Antibacterial Activity of Ginkgo biloba Extracts against Clavibacter michiganensis subsp. michiganensis, Pseudomonas spp., and Xanthomonas vesicatoria

Abstract

Phytopathogenic bacteria represent a risk to global food production by impacting a variety of crops. The aim of this study was to characterize the contents of bioactive constituents in extracts from Ginkgo biloba L. leaves and fruits and test their activity against six phytopathogenic bacteria that affect horticultural crops. Gas chromatography–mass spectrometry (GC−MS) was used for the chemical profiling of the aqueous methanol extracts, and their bacteriostatic activity against Clavibacter michiganensis subsp. michiganensis, Pseudomonas cichorii, Pseudomonas syringae pv. pisi, Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. tomato, and Xanthomonas vesicatoria (formerly Xanthomonas campestris pv. vesicatoria) was determined in vitro through the agar dilution method. The protective effect of the leaf extract was tested in vivo against the most relevant bacterial pathogens based on their economic/scientific importance, i.e., C. michiganensis subsp. michiganensis and P. syringae pv. pisi, in tomato (Solanum lycopersicum L.) and pea (Pisum sativum L.) plants, respectively, under greenhouse conditions. The GC−MS characterization of G. biloba extracts revealed the presence of dihydro-4-hydroxy-2(3H)-furanone, 2,4-dimethyl-3-hexanol, catechol, 3-O-methyl-D-fructose, 4,6-di-O-methyl-α-D-galactose, methyl 2-O-methyl-α-D-xylofuranoside, and 3-methyl mannoside. In vitro growth inhibition tests showed that, while the fruit extract had no activity, the leaf extract exhibited minimum inhibitory concentrations between 500 and 1000 μg mL⁻¹, which may be attributed to the presence of 2,4-dimethyl-3-hexanol and catechol. In vivo tests of the leaf extract demonstrated full protection in tomato and pea plants at 1000 and 1500 μg∙mL⁻¹, respectively. The results indicate that G. biloba leaves may be employed as a biorational source for integrated pest management in horticulture.

1. Introduction

Ginkgo biloba L. is a tree species belonging to the family Ginkgoaceae and the only living species of the Ginkgoales order. Ginkgo biloba trees are disease- and insect-resistant, making them exceptionally long-lived, with some specimens reportedly existing for over 2500 years. They generally reach a height of 20 to 35 m, possess an angular crown, and typically feature deep roots. The leaves are petiolated, with a fan-shaped blade and dichotomous and open venation. It is a dioecious species, where female trees produce fruit each year, while male trees do not (Figure 1). The fruits emit an unpleasant, musty odor due to the presence of butyric acid in the yellowish, fleshy part (sarcotesta), which is not consumed as it can be toxic; rather, the inner, softer portion of the seed (sclerotesta)—which has a greenish color—is eaten.

Ginkgo biloba is a widely used plant remedy throughout the world [1]. Its potential benefits for cardiovascular and neurodegenerative diseases have been substantiated [2,3]. Furthermore, the ginkgo nut has demonstrated potential in the prevention of neurological and cardiovascular diseases, though research in this area is limited [4].

Back in 1992, Gülz et al. initially documented the chemical composition of the waxes of G. biloba leaves [5], but it was not until 2008 that Lin et al. provided a more extensive report on the phytoconstituents of the aqueous methanol extract of the leaves, including forty-five glycosylated flavones and flavonols (predominantly hydroxyflavones), ten biflavones, three flavonol aglycones (myricetin, quercetin, and kaempferol), catechin, dihydroxybenzoic acid, and terpene lactones (ginkgolides and bilobalide, Figure 2) [6].

Regarding standardized G. biloba extract EGb 761 (a leaf extract frequently utilized as a dietary supplement), it is typically composed of flavone glycosides (24%), terpenes (6%), and other substances, such as proanthocyanidins and organic acids. Ginkgolides and bilobalide account for approximately 3.1% and 2.9% of the extract, respectively [7]. It has been suggested that some of the beneficial effects of EGb may be due to antioxidant activities, with an increasing body of evidence pointing to lipid peroxidation reactions and free radicals’ participation in neuronal damage and central and peripheral vascular diseases [8,9].

Ginkgo biloba has been reported to possess antifungal activity by Krauze-Baranowska and Wiwart [10] and Oh et al. [11], and antibacterial activity against Staphylococcus aureus Rosenbach, Bacillus thuringiensis Berliner, Clostridium perfringens (Veillon and Zuber, 1898) Hauduroy et al., 1937, Listeria monocytogenes (Murray et al., 1926) Pirie, 1940, and Haemophilus influenzae (Lehmann and Neumann, 1896) Winslow et al., 1917 [12]. However, to the best of our knowledge, no prior studies have been conducted to assess the antibacterial activity of this plant species on phytopathogenic taxa.

Given the significant global economic losses caused by bacterial diseases of plants—estimated at over one billion dollars worldwide annually [13]—there is an interest in investigating biorationals based on natural products for the integrated pest management of phytopathogenic bacteria that affect horticultural crops. For instance, these products could be used to control Gram-negative bacteria such as Pseudomonas syringae van Hall pathovars (e.g., Pseudomonas syringae pv. pisi (Sackett 1916) Young et al. 1978 Dye & Wilkie; Pseudomonas syringae pv. syringae van Hall 1902; Pseudomonas syringae pv. tomato (Okabe 1933) Young et al. 1978), Pseudomonas cichorii (Swingle 1925) Stapp, 1928; and Xanthomonas vesicatoria (Doidge) Vauterin et al., as well as Gram-positive bacteria such as Clavibacter michiganensis subsp. michiganensis corrig. (Smith 1918) Davis et al. 1984. Among these, P. syringae [14] and P. cichorii [15] can infect a wide range of plant species, while X. vesicatoria (formerly X. campestris pv. vesicatoria) is the causal agent of bacterial leaf spot on tomatoes (Solanum lycopersicum L.) and peppers (Capsicum annuum L.) [16] and C. michiganensis subsp. michiganensis causes bacterial canker of tomatoes, and can also affect other solanaceous plants [17]. It should be noted that P. syringae pathovars and C. michiganensis subsp. michiganensis are considered among the more relevant bacterial pathogens based on their scientific/economic importance [18].

The purpose of this study was twofold: (i) to characterize the phytoconstituents of Ginkgo biloba L. leaf and fruit extracts and (ii) to investigate their activity against the aforementioned six phytopathogenic bacteria that affect horticultural crops.

2. Materials and Methods

2.1. Plant Materials and Reagents

Ginkgo biloba leaf and fruit samples were collected from 20 female trees—grown under the same environmental conditions, soil type, and agronomic factors—along the Avenida de los Monegros in Huesca City, Aragon, northeastern Spain (42°7′46.9″ N 0°24′6.3″ W) in the late days of September 2021. For drying the samples, the freeze-drying method was chosen, in agreement with Boateng and Yang [19]: samples were initially frozen at −20 °C for 24 h and then freeze-dried at −50 °C.

Eight-week-old tomato (Solanum lycopersicum) cv. “Optima F1” plants and fourteen-week-old pea (Pisum sativum) cv. “Lincoln” plants used in the in vivo experiments were supplied by Agrodepa S.L. (Palencia, Spain).

Catechol (CAS no. 120-80-9) was acquired from Thermo Fisher Scientific (Madrid, Spain). Additionally, 2,4-dimethyl-3-hexanol (CAS no. 13432-25-2), tryptic soy broth (TSB, no. CAS 8013-01-2), and tryptic soy agar (TSA, CAS no. 91079-40-2) were procured from Sigma–Aldrich Química (Madrid, Spain).

2.2. Bacterial Strains

Pseudomonas cichorii (strain CITA Pci-5), P. syringae pv. pisi (strain CITA Pspi-12), P. syringae pv. syringae (strain CITA Psy-9), P. syringae pv. tomato (strain CITA Pst-7), X. vesicatoria (strain CITA Xv-5), and C. michiganensis subsp. michiganensis (strain CITA Cmm-30) were supplied by the Bacteriology Lab at the Agrifood Research and Technology Center of Aragon (CITA, Zaragoza, Spain) as TSA subcultures.

2.3. Extract Preparation

The preparation of the leaf, sarcotesta, and sclerotesta extracts was carried out according to the methodology described in [20]; the powdered sample was digested in a methanol/water (1:1, v/v) solution for 2 h, sonicated for 10 min in pulsed mode using a model UIP1000hdT probe-type ultrasonicator (20 kHz, 1000 W; Hielscher Ultrasonics, Teltow, Germany), and allowed to stand for 24 h. The solution was then centrifuged at 9000 rpm for 15 min, and the supernatant was filtered using Whatman No. 1 paper. Samples of the extracts were freeze-dried to obtain the solid residue. For gas chromatography–mass spectrometry (GC–MS) analyses, 25 mg of the freeze-dried extracts were dissolved in 5 mL of methanol (HPLC grade) to yield a 5 mg·mL⁻¹ solution, which was filtered before use.

2.4. Extract Characterization

The extracts were analyzed using GC–MS at the Research Support Services at the University of Alicante (Alicante, Spain). The equipment utilized was a model 7890A gas chromatograph coupled to a model 5975C quadrupole mass spectrometer (Agilent Technologies; Santa Clara, CA, USA). The conditions for chromatography included 3 injections per vial, 1 µL injection volume, an injector temperature of 280 °C, and an initial oven temperature of 60 °C. The temperature then increased at a rate of 10 °C·min⁻¹ to a final temperature of 300 °C, which was held for 15 min. The chromatographic column employed was an Agilent Technologies HP-5MS UI 30 m in length, 0.250 mm in diameter, and with a 0.25 µm film. The temperatures of the electron impact source of the mass spectrometer and of the quadrupole were set at 230 and 150 °C, respectively; ionization energy was set at 70 eV. For calibration, test mixture 2 for apolar capillary columns according to Grob and PFTBA tuning standards were used. Component identification was based on a comparison of their mass spectra and retention time to those of authentic compounds, and by computer matching using the National Institute of Standards and Technology (NIST11) database.

The infrared spectrum of the dried powdered leaves was acquired using a Nicolet iS50 Fourier-transform infrared spectrometer (Thermo Scientific; Waltham, MA, USA) with a diamond attenuated total reflectance system. The spectra were collected in the 400–4000 cm⁻¹ range, with a 1 cm⁻¹ spectral resolution, by co-adding 64 scans.

2.5. In Vitro Antibacterial Activity Assessment

The bacteriostatic activity was assessed according to the Clinical and Laboratory Standards Institute (CLSI) standard M07–11 [21], using the agar dilution method to determine the minimum inhibitory concentrations (MICs). Colonies of each species were incubated at 28 °C for 24 h in TSB. Serial dilutions of the cell suspension were then performed, beginning from a concentration of 10⁸ CFU·mL⁻¹, to obtain a final inoculum of 10⁴ CFU·mL⁻¹. The bacterial suspensions were then applied to the surface of the TSA plates to which the treatments had previously been added at concentrations between 62.5 and 1500 μg·mL⁻¹. The plates were incubated at 28 °C for 24 h, and readings were taken after 24 h. MICs were determined as the lowest concentrations of the bioactive products at which no bacterial growth was visible. All experiments were conducted in triplicate, with 3 plates per treatment/concentration. Statistical analyses of antibacterial activity data were conducted using IBM (Armonk, NY, USA) SPSS Statistics v.25.

For comparison, the MIC values of five antibiotics commonly used in clinical practice, viz. ampicillin (AM), benzylpenicillin or penicillin G (PG), ciprofloxacin (CI), gentamicin (GM), and tetracycline (TC), were determined using ETEST^® strips.

2.6. In Vivo Experiments on Tomato and Pea Plants under Greenhouse Conditions

Tomato plants cv. “Optima F1” were subjected to experiments in accordance with the guidelines in [22], with a few minor modifications. In a greenhouse cabin of biological containment level 2 (NCB2), tomato plants were grown in multi-pots (4 × 4 cm per pot) containing sterilized peat as substrate, at 20 ± 2 °C day/17 ± 2 °C night, with supplementary lighting to give a 12 h day. The last irrigation was carried out 48 h before inoculation, at which time the relative humidity was set at 100%. The G. biloba leaf extract was applied through spraying (3 mL per plant) at two concentrations (MIC and MIC × 2, i.e., 500 and 1000 μg·mL⁻¹, respectively, taking into consideration that some of the treatment may be lost due to evaporation, runoff, degradation, etc., thus necessitating a higher concentration to achieve the same effect as in vitro); control plants were sprayed with water. After two hours, an isolated colony of C. michiganensis subsp. michiganensis was inoculated to the node of the first true leaf with the stem, with the aid of a sterile entomological pin. The plants were then covered with polyethylene bags for five days, and the next irrigation was carried out 24 h after inoculation. Throughout the experiment, the plants were irrigated three times a week. For the pea assay, the experiments were carried out according to the guidelines in [23]. Pea plants were cultivated under the aforementioned greenhouse conditions in 4 × 4 cm pots that contained a mixture of sterilized peat and sand (2:1). The G. biloba leaf extract was sprayed at two concentrations (MIC and MIC × 2, i.e., 750 and 1500 μg·mL⁻¹, respectively) onto treated plants, and control plants were sprayed with water. Two hours after treatment, an isolated colony of P. syringae pv. pisi was inoculated using a sterile entomological pin, by pricking the back of the stem at the two youngest nodes of the plant, at its junction with the stipula. Inoculation of the leaflets or stipules took place at the same time as that of the stems, with 4–8 pricks in the youngest leaflets and stipules. The irrigation regime was similar to that described above for tomato plants.

In both experiments, five plants per treatment (negative control, positive control, MIC, and MIC × 2) were used, with two independent replicates. In regard to the tomato plant trials, the first replicate began on 31 January 2023 and concluded on 10 February 2023; the second replicate began on 3 February 2023, ending on 13 February 2023. Regarding the pea plant trials, the first replicate started on 27 January 2023, concluding on 6 February 2023. The second replicate started on 30 January 2023, ending on 9 February 2023. Concerning the experimental layout, a completely randomized design was used, where each experimental unit (plant) was randomly assigned to one of the treatments.

Disease incidence was evaluated after ten days of inoculation, using the following formula: I (%) = (Number of plants with symptoms/Total number of plants assayed) × 100.

3. Results

3.1. Extract Phytoconstituents Elucidation by GC−MS

In the GC−MS analysis of the leaf extract (Figure S1, Table S1, Figure 3a), the main identified compounds were: 3-O-methyl-D-fructose (18.1%); 2,4-dimethyl-3-hexanol (18.1%); acetic acid (16.2%); 3-methylmannoside (9.8%); acetic acid, 4-methylphenyl ester (4.4%); catechol (3.3%); 1-hydroxy-2-propanone (2.4%); and dihydro-4-hydroxy-2(3H)-furanone or β-hydroxy-γ-butyrolactone (1.2%).

Regarding the sarcotesta extract chromatogram (Figure S2, Table S2, Figure 3b), the following phytoconstituents were detected: acetic acid (11.9%); 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one and related compounds (8.7%); formic acid 2-methylpropyl ester and related compounds (5.4%); 1-hepten-4-ol (4.5%); catechol (4.1%); dihydroxyacetone (3.9%); 5-hydroxymethylfurfural (3.6%); 1-hydroxy-2-propanone (3.3%); 2-hydroxy-2-cyclopenten-1-one (3.1%); 4,6-di-O-methyl-α-D-galactose (1.6%); 4,5-dihydro-2-methyl-1H-imidazole (1.3%); 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one (1.3%); 2,5-dimethyl-4-hydroxy-3(2H)-furanone (1.0%); α-methyl mannofuranoside (1.3%); and azelaic acid (1.1%).

Lastly, GC−MS analysis of the sclerotesta extract (Figure S3, Table S3, Figure 3c) yielded the following main constituents: methyl 2-O-methyl-α-D-xylofuranoside (19.1%); dihydro-4-hydroxy-2(3H)-furanone (11.7%); 4,6-di-O-methyl-α-D-galactose (8.4%); catechol (7.9%); 3-methylmannoside (6.3%); 3-O-methyl-fructose (6.1%); tetrahydro-2-methyl-thiophene (3%); and 2-furanmethanol (2.5%).

3.2. Leaves Vibrational Spectra

Ginkgo biloba leaves showed absorption bands at 3309, 2915, 2849, 1731, 1712, 1630, 1605, 1515, 1465, 1371, 1243, 1166, 1104, 1020, and 832 cm⁻¹. The intense band at 3309 cm⁻¹ was attributed to OH stretching, while the one at 2915 cm⁻¹ was associated with methylene (CH₂) stretching, and 2849 cm⁻¹ with symmetric vibration of CH₂ groups. The bands at 1731 and 1712 cm⁻¹ were likely due to C = O stretching in unconjugated ketones, esters (cutin), and aldehydes (hemicelluloses); the one at 1605 cm⁻¹, to C = C aromatic skeletal vibrations (syringyl units); and the one at 1515 cm⁻¹, to aromatic skeletal vibrations (guaiacyl rings). The band at 1020 cm⁻¹ can be ascribed to the C–O stretching of secondary alcohols (in cellulose and lignin), despite the absence of the cellulose bands at 1032 cm⁻¹ and 1050 cm⁻¹ (assigned by [24] to the C–O stretching of secondary and primary alcohols, respectively). Other band assignments were: 1630 cm⁻¹ to carbonyl groups of unsaturated acids; 1465 cm⁻¹ to CH₂ in-plane bending; 1371 cm⁻¹ to CH₂ wagging in lipids and β-1,3 glucans; 1243 cm⁻¹ to cutin and other polysaccharides; 1166 and 1104 cm⁻¹ to asymmetric and symmetric C–O–C in esters (cutin, cellulose, pectin); and 832 cm⁻¹ to C–C or C–N ring stretching or to C–H, O–H, and C = O deformation.

3.3. Antibacterial Activity of the Extracts

The results of the bacterial growth inhibition tests of the G. biloba extracts against C. michiganensis, P. cichorii, the three pathovars of P. syringae, and X. vesicatoria are summarized in

Table 1. Minimum inhibitory concentrations (expressed in μg∙mL⁻¹) of G. biloba extracts against the horticultural pathogens.

Bacteria	Leaf Extract	Sarcotesta Extract	Sclerotesta Extract
Clavibacter michiganensis subsp. michiganensis	500	n.a.	n.a.
Pseudomonas cichorii	750	n.a.	n.a.
Pseudomonas syringae pv. pisi	750	n.a.	n.a.
Pseudomonas syringae pv. syringae	750	n.a.	n.a.
Pseudomonas syringae pv. tomato	750	n.a.	n.a.
Xanthomonas vesicatoria	1000	n.a.	n.a.

n.a.: no activity detected at the highest assayed dose.

. The hydromethanolic fruit (sarcotesta and sclerotesta) extracts were the least effective, with MIC values above 1500 μg·mL⁻¹. The leaf extract showed higher activity, with MIC values ranging from 500 to 1000 μg·mL⁻¹.

Table 2. Minimum inhibitory concentrations (expressed in μg∙mL⁻¹) of two G. biloba extract phytoconstituents against the horticultural pathogens.

Bacteria	2,4-Dimethyl-3-Hexanol	Catechol
Clavibacter michiganensis subsp. michiganensis	375	187.5
Pseudomonas cichorii	750	500
Pseudomonas syringae pv. pisi	750	375
Pseudomonas syringae pv. syringae	750	500
Pseudomonas syringae pv. tomato	500	500
Xanthomonas vesicatoria	250	187.5

presents the results of the bacteriostatic activity tests for two of the extract phytoconstituents (2,4-dimethyl-3-hexanol and catechol).

For comparison purposes, the MIC values of the five antibiotics commonly used in clinical practice (namely, benzylpenicillin, ampicillin, gentamicin, ciprofloxacin, and tetracycline) are provided in

Table 3. Minimum inhibitory concentrations (expressed in μg∙mL⁻¹) of conventional antibiotics (for clinical use) against the horticultural pathogens.

Bacteria	PG	AM	GM	CI	TC
Clavibacter michiganensis subsp. michiganensis	0.125	0.094	0.38	0.38	0.19
Pseudomonas cichorii	≥32	≥256	3	6	24
Pseudomonas syringae pv. pisi	≥32	48	1	0.25	1
Pseudomonas syringae pv. syringae	≥32	48	0.25	0.047	1
Pseudomonas syringae pv. tomato	≥32	16	0.125	0.047	0.75
Xanthomonas vesicatoria	≥32	≥256	0.75	0.125	1

PG: penicillin G; AM: ampicillin; GM: gentamicin; CI: ciprofloxacin; TC: tetracycline.

.

3.4. Tomato Plant Protection against C. michiganensis subsp. michiganensis

Given that G. biloba leaf extract was the most active in the in vitro tests, it was further tested as a protective treatment against bacterial canker in tomato plants cv. “Optima F1” (Figure 4). In the positive control (C. michiganensis subsp. michiganensis artificially inoculated on plants treated with bidistilled water only), all plants exhibited a typical vascular discoloration as brown streaks on the stem (Figure 4b). Plants treated with the extract at a concentration equal to the MIC (500 μg∙mL⁻¹) showed an incidence of 80%, although the stem discoloration was less pronounced compared to the positive control. On the other hand, plants treated with the highest concentration (twice the MIC, i.e., 1000 μg∙mL⁻¹) did not show symptoms of stem decay or phytotoxicity, appearing entirely similar to those of the negative control.

3.5. Pea Plant Protection against P. syringae pv. pisi

Figure 5 shows the results obtained for pea plants cv. “Lincoln” treated with different concentrations of G. biloba leaf extract, particularly MIC and MIC × 2 (i.e., 750 and 1500 μg∙mL⁻¹, respectively). The positive control showed a high susceptibility to bacterial blight, with an incidence of 100% of plants showing the characteristic brown and papery leaflet lesions. The plants treated with the extract at 750 μg∙mL⁻¹ exhibited a relatively lower susceptibility, with an incidence of 60% (i.e., 6 plants out of the 10 tested showed symptoms, albeit with less virulence than that of the positive control). In order to provide full protection, the dose had to be increased up to 1500 μg∙mL⁻¹. As in the case of tomato plants, no phytotoxicity symptoms were observed, with no visual differences between the negative control and the MIC × 2 treated plants.

4. Discussion

4.1. On the Identified Phytochemicals and Their Antimicrobial Activity

The main phytochemicals identified in G. biloba hydromethanolic extracts (Figure 3) were: dihydro-4-hydroxy-2(3H)-furanone (present in both leaf and sclerotesta extracts); 2,4-dimethyl-3-hexanol; catechol (present in the three extracts); 1-hydroxy-2-propanone; 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one; acetic acid (identified both in leaf and sarcotesta extracts); formic acid 2-methylpropyl ester; 1-hepten-4-ol; 3-O-methyl-D-fructose (present in both leaf and sclerotesta extracts); 4,6-di-O-methyl-α-D-galactose; 2-O-methyl-α-D-xylofuranoside; and 3-methylmannoside (present in both leaf and sclerotesta extracts). Their occurrence in other extracts, together with available information on their antimicrobial activity, is summarized below.

Dihydro-4-hydroxy-2(3H)-furanone was previously identified in Equisetum arvense L. and Urtica dioica L. extracts [25]. The presence of this phytochemical in G. biloba hydromethanolic extracts may be the result of the hydrolysis/breaking of ginkgolide and bilobalide components (Figure 2) [26]. Dihydro-4-hydroxy-2(3H)-furanone is a major constituent in the extract of Crocus sativus L. (22% content), which exhibited antifungal activity against Pyricularia oryzae Cavara, Cryptococcus neoformans (Sanfelice) Vuill., Trichophyton rubrum (Castell.) Sabour., and Aspergillus fumigatus Fresen. [27]. 2,4-dimethyl-3-hexanol, identified in leaf extracts of Silene uniflora Roth and Pentatropis microphylla (Roxb.) Wight & Arn. [28], has been shown to possess considerable biocontrol activity against Aspergillus flavus Link and the generation of aflatoxin in stored rice grains [29,30]. In the activity assays presented here (Table 2), 2,4-dimethyl-3-hexanol yielded MIC values in the 250 to 750 μg·mL⁻¹ range.

The presence of catechol should be attributed to the rupture of catechin (a constituent identified by Lin et al. [6] in the G. biloba leaves aqueous methanol extract) (Figure 2). Catechol had previously been identified in the extracts of Taxus baccata L. [31], Allium sativum L. [32], Sambucus nigra L. [33], and Quercus ilex L. [34], with concentrations ranging from 1.6 to 6.4% (compared to 3.3−7.9% in the extracts reported herein). Catechol oxidizes to melanoid pigments that are derivatives of benzoquinone, which is reported to have antimicrobial properties, slowing the decay of damaged fruits and other parts of plants. Catechol has been found to have antifungal activity against phytopathogenic taxa such as Penicillium italicum Wehmer and Fusarium oxysporum Schlecht. [35]. In our assays, catechol showed MIC values ranging from 187.5 to 500 μg·mL⁻¹ (Table 2).

1-hydroxy-2-propanone is an alpha-hydroxy ketone found, for instance, in T. baccata leaf aqueous ammonia extracts [31]. Hydroxyacetone has also been detected, but not quantified, in several different edible plants, such as black cabbages, pulses, mentha, onion family vegetables, and rape. Additionally, it was identified (alongside acetic acid) as a major constituent of bioactive metabolites from tomato endophytic fungi, which had antibacterial activity against X. vesicatoria [36]. Furthermore, asymmetric reduction of hydroxyacetone leads to (R)-1,2-propanediol, a building block for antibacterial agents [37], and methylglyoxal—produced non-enzymatically from dihydroxyacetone—exhibits high non-peroxide antibacterial activity [38].

2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one was identified in Hibiscus syriacus L. [39], S. nigra [33], Punica granatum L. [40], and T. baccata [31]. In terms of antifungal activity, it is effective against Phytophthora megasperma Drechsler, Verticillium dahliae Kleb., and Diaporthe amygdali (Delacr.) Udayanga, Crous & K.D. Hyde, with MIC values between 375 and 750 μg mL⁻¹ [33].

The presence of acetic acid in contents higher than those detected herein has been reported, for instance, in Artocarpus altilis (Parkinson) Fosberg 1941 methanolic extracts (34.7%) [41]. In the particular case of G. biloba, its presence has been documented in ginkgo vinegar (obtained from the ginkgo seed coat by fermentation) [42]. Research has proven that it has good antibacterial activity against Pseudomonas aeruginosa (J.Schröter) Migula [43] at 1660 μg·mL⁻¹. Concerning formic acid 2-methylpropyl ester (or isobutyl formate), it belongs to a family of esters which provide a sweet, floral, and fruity odor and flavor to foods and beverages, without any known antimicrobial properties.

1-hepten-4-ol was detected in leaf and bark extracts of Moringa concanensis Nimmo [44] and is a volatile constituent of Litchi chinensis Sonn. [45] and Aspalathus linearis (Burm.f.) R.Dahlgren [46]. It was tested against two barley fungal pathogens, Fusarium culmorum (Wm.G.Sm.) Sacc. and Cochliobolus sativus (S.Ito & Kurib.) Drechsler ex Dastur, but demonstrated low activity [47].

3-O-methyl-D-fructose has been found to be present in Ichnocarpus frutescens (L.) W.T.Aiton [48] and in T. baccata [31], and reports have indicated its antibacterial activity [49]; 4,6-di-methyl-α-D-galactose has been found in the Siberian ginseng Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. [50]; and 2-O-methyl-α-D-xylofuranoside has been reported in Alternanthera sessilis (L.) DC. [51] and T. baccata [31]. Additionally, 3-methylmannoside has been found in Psidium guajava L. [52]. None of these polysaccharides have been identified as bactericides. Nonanedioic acid (azelaic acid), however, has been shown to have an inhibitory effect on common dermatophytes, such as Scopulariopsis brevicaulis (Sacc.) Brainier, Candida glabrata (H.W.Anderson) S.A.Mey. & Yarrow, Candida albicans (C.P.Robin) Berkhout, and Pityrosporum ovale (Bizz.) Castell. & Chalm. [53].

4.2. On the Antimicrobial Activity of G. biloba Extracts

In agricultural systems, a study on the antifungal action of G. biloba outer seedcoat extracts against rice sheath blight (which is caused by Rhizoctonia solani J.G. Kuhn) reported significant inhibition at 500 μg·mL⁻¹ [11]. Regarding antibacterial activity, crude G. biloba extracts were tested by Ražná et al. [12] against human and veterinary pathogens, including Gram-positive bacteria such as B. thuringiensis, C. perfringens, H. influenzae, L. monocytogenes, and S. aureus, and Gram-negative bacteria such as Klebsiella pneumoniae (Schroeter, 1886) Trevisan, 1887, Escherichia coli (Migula, 1895) Castellani and Chalmers, 1919, Salmonella enterica (ex Kauffmann and Edwards, 1952) Le Minor and Popoff, 1987, Shigella sonnei (Levine, 1920) Weldin, 1927, and Yersinia enterocolitica (Schleifstein and Coleman, 1939) Frederiksen, 1964. Among these species, S. aureus, E. coli, K. pneumoniae, and Y. enterocolitica were the most susceptible, with the best results being obtained for S. aureus (MIC₅₀ = 64.2 and MIC₉₀ = 72.2 μg·mL⁻¹) and Y. enterocolitica (MIC₅₀ = 85.3 and MIC₉₀ = 99.6 μg·mL⁻¹). These outcomes were consistent with the ones reported herein, in which greater activity was observed against Gram-positive C. michiganensis subsp. michiganensis bacteria (MIC = 500 μg·mL⁻¹) than against Gram-negative P. syringae, P. cichorii, and X. vesicatoria bacteria (whose MIC values ranged from 750 to 1000 μg·mL⁻¹). However, the MIC values for our extracts were higher (i.e., their activity was lower) than those reported by Ražná et al. [12].

4.3. Comparison with Conventional Antibiotics

Upon comparison of the activity of the extract with those of gentamicin, tetracycline, and ciprofloxacin—representative of aminoglycosides, tetracyclines, and quinolones classes, respectively, that are commonly used in plant production in some countries outside of the European Union, as well as to treat human and animal diseases [54]—the efficacy of the conventional antibiotics was orders of magnitude higher (with MIC values in the 0.047 to 24 μg∙mL⁻¹ range, depending on the pathogen). Concerning the other two antibiotics tested, viz. penicillin G and ampicillin, both were very effective against the Gram-positive C. michiganensis subsp. michiganensis; however, resistance was observed for all the Gram-negative bacteria, given that some strains of P. syringae produce β-lactamases [55] (e.g., some reports have indicated that 58% of P. syringae pv. syringae strains are resistant to ampicillin [56]) and X. vesicatoria does too [57]. It is noteworthy that the utilization of conventional antibiotics in agriculture (including tetracyclines, aminoglycosides, β-lactams, lincosamides, macrolides, pleuromutilins, and sulphonamides) may involve risks related to the emergence of resistance. Consequently, while the effectiveness of the natural extracts is not as high, they might be seen as a promising alternative to meet the requirements of the Global Action Plan on Antimicrobial Resistance set out by the World Health Organization (WHO).

5. Conclusions

Bacteriostatic activity was only detected for the G. biloba leaf extract (with MIC values ranging from 500 to 1000 μg mL⁻¹ depending on the bacterial strain), in which 3-O-methyl-D-fructose and 2,4-dimethyl-3-hexanol were the main constituents. In vivo tests on tomato and pea plants artificially inoculated with C. michiganensis subsp. michiganensis and P. syringae pv. pisi, respectively, demonstrated the potential of G. biloba leaf extract as a protective treatment, although higher concentrations than the in vitro-determined MICs were required (1000 and 1500 μg·mL⁻¹, respectively). The reported findings suggest an alternative valorization pathway for G. biloba leaves as biorationals for crop protection in addition to their use as dietary supplements.