-078-5867), with peaks marked by an asterisk in Figure 1. This phase is due to the silver oxidation occurring during the pre-treatment stage of the analysis. The crystallite size determined using Equation (1) applied to the parameters of the (111) diffraction plane was 6.72 nm.
The electron microscopy evaluation ( Figure 2) revealed the formation of spherical and quasi-spherical nanoparticles with an average diameter of 17 nm, although larger particles and clusters of nanoparticles can also be observed. diffraction planes (110), (111), (220), and (221) (PDF card no. 01-078-5867), with peaks marked by an asterisk in Figure 1. This phase is due to the silver oxidation occurring during the pre-treatment stage of the analysis. The crystallite size determined using Equation (1) applied to the parameters of the (111) diffraction plane was 6.72 nm.
The electron microscopy evaluation ( Figure 2) revealed the formation of spherical and quasi-spherical nanoparticles with an average diameter of 17 nm, although larger particles and clusters of nanoparticles can also be observed. The obtained results support the formation of silver nanoparticles using fern extract. When comparing the results obtained with previous works, it can be noticed that, using the presented proposed method, larger NPs are obtained as compared to the ones phytosynthesized using classical temperature or microwave extracts of Asplenium scolopendrium L. leaves (under 14 nm) [46]. However, when comparing the results with the ones obtained using other types of vegetal materials, smaller dimensions were obtained in the present study when compared with Aconitum toxicum Reichenb rhizomes (over 50 nm) [58] and in the same range with those obtained using Raphanus sativus L. leaves [45].

In Vitro Antimicrobial Evaluation
Our previous works in the area of phytosynthesized nanoparticles [45,46,58] suggested that the silver nanoparticles obtained using ferns or other green leaves exhibit a potent antimicrobial activity, even against crops pathogens (such as Venturia inaequalis and Podosphaera leucotricha) [45]. In order to test this hypothesis, an in vitro evaluation of the antimicrobial potential of the two solutions (V1 and V2) was performed before field experiments on downy mildew (Plasmopara viticola). The obtained results are presented in Figure 3. The obtained results support the formation of silver nanoparticles using fern extract. When comparing the results obtained with previous works, it can be noticed that, using the presented proposed method, larger NPs are obtained as compared to the ones phytosynthesized using classical temperature or microwave extracts of Asplenium scolopendrium L. leaves (under 14 nm) [46]. However, when comparing the results with the ones obtained using other types of vegetal materials, smaller dimensions were obtained in the present study when compared with Aconitum toxicum Reichenb rhizomes (over 50 nm) [58] and in the same range with those obtained using Raphanus sativus L. leaves [45].

In Vitro Antimicrobial Evaluation
Our previous works in the area of phytosynthesized nanoparticles [45,46,58] suggested that the silver nanoparticles obtained using ferns or other green leaves exhibit a potent antimicrobial activity, even against crops pathogens (such as Venturia inaequalis and Podosphaera leucotricha) [45]. In order to test this hypothesis, an in vitro evaluation of the antimicrobial potential of the two solutions (V1 and V2) was performed before field experiments on downy mildew (Plasmopara viticola). The obtained results are presented in Figure 3.
The laboratory experiments suggest that the fern extract is not active against downy mildew (statistically similar results being obtained for V1 and negative control). A significant antimicrobial effect was observed for the V2 sample, for which an inhibition zone of approximately half of the positive control was observed.
Similar observations were made by Zheng et al. [59] (when applying Ag-SiO 2 coreshell nanoparticles against several phytopathogenic fungi) and by Wang et al. [60] (using a graphene oxide-Fe 3 O 4 nanocomposites). In the latter case, the effect was assigned by the authors to the blocking of sporangia's water channels through the surface adsorption of the nanocomposite [60,61]. Rashad et al. [62] used silica nanoparticles to control downy mildew and assigning the protective effect to the influence of the NPs on stomatal closure, stomatal area, and stomatal pore area.

Results of Field Experiments
The experimental observations on the occurrence of powdery mildew and downy mildew in the studied grapevine plants, together with the time and space disease evolution, were monitored for highlighting different levels of attack for each clone. On the studied grapevines, only powdery mildew appeared in the studies from 2019 and 2020. Thus, on 9 August, 19 August, and 26 August 2019, the F, I, and AD evaluations were performed for estimating powdery mildew on the grapevine plants studied (Figure 4). The symptoms were different from one genotype to another. For Feteasca alba Feteasca alba 97 St., a powdery mildew attack in all three groups (control and experimental) was registered (on 9 August 2019). Furthermore, after the first treatment application, considering the symptoms' appearance, the attack was stopped only in the case of V1 (when the plants were sprayed with alcoholic extract of D. filix-mas). After the third treatment, the attack was stopped in all three groups (control and experimental). The control plants of Feteasca neagra Feteasca neagra 6 St. did not register a powdery mildew attack. On the other hand, in the case of plants from V1 and V2 experimental groups, the attack of powdery mildew stopped after the last treatment application. . The values represent means ± SD. Means of the same parameter without a common lowercase letter (a to c) differ (p < 0.05) as analyzed by one-way ANOVA and the TUKEY test.
The laboratory experiments suggest that the fern extract is not active against downy mildew (statistically similar results being obtained for V1 and negative control). A significant antimicrobial effect was observed for the V2 sample, for which an inhibition zone of approximately half of the positive control was observed.
Similar observations were made by Zheng et al. [59] (when applying Ag-SiO2 coreshell nanoparticles against several phytopathogenic fungi) and by Wang et al. [60] (using a graphene oxide-Fe3O4 nanocomposites). In the latter case, the effect was assigned by the authors to the blocking of sporangia's water channels through the surface adsorption of the nanocomposite [60,61]. Rashad et al. [62] used silica nanoparticles to control downy mildew and assigning the protective effect to the influence of the NPs on stomatal closure, stomatal area, and stomatal pore area.

Results of Field Experiments
The experimental observations on the occurrence of powdery mildew and downy mildew in the studied grapevine plants, together with the time and space disease evolution, were monitored for highlighting different levels of attack for each clone. On the studied grapevines, only powdery mildew appeared in the studies from 2019 and 2020. Thus, on 9 August, 19 August, and 26 August 2019, the F, I, and AD evaluations were performed for estimating powdery mildew on the grapevine plants studied (Figure 4). The symptoms were different from one genotype to another. For Feteasca alba Feteasca alba 97 St., a powdery mildew attack in all three groups (control and experimental) was . The values represent means ± SD. Means of the same parameter without a common lowercase letter (a to c) differ (p < 0.05) as analyzed by one-way ANOVA and the TUKEY test.
Regarding the Feteasca regala Feteasca regala 72 St. clone, the plants of all three groups were affected by powdery mildew. After the second treatment, only those from the V1 experimental group were still affected. However, in their case, the attack of powdery mildew was stopped after the third treatment application.
In the case of the Cabernet Sauvignon 131 St., only the plants from control and V2 experimental groups were affected by powdery mildew and the attack was stopped after the second treatment. The attack of powdery mildew appeared after the second treatment only on new vegetative growths (tops and lateral shoots). Therefore, 5 days after the application of the last treatment (26 August 2019), the attack of powdery mildew was stopped on all grapevine plants (control, V1, V2).
The same observations regarding the appearance of the main pathogens were made in 2020. The symptoms were visible for the first time at the beginning of July on Feteasca neagra Feteasca neagra 6 St. for the experimental groups V1 and V2 due to higher plant growth (thus creating a favorable climate for the development of powdery mildew) as compared to the control (where the vegetative growths were reduced). After the application of 5 treatments, the attack was stopped for the plants in experimental groups V1 and V2, while for the control group the attack was stopped after the third treatment. The five treatments application led to the protection of Feteasca regala Feteasca regala 72 St. plants from V1 and V2 groups against the attack of powdery mildew, compared to the control where the attack was stopped after the application of the last chemical treatment. Following the application of treatments on Feteasca alba Feteasca alba 97 St., it was found that throughout the vegetation period of the plants belonging to the V2 experimental group, no disease-specific symptoms were formed. Furthermore, in the other two groups the degree of attack was similar, with 0.23% for control and 0.26% for V1. After the third treatment, the attack of powdery mildew stopped in both groups. At Cabernet Sauvignon 131 St., no downy mildew and powdery mildew attack was recorded during the entire vegetation period, regardless of the experimental group. Therefore, the two experimental groups (V1 and V2) ensured a very good phytosanitary condition, identical to the chemical treatments (control) ( Figure 5). Regarding the Feteasca regala Feteasca regala 72 St. clone, the plants of all three groups were affected by powdery mildew. After the second treatment, only those from the V1 experimental group were still affected. However, in their case, the attack of powdery mildew was stopped after the third treatment application.
In the case of the Cabernet Sauvignon 131 St., only the plants from control and V2 experimental groups were affected by powdery mildew and the attack was stopped after the second treatment. The attack of powdery mildew appeared after the second treatment only on new vegetative growths (tops and lateral shoots). Therefore, 5 days after the application of the last treatment (26 August 2019), the attack of powdery mildew was stopped on all grapevine plants (control, V1, V2).  The same observations regarding the appearance of the main pathogens were made in 2020. The symptoms were visible for the first time at the beginning of July on Feteasca neagra Feteasca neagra 6 St. for the experimental groups V1 and V2 due to higher plant growth (thus creating a favorable climate for the development of powdery mildew) as compared to the control (where the vegetative growths were reduced). After the application of 5 treatments, the attack was stopped for the plants in experimental groups V1 and V2, while for the control group the attack was stopped after the third treatment.  Similar results were obtained by other authors on Thompson Seedless grapevine, a significant reduction in the abnormal alterations induced by the downy mildew on grapevine leaves upon SiNPs spraying being recorded [62]. Additionally, nanoparticles of silicon and titanium were used to reduce powdery mildew infection and development at wheat plants [63], while silver nanoparticles were used for Phytophthora parasitica control [64]. For decades, pesticides have been an essential part of agriculture [65]. However, when applying a phytosanitary treatment, 30% of the applied substance is scattered in the atmo-sphere and about 30% falls on the ground, with these fractions contributing to air and soil pollution [66].
The attack degree of the U. necator pathogen was different from one genotype to another. Furthermore, during the studied years 2019 and 2020, it has been observed after the last treatment that the powdery mildew attack stopped on all grapevine plants, both in the control and in the case of the experimental group treated with V1 and V2 solutions. No downy mildew attack was recorded during the entire vegetation period, regardless of the experimental group.
Analyzing the wood from a morphological point of view, it was found that in 2019 and 2020 all the canes were well matured at all experimental groups. However, determinations at the base, middle, and top level of the cane for all the studied grapevine clones were performed. The purpose was to find out if the analyzed canes recorded differences in the P/W rapport in the same experimental groups along their length. control where the attack was stopped after the application of the last chemical treatment.
Following the application of treatments on Feteasca alba Feteasca alba 97 St., it was found that throughout the vegetation period of the plants belonging to the V2 experimental group, no disease-specific symptoms were formed. Furthermore, in the other two groups the degree of attack was similar, with 0.23% for control and 0.26% for V1. After the third treatment, the attack of powdery mildew stopped in both groups. At Cabernet Sauvignon 131 St., no downy mildew and powdery mildew attack was recorded during the entire vegetation period, regardless of the experimental group. Therefore, the two experimental groups (V1 and V2) ensured a very good phytosanitary condition, identical to the chemical treatments (control) ( Figure 5).  Similar results were obtained by other authors on Thompson Seedless grapevine, a significant reduction in the abnormal alterations induced by the downy mildew on grapevine leaves upon SiNPs spraying being recorded [62]. Additionally, nanoparticles of silicon and titanium were used to reduce powdery mildew infection and development at wheat plants [63], while silver nanoparticles were used for Phytophthora parasitica control [64]. For decades, pesticides have been an essential part of agriculture [65]. However, when applying a phytosanitary treatment, 30% of the applied substance is scattered in the atmosphere and about 30% falls on the ground, with these fractions contributing to air and soil pollution [66].
The attack degree of the U. necator pathogen was different from one genotype to another. Furthermore, during the studied years 2019 and 2020, it has been observed after the last treatment that the powdery mildew attack stopped on all grapevine plants, both in the control and in the case of the experimental group treated with V1 and V2 solutions. No downy mildew attack was recorded during the entire vegetation period, regardless of the experimental group.
Analyzing the wood from a morphological point of view, it was found that in 2019 and 2020 all the canes were well matured at all experimental groups. However, determinations at the base, middle, and top level of the cane for all the studied grapevine clones were performed. The purpose was to find out if the analyzed canes recorded differences in the P/W rapport in the same experimental groups along their length.
The study of the influence of biological products' application against downy and powdery mildew on the processes of cane's maturation is of high practical importance. This is due to the fact that the grapevine plants studied in this paper are used as initial propagating material. In the Feteasca regala Feteasca regala 72 St. and Cabernet Sauvignon 131 St. genotypes, the measurements on the pith/wood ratio did not reveal significant variations between the treatments with V1 and V2 solutions, as compared to the chemical treatment (control), during the two years of study on the entire length of the canes. In Feteasca alba Feteasca alba 97 St., for the V1 experimental group, the ratio  The study of the influence of biological products' application against downy and powdery mildew on the processes of cane's maturation is of high practical importance. This is due to the fact that the grapevine plants studied in this paper are used as initial propagating material. In the Feteasca regala Feteasca regala 72 St. and Cabernet Sauvignon 131 St. genotypes, the measurements on the pith/wood ratio did not reveal significant variations between the treatments with V1 and V2 solutions, as compared to the chemical treatment (control), during the two years of study on the entire length of the canes. In Feteasca alba Feteasca alba 97 St., for the V1 experimental group, the ratio increased significantly in the upper part of the canes after the first year of treatment only. However, the treatment with the V2 solution led to significant increasing or decreasing variations along the entire length of the cane in 2020 and only at the upper part in 2021. The Feteasca neagra 6 St. genotype behaved almost similarly (Table 2).
In the case of the Feteasca alba 97 St. clone (in 2020), the control recorded a decrease in the diameter of the top of the cane by 28.71% as compared to its base. However, for the plants treated with the V1 solution, the decrease was only 0.79%, and for the plants treated with the V2 solution there was an increase in its diameter by 41%. In the same year, Feteasca neagra 6 St. registered an increase of 8.65% in the case of the control, 20.67% for the plants treated with V1, and a slight decrease of 0.78% for plants treated with V2. The control of the Feteasca regala 72 St. registered an increase of 24.03%, while for the plants treated with V1 and V2, the increases were 10.26% and 19.74%. The fourth clone, Cabernet Sauvignon 131 St., registered an increase of 34% in the control, 28.01% for the treatment with V1, and a decrease of 7.35% for the treatment with V2. The pith/wood ratio had subunit values both in the case of chemical fungicide treatments (control) and in the experimental groups V1 and V2. Thus, by maintaining the health of plants, the normal physiological course of vegetative phenophases was favored. Similar results were obtained in other studies regarding the use of biostimulants for an increased tolerance to the virus diseases in grapevine [67].
Accumulation of the reserve substances in the canes wooden tissue is very important. Its maturation corresponds to the period when the total carbohydrates (soluble sugars and starch) reach the maximum. This period corresponds to the grapevine dormancy [56]. It varies with the variety vigor, the load of the shoots, and the climatic conditions. The maturation of the canes is a physiological process of reserve substances accumulation in the shoots (the maximum starch content when the leaves fall is about 11%). Meanwhile, the water content decreases from 80% to 45-50%, a level that ensures the viability of tissues until the spring of the next year [68].
In order to find out whether the treatments applied in 2019 and 2020 influenced the degree of wood maturation, biochemical determinations (total sugars, water and dry matter) were performed in February 2020 and February 2021. In the second year of the study, the biochemical analyses of the carbohydrate content (soluble sugars and starch), as well as the total water content of the canes collected from the studied grapevine genotypes, showed only significant sporadic differences in the experimental groups as compared to the control group. The treatment with nanoparticles solution (V2) led to a significant increase of the starch content in Feteasca alba 97 St., and an increase of soluble and total carbohydrates in Cabernet Sauvignon 131 St. An increase of the total water content in Feteasca neagra 6 St. was recorded for plants treated with both V1 and V2 solutions (Table 3). The experimental group V2 increased the frost resistance of the Cabernet Sauvignon 131 St. clone by the starch content increasement. Additionally, an increase in the soluble sugars was observed for the same clone (only after two years of treatment-2021). This variation could be explained by the NPs accumulation over the two treatment years and their involvement in the photosynthesis and sugar transformation processes. This has also been observed by other authors for the case of nanoparticles treatments [69][70][71][72]. In the canes, the conversion of starch into soluble carbohydrates is faster at resistant varieties. However, in the sensible ones, the starch content is kept at a higher level. Feteasca neagra, Feteasca regala, Cabernet Sauvignon, and Feteasca alba genotypes are considered resistant to frost [73]. Ruano-Rosa et al. [43] also used silver nanoparticles for pathogen control (against powdery mildew on the grapevine). As compared to the results obtained by them, the results presented in this manuscript are more promising. The starch content fluctuates in the roots and trunks during the vegetation period, with the lowest value being recorded between budbreak and flowering and the highest values between harvest and leaf fall [74].
The maturation of the grapevine canes is of major importance, being related to the frost resistance and to the quality of the scion and rootstock for the planting material production. The first studies on the cane maturation process are attributed to Gouin and Andouard. They studied the dry matter accumulation dynamics (in the year 1899) and nitrogen and starch content in canes (in the year 1901) [75].
Regarding the carbohydrates' dynamics during the dormancy, starting from December, the starch stored as a reserve substance was gradually hydrolyzed, reaching minimum values in February, while the reducing carbohydrates increase. According to several authors, when considering the starch cumulated with soluble sugars, it is estimated as a minimum level of 12% [76]. This provides nutritional support for starting a new vegetative cycle. In our study, the level of carbohydrates and total water includes the canes collected from plants subjected to treatments against downy and powdery mildew at an optimal level of maturation. This level was suitable for use in the propagation process, with total carbohydrates registering values between 16% and 20%. The same genotypes represented by grapevines stored under the same conditions (greenhouse) as those used in this study behaved similarly, with total carbohydrate values between 14 and 16%, depending on the climatic and cultivation conditions of that year [77].
In conclusion, the subunit ratio, the water content of over 46%, and the soluble carbohydrates of over 16% put the canes collected from the studied plants at an optimal level of maturation that makes them suitable in the multiplication process.
In order to establish the effect of the performed treatment on the productivity of the grapevine, the determination of young shoots length and grape number were performed over the year 2020 ( Figure 6).
During 2020, the growth of young shoots and the number of grapes were different from one genotype to another. Among the studied genotypes, the most significant increase in the shoot growth was recorded for the Feteasca neagra 6 St. treated with V2 (for which the lowest number of grapes among the treated genotypes was also recorded, although higher than the control). The experimental groups V1 and V2 registered a higher number of grapes compared to the control in all studied genotypes; for the genotypes F. alba 97 St and F. neagra 6 St., a higher quantity of grapes upon treatment with V2 was recorded as compared to V1, while for F. regala 72 St. and C. Sauvignon 131 St., treatment with V1 led to higher amounts of grapes compared to V2 ( Figure 6). As such, the performed treatments could have a biostimulant effect on some of the studied genotypes, an aspect which needs to be further studied. According to the available literature data, the nanoparticles have different pathogen inhibition pathways depending on the nature of the nanomaterial. For example, silica NPs were found to enhance the resistance of the plants to the action of the pathogens. More precisely, their application led to an increase in photosynthetic pigments and overexpression of plant hormones regulating the accumulation of relevant secondary metabolites. Similar observations were made by Farhat et al. [63] for SiNPs, while the action of the TiO 2 NPs was assigned by the authors to their photocatalytic properties. For AgNPs, literature data [64] suggest a complex mechanism, in which the inhibition of respiratory chain enzymes, the disruption of ribosome biogenesis, and the impairment of genes involved in oxidative stress all coexist. During 2020, the growth of young shoots and the number of grapes were different from one genotype to another. Among the studied genotypes, the most significant increase in the shoot growth was recorded for the Feteasca neagra 6 St. treated with V2 (for which the lowest number of grapes among the treated genotypes was also recorded, although higher than the control). The experimental groups V1 and V2 registered a higher number of grapes compared to the control in all studied genotypes; for the genotypes F. alba 97 St and F. neagra 6 St., a higher quantity of grapes upon treatment with V2 was recorded as compared to V1, while for F. regala 72 St. and C. Sauvignon 131 St., treatment with V1 led to higher amounts of grapes compared to V2 ( Figure 6). As such, the performed treatments could have a biostimulant effect on some of the studied genotypes, an aspect which needs to be further studied. According to the available literature data, the nanoparticles have different pathogen inhibition pathways depending on the nature of the nanomaterial. For example, silica NPs were found to enhance the resistance of the plants to the action of the pathogens. More precisely, their application led to an increase in photosynthetic pigments and overexpression of plant hormones regulating the In the case of the present study, the most probable explanation is a mixture of the two types of action. Thus, on the one hand, the silver nanoparticles are known to exhibit a potent antimicrobial effect against different types of microorganisms. These mechanisms have been previously presented by our group [47] and were found to be mainly based on disruption of cellular membrane functionality and generation of reactive oxygen species. These properties are well-connected to the properties of the NPs (size, morphology, etc.) and to the synthesis method, as the secondary metabolites acting as capping agents also enhance their properties. On the other hand, the applied treatments (either as fern extract or as silver nanoparticles phytosynthesized using the fern extract) led to an increase in several biochemical parameters (starch, sugars, and water content), as well as in productivity (grape number) and vigor parameters (length of young shoots) in a clone-dependent manner. These results would suggest a biostimulant effect which could contribute to the increase in plant resistance to pathogens, an aspect which needs further investigation.

Conclusions and Future Perspectives
The present study aimed to evaluate the protective effect on grapevine of a fern extract and of silver phytosynthesized using the same extract. Both types of materials revealed a significant decrease (by comparison with the chemical pesticide used in the experiments) of the frequency, intensity, and attack degree of the pathogen Uncinula necator (responsible for the apparition of powdery mildew). This observation was suggested by the laboratory results against Plasmopara viticola (pathogen responsible for the apparition of downy mildew).
The determinations regarding the pith/wood ratio of the canes showed that all the plants studied have developed very well-matured canes in all treatment options. Statistically significant differences were recorded for the Feteasca alba 97 St. clone treated with the nanoparticle solution in 2020 (lower for base and middle, higher for top level of cane, as compared to the control), while marginal differences were also recorded for 2021 (higher for all determination areas). Regarding the biochemical determinations, significant variations were observed for soluble sugars (increase for Feteasca neagra and Cabernet Sauvignon/second year), for starch (increase for Feteasca alba and Cabernet Sauvignon in 2021 for both clones), for total sugars (increase for Feteasca alba and Feteasca neagra in 2021 for both clones), and for total water content (increase for Feteasca alba and Feteasca neagra in 2021 for both clones), respectively. More than that, the biostimulant effect of the fern extract and of the silver nanoparticles solution was further confirmed by the determinations on the number of grapes produced by each clone and on the length of the young shoots.
Based on the findings of this study, in which positive results were obtained using hydroalcoholic extract of D. filix-mas and phytosynthesized nanoparticles mixture as preventive and curative treatments grapevine against downy mildew and powdery mildew in protected area, it is recommended to extend the research in vineyards. Further studies are also necessary in order to clarify the exact mechanisms involved in the clone-dependent protective potential of the proposed solutions.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ma15228188/s1, Figure S1: Obtaining the natural extracts in large scale reactors; Figure S2: The two types of recipes prepared for field application; Figure S3