Phytochemical Constituents and Antimicrobial Activity of Euphorbia serrata L. Extracts for Borago ofﬁcinalis L. Crop Protection

: The Euphorbia genus is the third-largest group of blooming plants, features a rich morpho-logical variability, has a near-cosmopolitan distribution, and diverse medicinal uses. Nonetheless, phytochemical information about Euphorbia serrata L. extracts is not available. The objective of this research was to examine the constituents of the hydromethanolic extract of its aerial parts and propose valorization pathways. The results of gas chromatography-mass spectroscopy (GC − MS) demonstrated that 3-methylbutyl formate, quinic acid, N1-(4-hydroxybutyl)-N3-methylguanidine acetate, and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one were the main phytocompounds, which have antimicrobial activity. Such activity was assayed against Pseudomonas cichorii , Botrytis cinerea , Fusarium oxysporum , and Sclerotinia sclerotiorum , four of the most destructive diseases of borage ( Borago ofﬁcinalis L.) crops, obtaining minimum inhibitory concentrations (MICs) of 750 and 1000 µ g · mL − 1 against the bacterium and the three fungal taxa, respectively, in in vitro tests. Conjugation of the extract with chitosan oligomers (COS) enhanced this activity, leading to MIC values of 187.5, 750, 500, and 500 µ g · mL − 1 for P. cichorii , B. cinerea , F. oxysporum , and S. sclerotiorum , respectively.


Introduction
Euphorbia serrata L. is an arvense, annual plant that grows up to 0.7 m tall ( Figure S1). It is known as serrate spurge or sawtooth spurge because of the serrate shape of the border of its leaves. Like other members of the family Euphorbiaceae, it is considered toxic and poisonous [1] because it exudes skin irritant and inflammatory, white milky latex (a complex emulsion consisting of starches, sugars, proteins, alkaloids, oils, tannins, resins, and gums, in which the presence of ingenol-3-palmitate has been documented [2]) when its stems or leaves are broken. However, according to Alves et al. [3], its medicinal use is documented in the treatment of conditions such as bronchitis, asthma, skin ailments, parasitic infections of the intestines, migraines, warts, gonorrhea, and dysentery.
A dried aerial part sample (11.4 g) was combined with 90 mL of a methanol/water solution (1:17 v/v). The mixture was heated for 30 min at 50 • C, sonicated using a probetype ultrasonicator (model UIP1000 hdT; Hielscher Ultrasonics; Teltow, Germany), and then centrifuged at 9000 rpm for 15 min. The resulting supernatant was filtered through Whatman No. 1 paper and subsequently subjected to freeze-drying to obtain the solid residue. The extraction yield was 52.6% (6 g). For subsequent gas chromatography-mass spectrophotometry (GC−MS) analysis, the freeze-dried extract was dissolved in HPLCgrade MeOH to obtain a 5 mg·mL −1 solution, followed by further filtration.
The method described in [19], with the modifications specified in [20], was followed to obtain chitosan oligomers (COS) with a molecular weight < 2000 Da in a solution with a pH of 4.5. COS and E. serrata extract solutions were mixed in a 1:1 (v/v) ratio (150 mL of each solution, at a concentration of 3000 µg·mL −1 ) and exposed to ultrasound for 15 min to form the conjugate complexes.

Physicochemical Characterization
A Thermo Scientific (Waltham, MA, USA) Nicolet iS50 Fourier-transform infrared (FTIR) spectrometer, with an in-built diamond attenuated total reflection (ATR) system, was used to register the infrared vibrational spectra of the shade-dried cyathia, shade-dried leaves, and latex samples. The spectral range of 400-4000 cm −1 was scanned with a 1 cm −1 resolution and 64 scans were co-added to generate the interferograms.
A gas chromatography-mass spectrometry (GC−MS) instrument comprising a model 7890A gas chromatograph coupled to a model 5975C quadrupole mass spectrometer (both from Agilent Technologies, Santa Clara, CA, USA) was used to elucidate the constituents of E. serrata aerial parts hydromethanolic extract. This analysis was outsourced to the Research Support Services (STI) at Universidad de Alicante (Alicante, Spain). The chromatographic conditions consisted of an injection volume of 1 µL, an injector temperature of 280 • C in splitless mode, an initial oven temperature of 60 • C held for 2 min, followed by a ramp of 10 • C·min −1 up to a final temperature of 300 • C held for 15 min. An HP-5MS UI chromatographic column (30 m length, 0.250 mm diameter, and 0.25 µm film), also from Agilent Technologies, was employed for the separation of the compounds. The mass spectrometer's electron impact source and quadrupole temperatures were 230 and 150 • C, respectively, with an ionization energy of 70 eV. Components were identified through comparisons of mass spectra and retention times with those of authentic compounds and computer matching with the database of the National Institute of Standards and Technology (NIST11). Test mixture 2 for apolar capillary columns according to Grob (Supelco 86501) and PFTBA tuning standards were used for equipment calibration.

In Vitro Antimicrobial Activity Assessment
The antibacterial activity was assessed using the agar dilution method, according to the Clinical and Laboratory Standards Institute (CLSI) standard M07-11 [21]. Initially, P. cichorii colonies were incubated at 28 • C for 24 h in TSB. Subsequently, serial dilutions were performed, starting from a concentration of 10 8 CFU·mL −1 , to achieve a final inoculum of 10 4 CFU·mL −1 . Next, the bacterial suspension was spread on the surface of TSA plates to which the extract had been previously added, at concentrations ranging from 62.5 to 1500 µg·mL −1 . The plates were then incubated at 28 • C for 24 h. The minimum inhibitory concentration (MIC) was determined as the lowest concentration of the extract at which no bacterial growth was visible. All experiments were replicated three times, with three plates per treatment and concentration.
The antifungal activity was determined using the agar dilution method as per the EUCAST antifungal susceptibility testing standard procedures [22]. Stock solution aliquots were incorporated into the PDA medium to produce final concentrations ranging from 62.5 to 1500 µg·mL −1 . Mycelial plugs (∅ = 5 mm), from the margin of 7-day-old PDA cultures of B. cinerea, S. sclerotiorum, and F. oxysporum were transferred to the center of PDA plates amended with the aforementioned concentrations (three plates per treatment and concentration, with two duplicates). Plates were incubated in the dark at 25 • C for seven days. The control consisted in replacing the extract with the solvent used for extraction (i.e., methanol:water 1:17 v/v) in the PDA medium. The inhibition of mycelial growth was estimated according to the formula in Equation (1): where d c and d t represent the mean diameters of the control and treated fungal colonies, respectively. The effective concentrations (EC 50 and EC 90 ) were determined via PROBIT analysis in SPSS Statistics v. 25 (IBM, New York, NY, USA). The synergy factor (SF), which measures the degree of interaction, was estimated using Wadley's method [23].
Since the Shapiro-Wilk and Levene tests indicated that the homogeneity and homoscedasticity requirements were fulfilled, the mycelial growth inhibition results were subjected to one-way analysis of variance (ANOVA) and subsequent post hoc comparison of means through Tukey's test at a significance level of p < 0.05 in IBM SPSS Statistics v. 25.

In Vivo Tests on Borage
Borage plants cv. "Movera" were used for in vivo experiments to assess the efficacy of the most potent in vitro treatment (i.e., COS-E. serrata) against artificially inoculated P. cichorii and F. oxysporum, following the methods described in [24] and [14], respectively, with minor modifications. Multiple 4 × 4 cm pots were used to grow the borage plants with sterile peat as substrate, with 5 plants per treatment and pathogen; two independent replicates were conducted. The treatment was applied at two concentrations (MIC and MIC × 2, i.e., 187.5 and 375 µg·mL −1 for P. cichorii, and 500 and 1000 µg·mL −1 for F. oxysporum). In the case of P. cichorii, it was applied through spraying (3 mL per plant). After 2 h, an isolated colony of P. cichorii was inoculated into the youngest leaflets by making 3-4 punctures using a sterile entomological pin. For the F. oxysporum assay, the fungus was previously grown in 250 mL flasks containing PDB for 3 days at 25 • C in the dark with constant shaking. Borage roots were then dipped in a suspension of 6 × 10 6 conidia·mL −1 for 2 min, and the plants were transferred to plastic pots with sterilized substrate. Non-inoculated plants sprayed with sterilized water or dipped in sterilized water were used as negative controls. All plants were incubated in a growth chamber (25 • C, 16/8 h photoperiod) for ten days.

Infrared Spectra
The main absorption bands in the FTIR spectra ( Figure S2) of E. serrata inflorescences (cyathia), leaves, and latex are listed in Table 1, alongside their functional group assignments. The symmetric C-H stretching vibrations of aliphatic groups (latex bands) are observed at 2916 ± 1 and 2848 ± 1 cm −1 , while the C=O stretching band at 1730 ± 2 cm −1 indicates ester bonds and carboxylic acid groups. In the fingerprint region of the spectrum, C−H and O−H deformation vibrations characteristic of carbohydrates can be observed between 1200 and 1462 cm −1 . Different types of C−H, C−O, and CH 3 vibrations, which cannot be identified more precisely, appear in the lower range of the fingerprint region below 1200 cm −1 [25]. Since the functional groups were found to be similar in the three organs (cyathia, leaves, and latex), a combined sample was utilized for the rest of the study. This approach is advantageous from a practical standpoint as it obviates the necessity of separating the aerial plant organs for subsequent extraction.

In Vitro Antimicrobial Activity
3.3.1. In Vitro Antibacterial Activity Some differences in terms of sensitivity to COS were observed among the four P. cichorii strains, with MIC values ranging from 500 to 750 µg·mL −1 , as shown in Table 3.

In Vitro Antibacterial Activity
Some differences in terms of sensitivity to COS were observed among the four P. cichorii strains, with MIC values ranging from 500 to 750 µg·mL −1 , as shown in Table 3. However, the MIC values obtained for E. serrata extract (comparable to/slightly higher than those of COS) and the COS-E. serrata conjugate complex (substantially lower than those of COS and E. serrata, with SF values > 1) were the same for all four strains. Table 3. Minimum inhibitory concentrations (in µg·mL −1 ) against four P. cichorii strains of chitosan oligomers (COS) and E. serrata aerial part extract alone, and their conjugate complex (COS-E. serrata). Synergy factors (SF) calculated for the conjugate complex are also indicated.  Figure S4 summarizes the results of the antifungal capacity testing. Higher doses resulted in reduced radial mycelium growth for the three tested treatments (viz., COS, E. serrata extract, and COS-E. serrata extract conjugate complex), with statistically significant differences in the case of the three pathogens. COS fully inhibited the mycelial growth of B. cinerea and S. sclerotiorum at 1500 µg·mL −1 and that of F. oxysporum at 1000 µg·mL −1 . Euphorbia serrata extract was more effective, resulting in complete inhibition of the three pathogens at 1000 µg·mL −1 . However, the antifungal activity was substantially increased by the application of conjugate complexes: the COS−E. serrata conjugate complex led to full inhibition at doses between 500 and 750 µg·mL −1 . Such activity enhancement was quantified using the effective concentration (EC) values of the separate bioactive products and those of the conjugate complexes (Table 4), obtaining the SF summarized in Table 5. Given that values close to two were obtained against the three phytopathogens, a clear synergistic action between COS and the extract may be inferred also in terms of antifungal activity. Taking into consideration that the COS-E. serrata conjugate complex was shown to be the most active in the in vitro tests, its bacteriostatic activity was further tested in in vivo tests. It was applied as a preventive treatment against bacterial blight on borage plants cv. "Movera" (as shown in Figure 2). Ten days post-inoculation, positive control plants (inoculated with P. cichorii only) displayed necrotic lesions that covered the entire leaf (Figure 2b). Plants sprayed with the COS-E. serrata conjugate complex at a concentration equal to the MIC (187.5 µg·mL −1 ) had necrotic lesions in the area where the pathogen was inoculated, which did not spread to the entire leaf, but showed some discoloration around these necrotic spots (Figure 2c). In turn, plants treated with a dose twice the MIC (375 µg· mL −1 ) showed barely any signs of disease attack (as shown in Figure 2d). No symptoms were observed in mockinoculated plants (sprayed with sterile distilled water) (Figure 2a).

In Vivo Antibacterial Activity against P. cichorii
Taking into consideration that the COS-E. serrata conjugate complex was shown to be the most active in the in vitro tests, its bacteriostatic activity was further tested in in vivo tests. It was applied as a preventive treatment against bacterial blight on borage plants cv. "Movera" (as shown in Figure 2). Ten days post-inoculation, positive control plants (inoculated with P. cichorii only) displayed necrotic lesions that covered the entire leaf ( Figure 2b). Plants sprayed with the COS-E. serrata conjugate complex at a concentration equal to the MIC (187.5 µg·mL −1 ) had necrotic lesions in the area where the pathogen was inoculated, which did not spread to the entire leaf, but showed some discoloration around these necrotic spots (Figure 2c). In turn, plants treated with a dose twice the MIC (375 µg·mL −1 ) showed barely any signs of disease attack (as shown in Figure 2d). No symptoms were observed in mock-inoculated plants (sprayed with sterile distilled water) (Figure 2a).    Figure 3 shows the results of the in vivo assays conducted with COS-E. serrata conjugate complex on borage cv. "Movera" plants to assess the protective (fungistatic) activity against Fusarium wilt. All positive control plants showed the characteristic severe wilting and yellowing symptoms, accompanied by dry necrosis of the central veins of some leaves, followed by plant death (Figure 3b). The disease incidence among plants treated with the extract at a concentration equal to the MIC (500 µg·mL −1 ) was high (80%), but the disease severity was lower (Figure 3c). To achieve complete protection, the dose had to be increased to 1000 µg·mL −1 : at twice the MIC, after which all plants remained asymptomatic (Figure 3d). It should be noted that at the highest concentration tested (1000 µg·mL −1 ), borage plants did not display symptoms of phytotoxicity, with no visual differences among plants treated at 1000 µg·mL −1 and the negative control (Figure 3a).

In Vivo Antifungal Activity against F. oxysporum
Horticulturae 2023, 9, x FOR PEER REVIEW 9 of 20 leaves, followed by plant death (Figure 3b). The disease incidence among plants treated with the extract at a concentration equal to the MIC (500 µg·mL −1 ) was high (80%), but the disease severity was lower (Figure 3c). To achieve complete protection, the dose had to be increased to 1000 µg·mL −1 : at twice the MIC, after which all plants remained asymptomatic ( Figure 3d). It should be noted that at the highest concentration tested (1000 µg·mL −1 ), borage plants did not display symptoms of phytotoxicity, with no visual differences among plants treated at 1000 µg·mL −1 and the negative control ( Figure 3a).

On the Phytochemical Profile of the Extract
3-Methylbutyl formate is a wax monoester related to 2-methylbutyl esters found in Chamaemelum nobile (L.) All. It was previously identified in red raspberries [26]. Mean-
Concerning quinic acid, this cyclohexanecarboxylic acid has been reported in the bark of Uncaria tomentosa (Willd. ex Schult.) DC. [31], as well as in the extracts of several parts of medicinal plants, including Achillea pseudoaleppica Hub.-Mor., Artemisia annua L., Coffea arabica L., Haematocarpus Validus (Miers) Bakh.fil. ex Forman, Hypericum empetrifolium Willd., Phagnalon saxatile (L.) Cass., Rumex nepalensis, and Ziziphus lotus L. [32]. Bai et al. [33] suggested that it could be used as an antibacterial agent in food preservation. The modulation of ribosome function, aminoacyl-tRNA synthesis, and alterations in the levels of glycerophospholipids and fatty acids, as well as interference with membrane fluidity by disrupting the oxidative phosphorylation pathway, are all significant contributors to its antibacterial effect [32]. Research on cellular functions has revealed that quinic acid leads to a notable decline in intracellular pH, lowers succinate dehydrogenase activity, and results in reduced intracellular ATP concentration. Lu et al. [34] indicated that the binding of quinic acid to RhlA, RhlR, and RhlB receptors interferes with the binding of signal molecules to these receptors, leading to the transcriptional regulation of signaling pathways.
Squalene is a triterpene with lipophilic properties and serves as a precursor to ergosterol, an important component of the plasmatic membrane in fungi and yeasts. Prior research has shown squalene to be a significant phytochemical in the bark of Quercus ilex subsp. ballota (Desf.) Samp. [46] and leaves of Hibiscus syriacus L. [47]. Furthermore, it is understood that squalene accumulation inside cells can lead to the formation of vesicles that contain squalene, thereby disrupting the fungal cell membrane. This process consequently removes essential membrane lipid components, weakening the fungal cells [48].
Hexadecanoic acid (or palmitic acid), found as the major component in A. maritima [44], has been shown to be highly suitable for the integrated control of phytopathogens [49].
It is worth noting that no correspondence between the main phytochemicals reported above and the phytoconstituents reported for E. hirta leaves extract by Mekam et al. [8] was observed. This difference is tentatively ascribed to the different analytical methods employed (GC−MS vs. LC−ESI−IT−MS/MS).
With regard to the activity of other Euphorbia spp. extracts tested against human and plant pathogens, a summary of the efficacies reported in the literature is presented in Table 6. It should be noted that direct comparisons are not possible because the pathogens are different, and, in those cases in which the same species were tested, the strains were different. Furthermore, results were not expressed as EC or as MIC values in all studies. Consequently, the comparisons presented below should be interpreted as a first approximation. Focusing on the data reported against phytopathogens, the antibacterial activity of the non-conjugated E. serrata extract would be comparable to that of Euphorbia cotinifolia L. [54]. As for its antifungal activity, it would be substantially higher than those of E. hirta and Euphorbia tirucalli L. against F. oxysporum [8,55,56] and higher than those of Euphorbia guyoniana Boiss. and Reut. and Euphorbia royleana Boiss. against other Fusarium spp. [57,58], while it would be comparable to that of Euphorbia macroclada Boiss. (for which full inhibition of F. oxysporum was reported at 1000 µg·mL −1 for chloroform, petroleum ether, and butanol flower extracts, as well as for a chloroform stem extract) [59].

Comparison with Synthetic Fungicides
A comparison of the activity of three conventional synthetic fungicides against the three fungal taxa is presented in Table 7. In the case of B. cinerea and S. sclerotiorum, the same isolates were used, while a different F. oxysporum isolate was used. A comparison with five clinical-grade antibiotics against P. cichorii (strain CITA Pci-5) is summarized in Table S2.
With respect to the antifungal activity, the MIC values for E. serrata extract and the COS-E. serrata conjugate complex (1000 and 500-750 µg·mL −1 , respectively) are lower than those of fosetyl-Al. Fosetyl-Al achieved full inhibition of B. cinerea and F. oxysporum at 2000 µg·mL −1 , but it did not fully inhibit S. sclerotiorum at this concentration. Furthermore, the MIC values for E. serrata extract and the COS-E. serrata conjugate complex are much lower than those of azoxystrobin, which did not fully inhibit any of the fungal taxa at 62,500 µg·mL −1 . However, the natural-based product would be less effective than mancozeb, for which full inhibition was achieved at 150 µg·mL −1 in all cases.
Regarding the antibacterial activity, E. serrata extract and the COS-E. serrata conjugate complex achieved full inhibition of P. cichorii strain CITA Pci-5 at 750 and 187.5 µg·mL −1 (Table 3). Therefore, at least the latter would be more active than ampicillin to which some Pseudomonas spp. are resistant due to their production of β-lactamases.

Conclusions
GC−MS characterization of the E. serrata hydromethanolic extract showed that the main constituents were 3-methylbutyl formate (15.5%), quinic acid (11.8%), N1-(4-hydroxybutyl)-N3-methylguanidine acetate (5.3%), and 2,3-dihydro-3,5-dihydroxy-6methyl-4H-pyran-4-one (or DDMP) (4%). With a view to its valorization for borage crop protection, the antimicrobial activity of the extract was tested in vitro against four P. cichorii strains and three isolates of B. cinerea, F. oxysporum, and S. sclerotiorum, respectively. Minimum inhibitory concentrations of 750 and 1000 µg·mL −1 were obtained against the bacteria and the three fungal taxa, respectively. Upon conjugation of the extract with COS, the antimicrobial activity improved, resulting in MIC values of 187.5, 750, 500, and 500 µg·mL −1 for P. cichorii, B. cinerea, F. oxysporum, and S. sclerotiorum, respectively. The most active treatment (i.e., COS-E. serrata extract conjugate complex) was further tested in in vivo assays against the two pathogens that currently have a higher impact in the main growing areas of borage in Aragón (Spain), confirming a strong protective action at a dose of 375 and 1000 µg·mL −1 for P. cichorii and F. oxysporum, respectively. These findings are noteworthy since the plant extract showed higher activity than synthetic fungicides such as azoxystrobin and fosetyl-Al. Therefore, this study calls for further research on the applicability of E. serrata extract as a biorational in integrated pest management of borage and other horticultural crops.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae9060652/s1, Figure S1: Euphorbia serrata aerial part and detail of the inflorescences (cyathia); Figure S2: Infrared spectra of E. serrata flowers, leaves, and latex; Figure S3: GC−MS chromatogram of E. serrata aerial parts hydromethanolic extract; Figure S4: Inhibition of the radial growth of the mycelium of B. cinerea, F. oxysporum, and S. sclerotiorum in the in vitro tests performed with PDA medium amended with different concentrations (in the range of 62.5-1500 µg·mL −1 ) of chitosan oligomers (COS), E. serrata aerial part extract, and their conjugate complex (COS-E. serrata). Table S1: Efficacy of plant extracts and essential oils reported in the literature against the phytopathogens under study; Table S2: Minimum inhibitory concentrations (expressed in µg·mL −1 ) of conventional antibiotics (for clinical use) against P. cichorii strain CITA Pci-5.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.