Holm Oak (Quercus ilex subsp. ballota (Desf.) Samp.) Bark Aqueous Ammonia Extract for the Control of Invasive Forest Pathogens

Holm oak (Quercus ilex subsp. ballota (Desf.) Samp.) bark is a commonly used remedy to treat gastrointestinal disorders, throat and skin infections, hemorrhages, and dysentery. It has also been previously reported that its methanol extracts possess antibacterial activity, which can be related to the richness of Quercus spp. extracts in phenolic compounds, such as flavonoids and tannins. However, there is no information on the antifungal (including oomycete) properties of the bark from Q. ilex or its subspecies (ilex and ballota). In this work, we report the characterization of the aqueous ammonia extract of its bark by FTIR and GC-MS and the results of in vitro and ex situ inhibition tests against three phytopathogens. The main phytochemical components identified were inositols (19.5%), trans-squalene (13%), 4-butoxy-1-butanol (11.4%), gulopyranose (9.6%), lyxose (6.5%), 2,4-dimethyl-benzo[H]quinoline (5.1%), catechol (4.5%), and methoxyphenols (4.2%). The efficacy of the extract in controlling forest phytopathogens was tested in vitro against Fusarium circinatum (responsible for pitch canker of Pinus spp.), Cryphonectria parasitica (which causes chestnut blight), and Phytophthora cinnamomi (which causes ‘root and crown rot’ in a variety of hosts, including Castanea, conifers, Eucalyptus, Fagus, Juglans, Quercus, etc.), obtaining EC90 values of 322, 295, and 75 μg·mL−1, respectively, much lower than those attained for a commercial strobilurin fungicide (azoxystrobin). The extract was further tested ex situ against P. cinnamomi on artificially inoculated, excised stems of ‘Garnem’ almond rootstock, attaining complete protection at a dose of 782 μg·mL−1. The results suggest that holm oak bark extract may be a promising source of bioactive compounds against invasive forest pathogens, including the oomycete that is causing its decline, the so-called ‘seca’ in Spain.


Introduction
From ancient times to the present, the usage of barks has changed and expanded in response to various socioeconomic circumstances as well as scientific and technological

In Vitro Antimicrobial Activity
Mycelial growth inhibition tests against the three phytopathogens were conducted for the bark extract and its two main constituents. The increase in the assayed dose resulted in statistically significant differences and in full inhibition in all cases ( Figure 2).

In Vitro Antimicrobial Activity
Mycelial growth inhibition tests against the three phytopathogens were conducted for the bark extract and its two main constituents. The increase in the assayed dose resulted in statistically significant differences and in full inhibition in all cases ( Figure 2). The inhibitory activity of Q. ilex subsp. ballota bark extract (Figures 2a and S2) was significantly higher against P. cinnamomi than against F. circinatum and C. parasitica (for which total inhibition was achieved at similar concentrations, as shown by the EC 90 effective concentration values summarized in Table 2). Upon testing the two main constituents, namely myo-inositol (Figures 2b and S3) and trans-squalene (Figures 2c and S4), it could be observed that the latter showed much higher efficacy than the former, attaining full inhibition of F. circinatum, C. parasitica, and P. cinnamomi at concentrations as low as 250, 187.5, and 85.9 µg·mL −1 , respectively (vs. 1000, 750 and 375 µg·mL −1 , respectively, for myo-inositol). The activity of the holm oak extract was compared with that of a commercial systemic, broad-spectrum fungicide frequently used in forestry and agriculture to control numerous plant diseases, viz. Azoxystrobin. This popular synthetic pesticide of the strobilurin family [11] inhibits pathogens' ability to respire their mitochondria and impairs a number of biological and biochemical functions of living cells (by preventing electron transfer, stopping the synthesis of adenosine triphosphate, and disrupting the flow of energy) [12]. At the manufacturer's recommended dose (62.5 mg·mL −1 ), 68%, 81.3%, and 92% inhibition was attained against F. circinatum, C. parasitica, and P. cinnamomi, respectively (corresponding to radial growth values of 24, 14, and 6 mm, respectively).

Protection of Excised Stems against P. cinnamomi
Ex situ tests conducted on almond rootstock 'Garnem' excised stems were conducted to assess the efficacy of the treatment against the phytopathogen for which the best in vitro results have been attained, viz. P. cinnamomi. At the lowest assayed dose, i.e., the MIC value obtained in the in vitro tests (78.2 µg·mL −1 ), no protection was observed, with canker lengths similar to those of the untreated stems. At five times the MIC dose (391 µg·mL −1 ), large cankers were also registered, with no significant differences vs. the control. It was necessary to increase the dosage up to 10 times the MIC (782 µg·mL −1 ) to obtain full protection of the excised stems, as shown in Figure 3. At this concentration, no signs of fungal colonization were observed in any of the replicates, neither in the outer bark nor in the cambium tissues. total inhibition was achieved at similar concentrations, as shown by the EC90 effective concentration values summarized in Table 2). Upon testing the two main constituents, namely myo-inositol (Figures 2b and S3) and trans-squalene (Figures 2c and S4), it could be observed that the latter showed much higher efficacy than the former, attaining full inhibition of F. circinatum, C. parasitica, and P. cinnamomi at concentrations as low as 250, 187.5, and 85.9 μg·mL −1 , respectively (vs. 1000, 750 and 375 μg·mL −1 , respectively, for myo-inositol).       To fulfill Koch's postulates, samples from both cankers of inoculated 'Garnem' stems and colonized PDA plates 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. These microscopical observations confirmed the presence of somatic and reproductive structures compatible with those of P. cinnamomi. To fulfill Koch's postulates, samples from both cankers of inoculated 'Garnem' stems and colonized PDA plates 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. These microscopical observations confirmed the presence of somatic and reproductive structures compatible with those of P. cinnamomi.

On the Phytochemical Composition
Differences in the phytochemical profiles between those reported in the literature [1,[3][4][5][6][7][8] and the one described in this study should be mainly ascribed to differences in extraction media and methodology, as well as in the method of acquisition [7], although slight differences between subspecies cannot be ruled out. Whereas the results of this study were obtained for an aqueous ammonia extract and applying GC-MS, the results by Meziti et al. [4] and Hadidi et al. [6] corresponded to hydromethanolic extracts characterized by high-performance liquid chromatography (HPLC) and high-performance liquid chromatography with a diode array detector (HPLC-DAD), respectively; the thorough study conducted by Sousa et al. [1] used dichloromethane as the extraction medium and GC-MS; Karioti et al. [5] chose diethylether for the extraction and used HPLC-DAD-MS; and Dallali et al. [8], also in diethylether medium, determined the constituents of the essential oils by gas chromatography with flame-ionization detection (GC−FID). However, the main constituents identified in the present study have been reported for other plant extracts in the literature: for instance, inositol was previously identified in Abrus precatorius L. seeds [13], Cocos nucifera L. [14], Crinum latifolium L. leaves [15], and Rhizophora apiculata Blume roots [16]. Myo-inositol has been found in the latex of C. bonplandianum L. [17]. Squalene is a component of Cuscuta reflexa Roxb., A. precatorius, Abutilon indicum L., Acalypha indica L., Ammannia baccifera L. [18], C. maxima Duchesne [19], Jasminum grandiflorum L. [20], and Leucas aspera (Willd.) Link [21]. Concerning the dialkyl ether 4-butoxy-1-butanol, it has been identified in Apium graveolens L. leaves by Nagella et al. [22].

Antimicrobial Activity Comparison
Regarding the antimicrobial activity of Q. ilex extracts, few studies are available in the literature (Table S1). As for leaf extracts, Boy et al. [23] evaluated the efficacy of Q. ilex leaf extracts obtained by ultrasonication in 90% (v/v) ethanol-water against yeasts responsible for food spoilage, finding a high inhibition capacity against Candida boidinii C.  [24] demonstrated the antibacterial activity of an aqueous extract of Q. ilex leaves obtained by a microwave-assisted extraction procedure against foodborne Gram-and Gram+ bacteria with inhibition values in the 1000 to 5000 µg·mL −1 range; and Güllüce et al. [25] tested in vitro a methanol extract of Q. ilex leaves against a wide range of human and plant-associated microorganisms, including Pseudomonas syringae Van Hall 1904, with a MIC of 250 µg·mL −1 . Berahou et al. [10] tested different solvents (ethyl acetate, n-butanol, or water) to prepare Q. ilex bark extracts, and assayed them against 11 reference bacterial strains, finding inhibition values ranging from 128 to 512 µg·mL −1 ; Merghache et al. [26] showed the inhibitory capacity of Q. ilex wood ashes against Candida albicans at a 5% concentration; and Bakour et al. [27] studied the antibacterial effect of the ethanolic extract of pollen from six botanicals, including Q. ilex, finding that it was the most efficient against multidrug-resistant bacteria.
To date, and to the best of the authors' knowledge, no data on the antifungal/antioomycete action of Q. ilex bark extracts have been reported. However, results on the antifungal activity of other species of the genus Quercus are available. For example, Q. suber L. bark acetone extract showed strong antifungal activity against Trichophyton verrucosum E. Bodin and T. mentagrophytes C.P. Robin) R. Blanch., with MIC values of 20 µg·mL −1 [28]. Antifungal activity against Aspergillus flavus Link, A. ochraceus Willhem, A. niger Tiegh., C. albicans (C.P. Robin) Berkhout, Penicillum feniculosum Thom, and P. ochrochloron Biourge has also been observed for methanolic extracts of Q. acutissima Carruth., Q. macrocarpa Michx., and Q. robur L. bark, with MIC values ranging from 160 to 2000 µg·mL −1 [29]. Dania et al. [30] found that Q. phillyreoides A. Gray bark aqueous extract, at a concentration of 3%, resulted in strong inhibition of mycelial growth (>77%) in in vitro tests against Rhizoctonia solani J.G. Kühn, Lasiodiplodia theobromae (Pat.) Griffon and Maubl., A. niger, Sclerotium rolfsii Sacc., P. oxalicum Currie and Thom, and F. oxysporum Schltdl. Inhibitions in the range of 60-80.5% against C. glabrata (H.W. Anderson) S.A. Mey. and Yarrow, C. albicans, A. niger, A. flavus, F. solani, and Microsporum canis E. Bodin ex Guég. have been reported for some compounds present in the methanolic extract of Q. incana W. Bartram bark at a concentration of 5000 µg·mL −1 [31]. Hence, the results obtained for the aqueous ammonia extract under study (with EC 90 values between 75 and 322 µg·mL −1 ) are among the lowest reported for Quercus spp. and may be regarded as very promising, especially in the case of P. cinnamomi. Table 3 summarizes a literature search on the inhibitory values of natural compounds evaluated against F. circinatum, C. parasitica, and P. cinnamomi with those obtained in this investigation. The MIC values given below should be taken with caution, given that the susceptibility profile depends on the isolates as well as on the testing methods used, and provided that units substantially differ (e.g., when essential oils are used as biofumigants, MICs are expressed in µg·mL −1 air, and the MICs of compounds tested by the agar/liquid dilution methods are expressed in µg·mL −1 ).   In the case of F. circinatum, the activity of the aqueous ammonia extract of Q. ilex subsp. ballota would be higher than those of cinnamon, fennel, clove [32], thyme, rose geranium, and lemon grass essential oils [33] (assuming that many essential oils have a density somewhere in the vicinity of 0.9 g·mL −1 ). As noted above, lower MIC values reported by Lee et al. [34] and Lee et al. [35] may not be compared, as in those studies the essential oils were used as biofumigants. The same applies to the MIC values reported by Lee et al. [34], Lee et al. [35], Lukovic et al. [36], and Kim et al. [37] against C. parasitica.

Comparison of Efficacy vs. Other Natural Compounds
As for P. cinnamomi, holm oak bark extract activity would be lower than that of ethanolic plant extracts of Larrea tridentata  [38], and higher than those of, for example, the most effective essential oils reported by Giamperi et al. [39] (those of Origanum vulgare L. and Thymus vulgaris L., with MICs ≥ 200 µg·mL −1 ) and of the essential oil of aerial parts of Beilschmiedia miersii (Gay) Kosterm. (MIC = 300 µg·mL −1 ) [40].

Comparison with a Conventional Fungicide
Concerning the chemical fungicide chosen as a reference, viz. azoxystrobin, it is one of the world's biggest selling fungicides [43]. Data on its efficacy against the three phytopathogens under study is scarce in the literature. Benalcázar Villalba [44] reported EC 50 values in the 49.8-263.8 µg·mL −1 range against Fusarium spp. associated with the death of Pinus radiata D. Don. and P. patula Schiede ex Schltdl. and Cham. seedlings in the nursery, and González-Varela and González [45] found that it was unable to stop C. parasitica growth at doses in the 1-16 µg·mL −1 range.
Based on the tests conducted in this study at the recommended dose (62.5 mg·mL −1 ), at which full inhibition was not reached, it may be inferred that the activity of the holm oak bark extract would be much higher (with EC 90 values ranging from 75 to 322 µg·mL −1 ).

Mode of Action
To provide a tentative explanation of the observed antifungal and anti-oomycete activity, a description of each of the main extract constituents and their previously reported activities is first presented. Inositol is a group of nine stereoisomers, but the name is usually used to describe the most common type of inositol, myo-inositol (cis-1,2,3,5-trans-4,6-cyclohexanehexol), considered a pseudovitamin. It has been reported to be an efficient adjuvant to antibiotic drugs, increasing their antimicrobial activity [46]. In a recent study, Ratiu et al. [47] examined 40 different plant species that contained variable quantities of myo-inositol. The fruits of blueberries, lettuce, and cinnamon had the greatest concentrations (0.96, 1.07, and 1.21 mg·g −1 dry plant material, respectively). Supraene (trans-squalene) is a linear triterpene synthesized in plants, animals, bacteria, and fungi as a precursor for the synthesis of secondary metabolites such as sterols, hormones, or vitamins. It is known to have active oxygen-scavenging activities, preventing oxidative damage. Concerning its fungicidal action, also reported for other supraene-rich natural products (summarized in Table S2 [48][49][50][51]), it is known that the intracellular accumulation of squalene disrupts fungal cell membranes, possibly by forming squalene vesicles that weaken fungal cells by extracting essential membrane lipid components [52]. In fact, the mechanism of action of terbinafine and other antifungal drugs is based on the inhibition of squalene peroxidase, resulting in the aforementioned squalene accumulation [53]. The dialkyl ether 4-butoxy-1-butanol has been identified in benzene/alcohol extractives of Artocarpus lingnanensis Merr. [54]. As regards 2,4-dimethyl-benzo[H]quinolone, it should exhibit antifungal activity, like other benzoquinoline derivatives [55]. Methoxyphenols as 2-methoxyphenol, 2,6-dimethoxyphenol, and 3,4,5-trimethoxy-phenol are among the phenolic compounds produced by alkaline destruction of poplar wood bark and exhibit antioxidant properties [56] and anti-quorum sensing effects [57,58]. For the minority component 2-hydroxy-2-cyclopent-2-en-1-one, Naika and Pavani [59] advocated the existence of antibacterial activity.
Based on the above-discussed activities and taking into consideration the EC 90 values for the two main constituents of the aqueous ammonia extract (between 321 and 710 µg·mL −1 for myo-inositol and ranging from 88 to 174 µg·mL −1 for squalene), the antimicrobial activity of the extract may be mainly ascribed to the trans-squalene content, whereas inositol would have a weaker activity and would act as an adjuvant of squalene. Nonetheless, contributions from other minor constituents such as 2,4-dimethyl-benzo[H]quinoline and 3,4,5-trimethoxyphenol cannot be ruled out (and their possible synergies may account for the higher efficacy of the bark extract against P. cinnamomi in comparison to that of pure squalene). Further research on the activity of these phytochemicals and their combinations is needed to elucidate the mechanism of action of the bark extract. The commercial fungicide used for comparison purposes, viz. Ortiva ® (azoxystrobin 25%; reg. no. 22000; Syngenta, Basel, Switzerland) was kindly provided by the Plant Health and Certification Service (CSCV) of Gobierno de Aragón (Zaragoza, Spain). This fungicide was selected due to its low risk of resistance, widespread use, favorable toxicological and environmental profile, and significant pathogen control ability.

Plant Material
The holm oak bark was torn from an old specimen from the holm oak forest of Nisano (Huesca, Spain). The sample was shade-dried and pulverized to a fine powder in a mechanical grinder.

Preparation of the Extract
An aqueous ammonia solution was chosen to dissolve the bioactive compounds of interest contained in the bark of holm oak. The bark extract was prepared according to the procedure described in [60]. Briefly stated, a probe-type ultrasonicator (model UIP1000hdT; 1000 W, 20 kHz; Hielscher Ultrasonics, Teltow, Germany) was used to sonicate the bark sample for 10 min in pulse mode with a 2 min break after every 2.5 min of sonication, and the sample was then left to settle for 24 h. Acetic acid was then used to change the pH to neutral. After 15 min of centrifuging the solution at 9000 rpm, the supernatant was filtered using Whatman No. 1 paper.

Characterization of the Extract
The aqueous ammonia extract of oak bark was analyzed by gas chromatography-mass spectrometry (GC-MS) at the Research Support Services (STI) at the University of Alicante (Alicante, Spain), using a gas chromatograph model 7890A coupled to a quadrupole mass spectrometer model 5975C (both from Agilent Technologies, Santa Clara, CA, USA). The operating conditions were: injector temperature = 280 • C, splitless mode; injection volume = 1 µL; initial temperature = 60 • C, 2 min, followed by a ramp of 10 • C·min −1 to a final temperature of 300 • C, 15 min. The chromatographic column used for the separation of the compounds was an Agilent Technologies HP-5MS UI of 30 m in length, 0.250 mm diameter, and 0.25 µm film. The mass spectrometer conditions were: mass spectrometer electron impact source temperature = 230 • C and quadrupole temperature = 150 • C; ionization energy = 70 eV. Test mixture 2 for apolar capillary columns according to Grob (Supelco 86501) and PFTBA tuning standards were used for calibration, purchased from Sigma Aldrich Química S.A. (Madrid, Spain). Comparison of mass spectra and retention times with those of reference compounds and computer matching with the databases of the National Institute of Standards and Techniques (NIST11) and the monograph by Adams [61] were used for compound identification.

In Vitro Antimicrobial Activity Assessment
The antimicrobial activity of the treatment was assessed according to EUCAST standard antifungal susceptibility testing techniques [62], using the agar dilution method. Aliquots of stock solution were mixed into a pouring PDA medium to obtain concentrations ranging from 62.5 to 1500 µg·mL −1 (albeit lower concentrations of 15.62 and 31.25 µg·mL −1 also had to be evaluated for the most efficient treatments to obtain reliable PROBIT fittings). Plugs (Ø = 5 mm) from the margins of one-week-old PDA cultures of F. circinatum, C. parasitica, and P. cinnamomi were transferred to plates integrating the above treatment concentrations (three plates per treatment and concentration, with two duplicates). Plates were incubated at 25 • C in the dark for one week. PDA media without any modification was employed as a control. Growth inhibition was estimated according to the formula ((d c − d t )/d c ) × 100, where d c and d t indicate the mean diameters of the control and treated colonies, respectively. The effective concentrations (EC 50 and EC 90 ) were determined using PROBIT analysis in IBM SPSS Statistics v.25 software (IBM; Armonk, NY, USA).

Protection Tests on Artificially Inoculated Excised Stems
Given the restrictions that apply to in vivo assays involving P. cinnamomi, the efficacy of the treatment was tested by artificial inoculation of excised stems in controlled laboratory conditions. Inoculation was performed according to the procedure proposed by Matheron [63], with modifications. Using a grafting knife, young stems of healthy 'Garnem' (Prunus amygdalus × P. persica) rootstock with a 1.5 cm diameter were cut into 10 cm-long sections. The excised stem pieces were immediately wrapped in moistened sterile absorbent paper, and the wounds produced were painted with Mastix ® .
In the laboratory, the freshly excised stem segments were first immersed in ethanol for 1 min, then immersed in a NaClO solution for 8 min, and then thoroughly rinsed with distilled water [64]. Some of the stem segments (n = 15) were soaked for 1 h in distilled water as a control, and the remaining stem segments were soaked for 1 h in aqueous solutions to which an appropriate amount of the bark extract had been added to obtain MIC, MIC × 5, and MIC × 10 concentrations (n = 15 segments/concentration). Alkir ® co-adjuvant (1% v/v) was added to all solutions (including the control) to facilitate bark penetration of the treatment.
The stem pieces were allowed to dry, placed on an agar Petri dish, and subsequently inoculated by placing a plug (Ø = 5 mm) from the margin of one-week-old PDA cultures of P. cinnamomi on the center of the outer surface of the bark. After inoculation, the stem segments were incubated in a humid chamber for 4 days at 24 • C, 95-98% RH.
The efficacy of the treatments was assessed by visual inspection of the presence of rotting at the inoculation sites, confirmed under the microscope, both on the outer bark and on the inner bark after careful removal with a scalpel to reveal the cambium. Then, the oomycete was re-isolated and morphologically identified from the lesions to fulfill Koch's postulates.

Statistical Analysis
The results of the postharvest protection study were statistically analyzed in IBM SPSS Statistics v.25 software by analysis of variance (ANOVA), followed by a post hoc comparison of means by Tukey's test (because the requirements of homogeneity and homoscedasticity were met, according to the Shapiro-Wilk and Levene tests).

Conclusions
In vitro tests on the antifungal and anti-oomycete activities of the aqueous ammonia extract of holm oak bark aimed at controlling important forest phytopathogens such as F. circinatum, C. parasitica, and P. cinnamomi, have resulted in EC 90 values of 322, 295, and 75 µg·mL −1 , respectively. Such activity, which may be ascribed to trans-squalene content (13%), was shown to be much higher than that of azoxystrobin, tested for comparison purposes, and higher than most of those reported in the literature for other natural products against these forest pathogens. Although a higher dose (782 µg·mL −1 ) was needed to attain full inhibition in further tests conducted on excised almond 'Garnem' stems artificially infected with P. cinnamomi, the results suggest that Q. ilex subsp. ballota bark extract may be a promising source of bioactive compounds against phytopathogens.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to their relevance to an ongoing Ph.D. thesis.