Phytochemical profiling of Sambucus nigra L. flower and leaf extracts and their antimicrobial potential against almond tree pathogens

Citation: Sánchez-Hernández, E.; Balduque-Gil, J.; González-García, V.; Barriuso-Vargas, J.J.; Casanova-Gascón, J.; Martín-Gil, J.; Martín-Ramos, P. Phytochemical Profiling of Sambucus nigra L. Flower and Leaf Extracts and Their Antimicrobial Potential against Almond Tree Pathogens. Int. J. Mol. Sci. 2023,24, 1154. https://doi.org/ 10.3390/ijms24021154 Academic Editors: Cristina Elena Dinu-Pîrvu and Robert Ancuceanu Received: 6 December 2022 Revised: 3 January 2023 Accepted: 4 January 2023 Published: 6 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Phytochemical Profiling of Sambucus nigra L. Flower and Leaf Extracts and Their Antimicrobial Potential against Almond Tree Pathogens Eva Sánchez-Hernández 1, Joaquín Balduque-Gil 2, Vicente González-García3, Juan J. Barriuso-Vargas 2, JoséCasanova-Gascón2, Jesús Martín-Gil 1and Pablo Martín-Ramos 1,4,* 1Department of Agricultural and Forestry Engineering, ETSIIAA, Universidad de Valladolid, 34004 Palencia, Spain 2AgriFood Institute of Aragon (IA2), Universidad de Zaragoza–CITA, 50059 Zaragoza, Spain 3 Department of Agricultural, Forest and Environmental Systems, Agrifood Research and Technology Centre of Aragón, Instituto Agroalimentario de Aragón—IA2, CITA–Universidad de Zaragoza, 50059 Zaragoza, Spain 4Instituto Universitario de Investigación en Ciencias Ambientales de Aragón, EPS, Universidad de Zaragoza, 22071 Huesca, Spain *Correspondence: [email protected] Abstract: Despite extensive research on the chemical composition of elderberries and their numerous uses in pharmaceutical, beverage, and food production, there is still a lack of knowledge about Sambucus nigra leaves and flowers’ antimicrobial activity against plant pathogens. In this study, the phytoconstituents of their aqueous ammonia extracts were first characterized by infrared spectroscopy and gas chromatography–mass spectrometry. The major phytocompounds identified in the flower extract were octyl 2-methylpropanoate; 3,5-dihydroxy-6-methyl-2,3-dihydropyran-4-one; propyl malonic acid; adenine; and 1-methyl-2-piperidinemethanol. Concerning the leaf extract, 1,6-anhydroβ -D-glucopyranose; oleic acid; 2,1,3-benzothiadiazole; 2,3-dihydro-benzofuran; and 4-((1E)-3-hydroxy-1-propenyl)-2-methoxyphenol and other phenol derivatives were the main constituents. The potential of the extracts to act as bioprotectants was then investigated against three almond tree pathogens: Diaporthe amygdali,Phytophthora megasperma, and Verticillium dahliae. In vitro tests showed higher activity of the flower extract, with EC 90 values in the 241–984 µ g · mL −1 range (depending on the pathogen) vs. 354–1322 µ g · mL −1 for the leaf extract. In addition, the flower extract led to full protection against P. megasperma at a dose of 1875 µ g · mL −1 in ex situ tests on artificially-infected excised almond stems. These inhibitory concentrations were lower than those of commercial fungicides. These findings suggest that S. nigra aerial organs may be susceptible to valorization as an alternative to synthetic fungicides for the protection of this important crop. Keywords: black elderberry; Diaporthe amygdali; integrated pest management; Phytophthora megasperma; Prunus dulcis;Verticillium dahliae 1. Introduction Sambucus nigra L. (black elderberry or common elderberry) is a species of the Adoxaceae family (syn. Caprifoliaceae). It is a medicinal shrub or small tree native to the western and southern parts of Europe and North Africa. The species has also been introduced into other parts of the world, including North America, South East Asia, and Australia [1]. It has pinnate leaves, with five leaflets, short-stalked, elliptic, 4–12 cm long (Figure 1a). Inflorescences (Figure 1b) are small, yellowish-white, with three small bracts, and may have a peduncle; calyx minute, five-lobed; corolla five-cleft gamopetalous; five yellow stamens; and a tricarpellate pistil of three stigmas. Flowers have a strong pleasant odor, although they may have a fetid note [ 2 ], and are traditionally used to flavor wine, make tea and a nonalcoholic cordial, and are added to the batter used to prepare pastry [ 3 ]. Widely Int. J. Mol. Sci. 2023,24, 1154. https://doi.org/10.3390/ijms24021154 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023,24, 1154 2 of 17 used in herbal medicine and the food industry, the fruits (Figure 1c) are shiny metallic, purple-black, 6–8 mm in diameter drupes that grow in corymbs containing several hundred pieces each [4]. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 2 of 18 odor, although they may have a fetid note [2], and are traditionally used to flavor wine, make tea and a nonalcoholic cordial, and are added to the batter used to prepare pastry [3]. Widely used in herbal medicine and the food industry, the fruits (Figure 1c) are shiny metallic, purple-black, 6–8 mm in diameter drupes that grow in corymbs containing several hundred pieces each [4]. Figure 1. (a) Inflorescences and leaves, (b) flowers, and (c) fruits of Sambucus nigra. The health benefits of the different parts of the S. nigra plant have been investigated by different authors. It has been found that elder flowers are antipyretic, diaphoretic, emetic, expectorant, anti-inflammatory, immunostimulant, antiviral, and antibacterial, while the leaves are disinfectant, diuretic, laxative, purify the blood, and have detoxifying properties [5,6]. The chemical components of S. nigra and its biological properties are tightly connected. The majority of the constituents of S. nigra flower aroma compounds are of terpenoid origin and include monoterpenes (phelandrene, αand γ-terpinene, terpinolene, and safranal), terpenoid alcohols and oxides (terpinen-4-ol, hotrienol, α-terpineol, linalool, hydroxylinalool linalool oxides, cisand trans-rose oxide, and nerol oxide), and a sesquiterpene (β-caryophyllene). Other terpenes—likely derivatives of carotenoids—are 6-methyl5-hepten-2-one, 6-methyl-5-hepten-2-ol, β-ionone, β-damascenone, and 1,1,6-trimethyl−1,2-dihydronaphthalene (1,1,6-TDN). Another major group of the constituents arises from autoxidation of fatty acids, being primarily constituted of butanol, 2-hexenol, 3-hexenol, 3-hydroxy-2-butanone, 1-penten-3-one, 1-octen-3-one, 2,4-heptadienal, and 2-octenal [7]. Both flowers and leaves of black elder are a rich source of basic substances such as rutin, quercetin, protocateuchic acid, 3,5-dicaffeoylquinic acid, neochlorogenic acid, and tannins, as well as vitamin E [3]. On the other hand, sambunigrin and prunasin are the cyanogenic glycosides found in S. nigra that are present in the greatest amounts in all of its sections. Specifically, S. nigra leaves contain the highest concentrations of sambunigrin (27.68–209.61 μg·g−1 fresh weight), followed by flowers (1.23−18.88 μg·g−1 FW) and berries (0.08–0.77 μg·g−1 FW) [8]. Sambucus nigra also has m-hydroxysubstituted glycosides, such as zierin and holocalin. All these substances have the potential to be poisonous and lethal since they produce cyanide after hydrolysis [9]. Concerning the antimicrobial activity of S. nigra, and on the basis of the fact that all organs of the black elder contain the aforementioned cyanogenic glycosides prunasin and sambunigrin, it has been suggested that their plant extracts may possess antifungal activity [10]. Álvarez et al. [11] extracted cysteine-rich peptides from the flowers in a mixture of dicloromethane/methanol, which exhibited activity against different fish pathogens of aquaculture interest (viz., Escherichia coli (Migula) Castellani and Chalmers, Vibrio anguillarum Bergeman, Vibrio ordalii Schiewe et al., Flavobacterium psychrophilum (Bernardet and Grimont) Bernardet et al., and Aeromonas salmonicida (Lehmann and Neumann) Griffin et al.). It was postulated that their antimicrobial activity was related to bacterial cell membrane disruption. In 2013, a brief report regarding the antifungal activity of S. nigra fruit ethanolic extracts concluded the existence of in vitro inhibitory activity against Phytophthora infestans (Mont.) de Bary [12]. Binding of antifungal PR 1 proteins to the protein Figure 1. (a) Inflorescences and leaves, (b) flowers, and (c) fruits of Sambucus nigra. The health benefits of the different parts of the S. nigra plant have been investigated by different authors. It has been found that elder flowers are antipyretic, diaphoretic, emetic, expectorant, anti-inflammatory, immunostimulant, antiviral, and antibacterial, while the leaves are disinfectant, diuretic, laxative, purify the blood, and have detoxifying properties [5,6]. The chemical components of S. nigra and its biological properties are tightly connected. The majority of the constituents of S. nigra flower aroma compounds are of terpenoid origin and include monoterpenes (phelandrene, α -and γ -terpinene, terpinolene, and safranal), terpenoid alcohols and oxides (terpinen-4-ol, hotrienol, α -terpineol, linalool, hydroxylinalool linalool oxides, cisand trans-rose oxide, and nerol oxide), and a sesquiterpene ( β -caryophyllene). Other terpenes—likely derivatives of carotenoids—are 6-methyl-5hepten-2-one, 6-methyl-5-hepten-2-ol, β -ionone, β -damascenone, and 1,1,6-trimethyl − 1,2dihydronaphthalene (1,1,6-TDN). Another major group of the constituents arises from autoxidation of fatty acids, being primarily constituted of butanol, 2-hexenol, 3-hexenol, 3-hydroxy-2-butanone, 1-penten-3-one, 1-octen-3-one, 2,4-heptadienal, and 2-octenal [ 7 ]. Both flowers and leaves of black elder are a rich source of basic substances such as rutin, quercetin, protocateuchic acid, 3,5-dicaffeoylquinic acid, neochlorogenic acid, and tannins, as well as vitamin E [ 3 ]. On the other hand, sambunigrin and prunasin are the cyanogenic glycosides found in S. nigra that are present in the greatest amounts in all of its sections. Specifically, S. nigra leaves contain the highest concentrations of sambunigrin ( 27.68–209.61 µg·g−1 fresh weight), followed by flowers (1.23–18.88 µ g · g −1 FW) and berries ( 0.08–0.77 µg·g−1FW ) [ 8 ]. Sambucus nigra also has m-hydroxysubstituted glycosides, such as zierin and holocalin. All these substances have the potential to be poisonous and lethal since they produce cyanide after hydrolysis [9]. Concerning the antimicrobial activity of S. nigra, and on the basis of the fact that all organs of the black elder contain the aforementioned cyanogenic glycosides prunasin and sambunigrin, it has been suggested that their plant extracts may possess antifungal activity [ 10 ]. Álvarez et al. [ 11 ] extracted cysteine-rich peptides from the flowers in a mixture of dicloromethane/methanol, which exhibited activity against different fish pathogens of aquaculture interest (viz., Escherichia coli (Migula) Castellani and Chalmers, Vibrio anguillarum Bergeman, Vibrio ordalii Schiewe et al., Flavobacterium psychrophilum (Bernardet and Grimont) Bernardet et al., and Aeromonas salmonicida (Lehmann and Neumann) Griffin et al.). It was postulated that their antimicrobial activity was related to bacterial cell membrane disruption. In 2013, a brief report regarding the antifungal activity of S. nigra fruit ethanolic extracts concluded the existence of in vitro inhibitory activity against Phytophthora infestans (Mont.) de Bary [ 12 ]. Binding of antifungal PR 1 proteins to the protein channels in cell membranes, affecting the release of Ca 2+ ions, was proposed as the mechanism behind such antifungal activity [13]. Int. J. Mol. Sci. 2023,24, 1154 3 of 17 As previously mentioned, there is still a lack of knowledge about the leaves’ and flowers’ bioactive compound content and potential to act as bioprotectants in fruticulture, specifically to control diseases associated with plants of the genus Prunus L. (cherry, almond, peach, and plum). In farmed Prunus plants, more than ten Phytophthora species have been found to cause root rot, crown rot, and stem and scaffold cankers. Among them, the soil-borne infection caused by Phytophthora megasperma Drechs. is frequently linked to root and crown rots, as well as trunk cankers [ 14 ], causing tree losses, especially of young plants in poorly drained soils. Vascular wilts are among the most severe fungal diseases in the world, and certain species of the genus Verticillium are responsible for them. These soil-borne, cosmopolitan, ascomycetous fungi possess a wide range of plant hosts, causing significant yield losses [ 15 ]. Verticillium dahliae Kleb., one of the many species of the genus, can infect more than 200 plant species worldwide [ 16 ]. Verticillium wilt of almond (Prunus dulcis (Mill.) D.A.Webb) can damage developing orchards and reduce productivity. Although historical reports of Verticillium wilt of Prunus spp. usually describe individual branch or limb death, with rare losses of entire trees (usually in the second or third growing season), they result in substantial economic losses primarily due to tree removal and replacement cost, extra pruning, and lost production from weakened plant stands [17]. As for Diaporthe species—which can either be plant pathogens, endophytes, or saprobes—some have been linked to twig canker, bud and shoot blight, dieback, wood decay, and fruit rot in almond trees [ 18 ]; canker, shoot dieback, and bud and shoot blight in peaches [ 19 ]; cankers and shoot blight in apples [ 20 ]; and dieback and canker in pear and plum trees [ 21 ]. In particular, Diaporthe amygdali (Delacr.) Udayanga, Crous and K.D. Hyde has been identified as the causal agent of twig canker and blight in almond and peach trees, being associated with fruit rot of peaches and fruit rot and branch dieback (‘Fusicoccum canker’) of almond [ 18 ]. The rapid desiccation of buds, blooms, and leaves in late winter or early spring is one of the disease’s symptoms. Brown lesions, which first appear around green shoot buds, progress into yearly sunken cankers, which can occasionally have a sticky exudate as well as twig withering [22]. According to Article 14 of Directive 2009/128/EC, which promotes the use of formulations based on natural products in the frame of Integrated Pest Management programs, the current work suggests the use of aqueous ammonia extracts of the flowers or leaves of S. nigra as biorationals for the protection of certain important fungal pathogens of P. dulcis. Both extracts (whose main constituents were determined by GC–MS) were first studied in vitro against the aforementioned three pathogens, and the most active extract was subsequently tested in ex situ assays on excised stems to confirm its anti-oomycete activity against P. megasperma. 2. Results 2.1. Vibrational Characterization The main infrared absorption bands present in the spectra of S. nigra flowers and leaves, as well as in those of their aqueous ammonia extracts (Figure S1), are summarized in Table 1. Int. J. Mol. Sci. 2023,24, 1154 4 of 17 Table 1. Main bands in the infrared spectra of S. nigra flowers and leaves, and those of their aqueous ammonia extracts. Flower Flower Extract Leaf Leaf Extract Assignment 3369 O–H stretching of free hydroxyl groups 3288 3231 3239 3239 bonded O–H stretching (cellulose, hemicellulose, lignin) 2921 2923 2924 2930 –CH2asymmetric stretching of alkyls (cutine, wax, pectin) 2851 2852 2850 –CH2sym. stretch (cutine and wax)/CH2–(C6) bend (cellulose) 1733 1733 1733 C=O stretching of alkyl ester 1685 1685 1686 α,β-unsaturated carbonyls 1633 1636 skeletal aromatic C=C ring stretching and C=O stretching 1605 1602 1588 1582 aromatic C=C stretching 1519 1516 1522 aromatic skeletal 1455 1462 CH2, CH3bending/C=C stretching, furan ring (furfural)/O–CH3str. 1388 1376 1387 1375 1389 1389 CH3antisymmetric bending aliphatic C–H stretching in methyl and phenol OH 1312 1315 1314 C–H vibration of the methyl group 1254 1254 1250 1263 C–O–C symmetric stretching/C–O stretching 1143 1164 1163 1154 C–O–C asym. stretching in celluloses; C–O stretching; C–C in-plane 1100 1121 1100 in plane =C–H bending/C=C stretching/C−O−C stretching in the pyranose ring skeletal (cellulose) 1047 1045 1046 O–H out plane bending/C–O stretching 1015 1026 1010 C–H bending 919 918 918 914 β-glycosidic linkages (glucose units of cellulose chains) 872 873 C–H bending The spectral profiles are compatible with the presence of the functional groups of alkaloids, polyphenols, and organic acid esters. For example, the key vibrations bands at 3369 cm −1 (indicative of O–H stretching of free hydroxyl groups), 2922 cm −1 (indicative of C–H stretching of alkanes), and 1602 cm −1 (indicative of α and β unsaturated ketone groups) are consistent with the functional groups found on DDMP (2,3-dihydro-3,5-dihydroxy-6methyl-4H-pyran-4-one or 3,5-dihydroxy-6-methyl-2,3-dihydropyran-4-one), in agreement with the work by Olaniyan et al. [23]. 2.2. Analysis of the Constituents of the Flower and Leaf Extracts by GC-MS The results obtained from the phytochemical analyses of S. nigra flower extract ( Table S1 , Figure S2) show relative high concentrations of octyl 2-methylpropanoate (also named octyl isobutyrate and caprylyl isobutyrate; 10.5%); DDMP (5.1%); 2-propyl malonic acid (or 2-propylpropanedioic acid) and dimethylmalonic acid, ethyl isohexyl ester (4.9%); heptanal-related compounds (4.6%); glycerin (4.2%); adenine (4.2%); and 1methyl-2-piperidinemethanol (3.5%). Other minor constituents include 2-cyclopentylidenecyclopentanone (3%); benzoic acid derivatives (2.5%); N,N-diamylmethylamine (2.3%); geranic acid (1.7%); catechol (1.6%); and 2,3-dihydro-benzofuran (1.6%). In turn, the main phytochemicals identified from S. nigra leaf extract (Table S2, Figure S3 ) were 1,6-anhydroβ -D-glucopyranose (11.6%); oleic acid (6.3%); butanoic acid pentyl ester or pentyl butyrate (8%); 2,1,3-benzothiadiazole (4.5%); trans-3-penten-2-ol (3.9%); 2,3-dihydro-benzofuran (3.5%); 4-((1E)-3-hydroxy-1-propenyl)-2-methoxyphenol and other phenol derivatives (3.1%); 6-hydroxy-4(1H)-pyrimidinone (2.9%); catechol (2.8%); inositol derivatives (2.2%); DDMP (2.1%); 2,3-dihydroxycyclohexanone (2.0%); glycerin (1.8%); and hydroquinone (1.7%). Hence, the flower and leaf extracts shared the presence of glycerin, DDMP, 2,3-dihydrobenzofuran, catechol, and hydroquinone. Int. J. Mol. Sci. 2023,24, 1154 5 of 17 2.3. Antifungal and Anti-Oomycete Activity Assessment 2.3.1. In Vitro Activity The aqueous ammonia extracts of flowers and leaves of S. nigra were evaluated for their capacity to inhibit the mycelial development of D. amygdali,P. megasperma, and V. dahliae strains (Figure 2). The flower extract of S. nigra was more effective than the leaf extract against D. amygdali, with inhibition values of 1000 and 1500 µ g · mL −1 , respectively, and equally effective against the oomycete P. megasperma and the soil-borne ascomycete V. dahliae, inhibiting their mycelial growth at concentrations of 375 and 1500 µ g · mL −1 , respectively. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 18 inositol derivatives (2.2%); DDMP (2.1%); 2,3-dihydroxycyclohexanone (2.0%); glycerin (1.8%); and hydroquinone (1.7%). Hence, the flower and leaf extracts shared the presence of glycerin, DDMP, 2,3-dihydro-benzofuran, catechol, and hydroquinone. 2.3. Antifungal and Anti-Oomycete Activity Assessment 2.3.1. In Vitro Activity The aqueous ammonia extracts of flowers and leaves of S. nigra were evaluated for their capacity to inhibit the mycelial development of D. amygdali, P. megasperma, and V. dahliae strains (Figure 2). The flower extract of S. nigra was more effective than the leaf extract against D. amygdali, with inhibition values of 1000 and 1500 μg·mL−1, respectively, and equally effective against the oomycete P. megasperma and the soil-borne ascomycete V. dahliae, inhibiting their mycelial growth at concentrations of 375 and 1500 μg·mL−1, respectively. Figure 2. Radial growth of the mycelium of (a) D. amygdali, (b) P. megasperma, and (c) V. dahliae in in vitro assays performed on PDA medium with different concentrations (in the 62.5–1500 μg·mL−1 range) of S. nigra flower and leaf extracts, as well as some of the main constituents of flower extract. The same letters above concentrations mean that they are not significantly different at p < 0.05. Standard deviations are represented by error bars. DDMP = 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-; piperidinemethanol = 1-methyl-2-piperidinemethanol. Figure 2. Radial growth of the mycelium of ( a )D. amygdali, ( b )P. megasperma, and ( c )V. dahliae in in vitro assays performed on PDA medium with different concentrations (in the 62.5–1500 µ g · mL −1 range) of S. nigra flower and leaf extracts, as well as some of the main constituents of flower extract. The same letters above concentrations mean that they are not significantly different at p< 0.05 . Standard deviations are represented by error bars. DDMP = 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy6-methyl-; piperidinemethanol = 1-methyl-2-piperidinemethanol. Because of the higher activity of the flower extract, and in a first approach to investigate its mode of action, three of its constituents (viz., DDMP, octyl isobutyrate, and 1-methyl2-piperidine methanol, shown in Figure 3) were also tested. DDMP was equally effective against P. megasperma and V. dahliae, with inhibition values of 375 µ g · mL −1 , compared to Int. J. Mol. Sci. 2023,24, 1154 6 of 17 750 µ g · mL −1 for D. amygdali. The most abundant compound in the flower extract, octyl isobutyrate, was most effective against P. megasperma (MIC = 250 µ g · mL −1 ), followed by V. dahliae (MIC = 500 µ g · mL −1 ) and D. amygdali (MIC = 750 µ g · mL −1 ). Finally, 1-methyl-2piperidinemethanol inhibition was higher in P. megasperma ( MIC = 375 µg·mL−1 ) compared to that obtained against V. dahliae (MIC = 500 µ g · mL −1 ) and D. amygdali ( MIC = 750 µg·mL−1 ). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 18 Because of the higher activity of the flower extract, and in a first approach to investigate its mode of action, three of its constituents (viz., DDMP, octyl isobutyrate, and 1methyl-2-piperidine methanol, shown in Figure 3) were also tested. DDMP was equally effective against P. megasperma and V. dahliae, with inhibition values of 375 μg·mL−1, compared to 750 μg·mL−1 for D. amygdali. The most abundant compound in the flower extract, octyl isobutyrate, was most effective against P. megasperma (MIC = 250 μg·mL−1), followed by V. dahliae (MIC = 500 μg·mL−1) and D. amygdali (MIC = 750 μg·mL−1). Finally, 1-methyl2-piperidinemethanol inhibition was higher in P. megasperma (MIC = 375 μg·mL−1) compared to that obtained against V. dahliae (MIC = 500 μg·mL−1) and D. amygdali (MIC = 750 μg·mL−1). Table 2 summarizes the effective concentrations (EC50 and EC90) obtained for the two extracts and each of the pure compounds against each pathogen. Figure 3. Some of the main phytochemicals identified in S. nigra flower aqueous ammonia extract. Table 2. Effective concentrations (expressed in μg·mL−1) against D. amygdali, P. megasperma, and V. dahliae of S. nigra flower and leaf aqueous ammonia extracts, together with those of three of the main constituents of the flower extract. Treatment Effective Concentration (µg·mL−1) D. amygdali P. megasperma V. dahliae S. nigra flower extract EC50 720.5 193.9 516.1 EC90 981.7 241.2 984.1 S. nigra leaf extract EC50 860.3 269.3 539.2 EC90 1322.4 354.6 975.2 DDMP EC50 152.7 63.6 159.9 EC90 482.4 211.1 314.1 Octyl isobutyrate EC50 164.4 87.0 142.4 EC90 647.5 177.2 471.2 Piperidinemethanol EC50 233.4 76.4 185.8 EC90 635.5 225.6 447.0 DDMP = 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-; piperidinemethanol = 1-methyl-2piperidinemethanol. For comparison purposes, three commercial synthetic fungicides (viz., azoxystrobin, mancozeb, and fosetyl-Al) were also tested at three concentrations (the recommended dose, 1/10th of the recommended dose, and 10 times the recommended dose). Radial growth inhibition results are summarized in Table 3. The highest activity was attained by mancozeb, which fully inhibited the growth of V. dahliae, D. amygdali, and P. megasperma at doses of 150, 1500, and 15,000 μg·mL−1, respectively. Fosetyl-Al showed an intermediate activity, achieving full inhibition at 2000 μg·mL−1 against V. dahliae, and at 20,000 μg·mL−1 against the other two pathogens. The lowest activity was attained by azoxystrobin, which did not fully inhibit the growth of P. megasperma at ten times the recommended dose, and for which MIC values of 625,000 μg·mL−1 were obtained against D. amygdali and V. dahliae. Figure 3. Some of the main phytochemicals identified in S. nigra flower aqueous ammonia extract. Table 2summarizes the effective concentrations (EC 50 and EC 90 ) obtained for the two extracts and each of the pure compounds against each pathogen. Table 2. Effective concentrations (expressed in µ g · mL −1 ) against D. amygdali,P. megasperma, and V. dahliae of S. nigra flower and leaf aqueous ammonia extracts, together with those of three of the main constituents of the flower extract. Treatment Effective Concentration (µg·mL−1)D. amygdali P. megasperma V. dahliae S. nigra flower extract EC50 720.5 193.9 516.1 EC90 981.7 241.2 984.1 S. nigra leaf extract EC50 860.3 269.3 539.2 EC90 1322.4 354.6 975.2 DDMP EC50 152.7 63.6 159.9 EC90 482.4 211.1 314.1 Octyl isobutyrate EC50 164.4 87.0 142.4 EC90 647.5 177.2 471.2 Piperidinemethanol EC50 233.4 76.4 185.8 EC90 635.5 225.6 447.0 DDMP = 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-; piperidinemethanol = 1-methyl-2-piperidinemethanol. For comparison purposes, three commercial synthetic fungicides (viz., azoxystrobin, mancozeb, and fosetyl-Al) were also tested at three concentrations (the recommended dose, 1/10th of the recommended dose, and 10 times the recommended dose). Radial growth inhibition results are summarized in Table 3. The highest activity was attained by mancozeb, which fully inhibited the growth of V. dahliae,D. amygdali, and P. megasperma at doses of 150, 1500, and 15,000 µ g · mL −1 , respectively. Fosetyl-Al showed an intermediate activity, achieving full inhibition at 2000 µ g · mL −1 against V. dahliae, and at 20,000 µ g · mL −1 against the other two pathogens. The lowest activity was attained by azoxystrobin, which did not fully inhibit the growth of P. megasperma at ten times the recommended dose, and for which MIC values of 625,000 µ g · mL −1 were obtained against D. amygdali and V. dahliae. Int. J. Mol. Sci. 2023,24, 1154 7 of 17 Table 3. Radial growth of mycelium of D. amygdali, P. megasperma, and V. dahliae in in vitro assays performed on a PDA medium amended with different concentrations (the recommended dose, 1/10th of the recommended dose, and 10 times the recommended dose) of three commercial synthetic fungicides. Commercial Fungicide Pathogen Radial Growth of Mycelium (mm) Inhibition (%) Rd/10 Rd * Rd ×10 Rd/10 Rd * Rd ×10 Azoxystrobin D. amygdali 33.2 14.2 0 55.7 81.1 100 P. megasperma 48.1 34.3 14.5 35.9 54.3 80.6 V. dahliae 26 24 0 65.3 68 100 Mancozeb D. amygdali 13.4 0 0 82.2 100 100 P. megasperma 61.3 31.3 0 18.3 58.3 100 V. dahliae 0 0 0 100 100 100 Fosetyl-Al D. amygdali 72 9.9 0 4 86.9 100 P. megasperma 75 16.1 0 0 78.6 100 V. dahliae 36 0 0 52 100 100 * Rd stands for recommended dose, i.e., 62.5 mg · mL −1 of azoxystrobin (250 g · L −1 for Ortiva ® , azoxystrobin 25%), 1.5 mg · mL −1 of mancozeb (2 g · L −1 for Vondozeb ® , mancozeb 75%), and 2 mg · mL −1 of fosetyl-Al (2.5 g · L −1 for Fosbel ® , fosetyl-Al 80%). The radial growth of the mycelium for the control (PDA only) was 75 mm. All mycelial growth values (in mm) are average values (n= 3). 2.3.2. Ex Situ Activity for the Protection of Excised Stems Because of the high activity of S. nigra extracts, ex situ tests were conducted on almond rootstock ‘Garnem’ excised stems to evaluate the efficacy of the most active treatment (the flower extract) against the most resistant pathogen, viz., P. megasperma. At the lowest assayed concentration, i.e., the MIC value obtained in the in vitro tests (375 µ g · mL −1 ), no protection was observed, with canker lengths similar to those of the untreated stems. When the dose was increased by a factor of 2.5 (937.5 µ g · mL −1 ), large cankers were still registered, with no significant differences versus the control. Only when the protective treatment concentration was five times the MIC value (1875 µ g · mL −1 ) was full protection of the excised stems achieved, with no signs of fungal colonization in the outer bark or in the cambium tissues of any of the replicates (Figure 4). To fulfill Koch’s postulates, samples of the inoculated ‘Garnem’ stems showing cankers were taken apart and mounted on a microscope slide with 3% KOH as mounting media and morphologically inspected to confirm the identity of the microorganism responsible for the lesions. Such microscopical observations confirmed the presence of somatic and reproductive structures compatible with those of P. megasperma (Figure 5). Int. J. Mol. Sci. 2023,24, 1154 8 of 17 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 18 Figure 4. ‘Garnem’ stem segments artificially inoculated with P. megasperma after 4 days of incubation: (left) untreated samples (top: outer bark; bottom: cambium); (right) samples (top: outer bark; bottom: cambium) treated with S. nigra flower aqueous ammonia extract at 1875 μg·mL−1 (MIC×5). Figure 5. Re-isolation and characterization of the P. megasperma strain previously inoculated in ‘Garnem’ stem segments. (a) Mycelium in PDA plates subjected to microscopic observations; (b) microscopical aspect of somatic hyphae; (c) zoosporangia; (d) oospore. Figure 4. ‘Garnem’ stem segments artificially inoculated with P. megasperma after 4 days of incubation: ( left ) untreated samples ( top : outer bark; bottom : cambium); ( right ) samples ( top : outer bark; bottom: cambium) treated with S. nigra flower aqueous ammonia extract at 1875 µ g · mL −1 (MIC × 5). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 18 Figure 4. ‘Garnem’ stem segments artificially inoculated with P. megasperma after 4 days of incubation: (left) untreated samples (top: outer bark; bottom: cambium); (right) samples (top: outer bark; bottom: cambium) treated with S. nigra flower aqueous ammonia extract at 1875 μg·mL−1 (MIC×5). Figure 5. Re-isolation and characterization of the P. megasperma strain previously inoculated in ‘Garnem’ stem segments. (a) Mycelium in PDA plates subjected to microscopic observations; (b) microscopical aspect of somatic hyphae; (c) zoosporangia; (d) oospore. Figure 5. Re-isolation and characterization of the P. megasperma strain previously inoculated in ‘Garnem’ stem segments. ( a ) Mycelium in PDA plates subjected to microscopic observations; ( b ) microscopical aspect of somatic hyphae; (c) zoosporangia; (d) oospore. Int. J. Mol. Sci. 2023,24, 1154 9 of 17 3. Discussion 3.1. Phytochemical Profile As indicated above, previous works on S. nigra flower aqueous extracts mainly reported the presence of terpenes—including monoterpenes, terpenoids alcohols and oxides, sesquiterpenes, and derivatives of carotenoids—and compounds arising from the autoxidation of fatty acids [ 7 ], none of them present in the extract studied herein (except for terpinen-4-ol and heptanal). Other reports on aqueous, ethanolic, and methanolic flower and leaf extracts detected rutin, quercetin, protocateuchic acid, 3,5-dicaffeoylquinic acid, neochlorogenic acid, tannins, vitamin E, and glycosides [ 3 , 8 , 9 ], out of which only the latter were found in the leaf aqueous ammonia extract (e.g., 1,6-anhydroβ -D-glucopyranose) here analyzed. Although the composition of S. nigra’s components is known to be influenced by a number of variables, including variety, cultivar, and environmental and climatic circumstances [ 24 ], in our view, differences, in this case, should be mainly ascribed to the choice of the extraction solvent. Nonetheless, as discussed below, the presence of the main compounds identified herein has also been reported in other plant extracts. Concerning the flower extract constituents, octyl isobutyrate, the octyl ester of isobutyric acid, was identified as one of the major phytochemicals in essential oils with antimicrobial potential from Malabaila aurea Boiss. aerial parts (40%) [ 25 ], and is also present in large amounts in the bark from Uncaria tomentosa (Willd. ex Schult.) DC. (30.7%) [ 26 ]; in Heracleum sphondylium subsp. ternatum (Velen.) Brummitt (24.6%) [ 27 ]; in Heracleum persicum Desf. (17.82%) [ 28 ]; and—in smaller proportions—in the essential oils from roots of Caucalis platycarpos L. (8.5%), Elaeosticta glaucescens Boiss. (4.0%), and Eryngium caucasicum Trautv. (2.8%) [29]. Due to their extensive biological and pharmaceutical properties, 4H-pyrans belong to a significant class of heterocyclic compounds. In particular, the flavonoid named 4H-pyran4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- (or 3,5-dihydroxy-6-methyl-2,3-dihydropyran4-one) has been isolated from Chuanminshen violaceum Sheh and Shan (25%) [ 30 ], Punica granatum var. nana L. (9.7%) [ 31 ], Nephrolepis biserrata (Sw.) Schott (9.3%) [ 32 ], Hibiscus syriacus L. (4.2%) [ 33 ], Cyperus rotundus L. (3.8%) [ 34 ], Euphorbia serrata L., Camellia japonica L., Acalypha indica L., Ammannia baccifera L., Borassus flabellifer L., Cocculus hirsutus (L.) Diels, Cucumis sativus L., Leucas aspera (Willd.) Link, Litchi chinensis Sonn., Marsilea quadrifolia L., and Rumex vesicarius L. [ 35 ]. It has been reported to display antioxidant, antimicrobial, anticancer, anti-inflammatory, and cytotoxic activities [31,36,37]. Although acid 2-propylmalonic acid (a fatty acid derivative) has not been reported to display antimicrobial activity, its diisopropyl malonate derivative is used for the synthesis of the fungicide isoprothiolane [ 38 ]. Due to their numerous applications, piperidine alkaloid compounds are among the chemical classes that are often studied in medicine as raw ingredients to create new medications or as medications themselves to treat certain ailments [39]. The presence of glycerin in plant extracts has been reported, for instance, in Plantago major L. leaf extracts [ 40 ]; in Allamanda cathartica L. [ 41 ]; in Cynodon dactylon (L.) Pers. (with an activity comparable to that of streptomycin against Staphylococcus aureus Rosenbach, E. coli, Salmonella typhi (Schroeter) Warren and Scott, Proteus mirabilis Hauser, and Streptococcus pyogenes Rosenbach) [ 42 ]; in Salvadora persica L. (with activity against S. aureus and Aspergillus terreus Thom) [ 43 ]; and in Aphelandra squarrosa Nees (with a glycerin content as high as 46% and strong activity against E. coli) [44]. As for 1-methyl-2-piperidinemethanol, it has been previously isolated from young stems of Lobelia polyphylla Hook. and Arn. 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