Characterization and Antimicrobial Activity of a Halophyte from the Asturian Coast (Spain): Limonium binervosum (G.E.Sm.) C.E.Salmon

The work presented herein deals with the characterization and valorization of a halophyte from the cliffs of the Asturian coast: Limonium binervosum (G.E.Sm.) C.E.Salmon (rock sea-lavender). Its biomass and hydromethanolic extracts were studied by elemental and thermal analysis, infrared spectroscopy and gas chromatography–mass spectroscopy. Tetradecanoic acid/esters and 1,2-tetradecanediol were identified in its flower extract, while the leaf extract was rich in linolenic and linoleic acids and their esters, hexadecanoic acid and its esters, and phytol. Both flower and leaf hydromethanolic extracts contained eicosane, sitosterol and tocopherols in significant amounts. With a view to its valorization, the antimicrobial activity of these extracts was investigated against three apple tree and grapevine phytopathogens. Both the hydroalcoholic extracts and their main constituents, alone or in combination with chitosan oligomers (COS), were tested in vitro. A remarkable antibacterial activity was observed for the conjugated complexes of the flower extract with COS, both against Xylophilus ampelinus (MIC = 250 μg·mL−1) and Erwinia amylovora (MIC = 500 μg·mL−1), and complete inhibition of the mycelial growth of Diplodia seriata was found at concentrations <1000 μg·mL−1. In view of these results, this extremophile plant can be put forward as a promising source of bioactive metabolites.


Introduction
Limonium is one of the most important species-rich genera in the Plumbaginaceae family. This widespread genus of halophytes and taxa includes sexual diploids of the L. ovalifolium (Poir.) Kuntze complex, the triploid L. algarvense Erben and the agamospermous tetraploids of the L. binervosum (G.E.Sm.) C.E.Salmon complex [1]. The L. binervosum aggregate is a species group that has not been assigned to any of the subsections of L. sect. Limonium [2] and was first reported in 1922 by Salmon [3].
The habitat of L. binervosum includes coastal cliffs, pebble beach margins, steppes, meadows and lagoons. It grows on the Atlantic coasts of Europe, from the south-west United Kingdom and north-west France to northern Spain, with a number of geographically restricted segregate taxa (Figure 1a). A review of the bioactive components in several species of the Limonium genus suggests that they are a good source of antioxidants. For instance, in L. algarvense flowers, the antioxidants are related to gallic acid, catechin, salicylic and rosmarinic acids, and epigallocatechin gallate [5], similar to those found in L. brasiliense (Boiss.) Kuntze (viz. gallic acid, gallocatechin, epigallocatechin, PDE gallate, etc.) [6]. In L. aureum (L.) Hill, the antioxidants identified were myricetin (or cannabiscetine), myricetin-3-O-glucoside, myricetin-3-O-β-D-glucopyranoside, myricitrin, erioictyol, homoeridictyol, and eriodictyol-7-O-glucoside [7]. In a study on the leaves of L. delicatulum (Girard) Kuntze and L. quesadense Erben it was found that the former is rich in myricetin glycosides, whereas in the latter epigallocatechin gallate and its dimer are some of the most abundant compounds [8]. Consequently, L. binervosum-whose phytochemical constituents have not been studied to date-may also be a promising source of antioxidants.
Taking into consideration that antioxidant activity is generally associated with antibacterial, antifungal and antimycotoxigenic biological activities [9], potential valorization strategies for L. binervosum as a source of bioactive products may be envisaged, aligned with the premises of current EU regulation (Directive 2009/128/EC on the sustainable use of pesticides, Council Regulation (EC) 834/2007 on organic production and labeling of organic products, Regulation (EU) 2019/1009 on the market of EU fertilizing products, etc.), in which the replacement of conventional phytosanitary products with formulations based on natural products is encouraged.
In particular, in this study, its application to the control of grapevine (Vitis vinifera L.) and apple tree (Malus domestica Borkh.) pathogens was explored by assessing its antibacterial activity against Xylophilus ampelinus (Panagopoulos 1969) Willems et al. 1987 and Erwinia amylovora (Burrill), and its antifungal activity against Diplodia seriata De Not.
X. ampelinus (syn. Xanthomonas ampelina and Erwinia vitivora [10]), a quarantine A2 organism according to the European and Mediterranean Plant Protection Organization (EPPO), causes the bacterial necrosis of grapevines ("mal nero" or "maladie d'Oléron"), resulting in yield losses of up to 70% [11]. E. amylovora, also cataloged as a quarantine organism, causes fire blight, which poses a serious threat to pear and apple production [12]. In turn, D. seriata, a Botryosphaeriaceous fungus, causes dieback, canker, leaf spot and fruit rot in a wide range of hosts, including grapevine [13,14] and apple trees [15][16][17]. Plants of L. binervosum can grow up to 20 cm, and have regular, straight spikes, which are not aggregated into a corymbose head (Figure 1b). Stems divide several times. Leaves are narrow oblanceolate, greyish-green in color, with a midvein. The flowers form in compact clusters along the leafless stem branches and are pink, formed of five notched petals, with five short stamens topped by white anthers and a purplish calyx (Figure 1c) [4].
A review of the bioactive components in several species of the Limonium genus suggests that they are a good source of antioxidants. For instance, in L. algarvense flowers, the antioxidants are related to gallic acid, catechin, salicylic and rosmarinic acids, and epigallocatechin gallate [5], similar to those found in L. brasiliense (Boiss.) Kuntze (viz. gallic acid, gallocatechin, epigallocatechin, PDE gallate, etc.) [6]. In L. aureum (L.) Hill, the antioxidants identified were myricetin (or cannabiscetine), myricetin-3-O-glucoside, myricetin-3-O-β-Dglucopyranoside, myricitrin, erioictyol, homoeridictyol, and eriodictyol-7-O-glucoside [7]. In a study on the leaves of L. delicatulum (Girard) Kuntze and L. quesadense Erben it was found that the former is rich in myricetin glycosides, whereas in the latter epigallocatechin gallate and its dimer are some of the most abundant compounds [8]. Consequently, L. binervosum-whose phytochemical constituents have not been studied to date-may also be a promising source of antioxidants.
Taking into consideration that antioxidant activity is generally associated with antibacterial, antifungal and antimycotoxigenic biological activities [9], potential valorization strategies for L. binervosum as a source of bioactive products may be envisaged, aligned with the premises of current EU regulation (Directive 2009/128/EC on the sustainable use of pesticides, Council Regulation (EC) 834/2007 on organic production and labeling of organic products, Regulation (EU) 2019/1009 on the market of EU fertilizing products, etc.), in which the replacement of conventional phytosanitary products with formulations based on natural products is encouraged.
In particular, in this study, its application to the control of grapevine (Vitis vinifera L.) and apple tree (Malus domestica Borkh.) pathogens was explored by assessing its antibacterial activity against Xylophilus ampelinus (Panagopoulos 1969) Willems et al. 1987 and Erwinia amylovora (Burrill), and its antifungal activity against Diplodia seriata De Not.
With a view to a possible valorization of this halophyte (L. binervosum), a physicochemical characterization is presented, together with an in vitro evaluation of the antimicrobial Plants 2021, 10, 1852 3 of 16 activity of its extracts-alone and in combination with chitosan oligomers-against aforementioned phytopathogens.

Elemental Analysis and Calorific Values Calculation
The C, H, N and S percentages of L. binervosum components (wt% of dry material) were in the 40.5-44.7%, 6.4-6.5%, 1.2-2.6% and 0.2-0.9% range, respectively (Table 1). Higher heating values derived from elemental analysis data resulted in heating values for flowers and leaves of 18 and 16 kJ·g −1 , respectively.

Thermal Analyses
The TG, DTG and DSC curves of flowers and leaves are shown in Figures S1 and S2, respectively. In the case of flowers, exothermal effects were detected at 329, 420 and 470 • C; the ash content (at 550 • C) was 5.6%. Concerning leaves, exothermal effects were registered at 320 and 470 • C, and the ash content (at 580 • C) reached 17%. For comparison purposes, the total ash content reported L. stocksii (Boiss.) Kuntze was 11.83% [18].

Phenolic Contents
The total phenolic content of the flower and leaf extracts were 162 ± 7 and 58 ± 2 mg GAE/g DW, respectively. In-plane bending or rocking of the methylenes (-CH 2 -) 668 C-C out-of-plane bending

In Vitro Antibacterial Activity
The inhibition of flower and leaf extracts against X. ampelinus and E. amylovora were similar and comparable to that attained with COS (Table 5). Regarding the activities of the main active principles present in the extracts, differences were observed as a function of the pathogen: while tetradecanoic acid, linolenic acid and vitamin E showed similar activity against X. ampelinus (MIC = 500 µg·mL −1 ), tetradecanoic acid was the most effective against E. amylovora (MIC = 500 µg·mL −1 ), and linolenic acid and vitamin E were less effective (MIC = 750 µg·mL −1 ). β-sitosterol showed worse performance than the former three (MIC = 1000 and 1500 µg·mL −1 against X. ampelinus and E. amylovora, respectively), and eicosane was the least effective (MIC = 1000 and >1500 µg·mL −1 against X. ampelinus and E. amylovora, respectively). Upon conjugation with COS, a synergistic behavior was observed for all phytochemicals. The best results against X. ampelinus were attained with the COS-flower extract conjugate complex (MIC = 250 µg·mL −1 ), comparable to those attained for the COStetradecanoic acid, COS-linolenic acid and COS-vitamin E conjugate complexes, while the effectiveness of the COS-leaf extract was lower (MIC = 500 µg·mL −1 ). In the case of E. amylovora, the COS-flower extract conjugate complex was more effective than the leaf-based one (MIC = 500 µg·mL −1 vs. 750 µg·mL −1 , respectively), but less effective than the COS-tetradecanoic acid, COS-linolenic acid and COS-vitamin E conjugate complexes (MIC = 250 µg·mL −1 , similar to those observed against X. ampelinus).

In Vitro Antifungal Activity
The results from the D. seriata mycelial growth inhibition tests are shown in Figures 2  and S5. At the highest dose (1500 µg·mL −1 ), the flower and the leaf extracts resulted in 82% and 71% inhibition, respectively, while full inhibition was attained at 750 µg·mL −1 for tetradecanoic acid, linolenic acid and vitamin E constituents, and at 250 µg·mL −1 for β-sitosterol. In the case of eicosane, 93% inhibition was observed at the highest dose.
The formation of conjugate complexes improved the activity in all cases, with remarkable improvements in COS-tetradecanoic and COS-linolenic (from 750 down to 187.5 µg·mL −1 ). Concerning flower and leaf extracts, full inhibition was attained at 1000 µg·mL −1 in both cases.
Determination of EC 50 and EC 90 values (50% and 90% maximal effective concentration, respectively), summarized in Table 6, and calculation of synergy factors, presented in Table 7, confirmed the strong synergistic behavior previously mentioned for COS and tetradecanoic and linolenic acids (with SFs of 4.55 and 5.75 for the EC 90 , respectively). In all the other cases, SFs > 1 (i.e., indicative of a synergistic behavior) were also obtained, albeit more moderate.
"+" and "−" indicate presence and absence of bacterial growth, respectively. 2 and S5. At the highest dose (1500 μg·mL −1 ), the flower and the leaf extracts resulted in 82% and 71% inhibition, respectively, while full inhibition was attained at 750 μg·mL −1 for tetradecanoic acid, linolenic acid and vitamin E constituents, and at 250 μg·mL −1 for βsitosterol. In the case of eicosane, 93% inhibition was observed at the highest dose. The formation of conjugate complexes improved the activity in all cases, with remarkable improvements in COS-tetradecanoic and COS-linolenic (from 750 down to 187.5 μg·mL −1 ). Concerning flower and leaf extracts, full inhibition was attained at 1000 μg·mL −1 in both cases.

Elemental Analysis and Calorific Values Calculation
In relation to the elemental analysis results, the carbon content is close to that reported by Park et al. [24] for L. tetragonum (Thunb.) Bullock (45.5%), while the nitrogen content in leaves is in good agreement with that reported for L. echioides (L.) Mill. (ca. 2.4%) for complete shoots [25]. The fact that the values of the C/N ratios for flowers are twice those obtained for leaves is consistent with the higher percentage of carbonitrogenated compounds in leaves (viz. n-methyl-1-adamantaneacetamide, and 2-(4-fluoro-phenyl)-4-(3-methyl-benzylidene)-4h-oxazol-5-one, which account for ca. 3% according to GC-MS results) than in flowers (viz. 2-ethylacridine, 1.59%).
The calorific values obtained from elemental analysis data, below the 18.82 kJ·g −1 limit required in EN 14961-2 [26], and the high ash contents (above the 2% limit), preclude the valorization of this halophyte as solid biofuel. Nonetheless, it is worth noting that the fatty acid profile (discussed below), rich in linolenic and linoleic acids, can make L. binervosum a promising biofuel feedstock, according to Patel et al. [27].

Antimicrobial Activity of the Main Identified Phytochemicals
All the main phytochemicals found in the L. binervosum flower and leaf extracts have been reported to have both antimicrobial and antifungal activity (albeit not against any of the phytopathogens referred herein).
Concerning fatty acids, which are the major constituents of L. binervosum extracts, it was demonstrated that the antibacterial action of long-chain unsaturated fatty acids is mediated by the inhibition of fatty acid synthesis [53], and it was shown that both saturated and unsaturated fatty acids have antifungal activity, although saturated fatty acids would show a stronger activity [54]. In particular, antimicrobial properties of tetradecanoic acid were referred to in the literature (against, for instance, Listeria monocytogenes [55] and C. albicans [56]), as well as for its derivatives, such as methyl 2-hydroxytetradecanoate (against C. albicans, Cryptococcus neoformans and A. niger [57]). Regarding linolenic acid, Lee et al. [58] concluded that this fatty acid has a strong antibacterial activity against B. cereus and S. aureus, and Walters et al. [59] showed its activity against R. solani, Pythium ultimum, Pyrenophora avenae and Crinipellis perniciosa.
With regard to vitamin E, its antibacterial activity against E. coli, S. aureus, S. epidermidis, P. aeruginosa, Proteus spp., Klebsiella spp., and Enterobacter spp. was evidenced by Al-Salih et al. [60], and it was reported that-in combination with fluconazole-it results effective in the treatment of some human fungal diseases [61].

On the Synergistic Behavior Observed for the Conjugate Complexes
The combination of chitosan with several of the main constituents of L. binervosum extracts has precedents in the literature. For instance, combinations of chitosan with vitamin E were studied by Yeamsuksawat and Liang [62], Martins et al. [63] and Raza et al. [64]. The rationale behind such choice is that, while α-tocopherol has feeble stability, it is improved by encapsulation in chitosan as a capping agent, as well as its release when required over a sustained period. Similarly, Liu et al. [65] reported the formation of self-assembled nanoparticles by coupling chitosan with linolenic acid, taking advantage of the fact that chitosan is known to inhibit the linoleic (and linolenic) acid oxidation process [66]. In the case of tetradecanoic acid, chitosan-tetradecanoic acid nanogels with MIC values of 10 mg·mL −1 against S. enterica were reported by Rajaei et al. [67].
Nonetheless, none of the aforementioned combinations are conjugated complexes, and the existence of interactions between the two components in terms of antimicrobial activity was not explored. Albeit for other phytochemicals different from the ones present in L. binervosum, a synergistic behavior upon conjugation with COS was reported in the literature against phytopathogens: e.g., for horsetail (Equisetum arvense L.) and nettle (Urtica dioica L.) extracts against eight fungal species involved in grapevine trunk diseases [68], with EC 90 values in the 208-1000 µg·mL −1 range (depending on the extract and on the Botryosphaeriaceae taxa). The value reported in this work for the COS-flower extract complex (914 µg·mL −1 ) would be on the upper limit.
The mechanism of synergistic action of such COS-phytochemical conjugates has not been dilucidated yet. Nonetheless, it was suggested that it might be the result of an enhanced additive antimicrobial effect, per se, and/or via a concurrent action on diverse microbial metabolic sites. An increase in the cationic surface charge of COS may also result from conjugation with phytochemicals, which would enhance the linkage to negatively charged site-specific binding receptors on the bacterial/fungal membranes [70][71][72][73].
Eicosane  L. binervosum flower samples were mixed (1:20 w/v) with a 1:1 v/v methanol:water solution and heated for 30 min in a water bath at 50 • C. Subsequently, they were sonicated for 5 min in pulse mode with a 1 min stop for each 2.5 min, using a Hielscher Ultrasonics (Teltow, Germany) probe-type ultrasonicator (model UIP1000hdT; 1000 W, 20 kHz). The solution was subjected to centrifugation at 9000 rpm for 15 min, and the supernatant was filtered through Whatman No. 1 paper. Aliquots were lyophilized for CHNS and FTIR analyses. The extraction procedure for leaf samples was identical.
Each extraction procedure was replicated three times (on subsamples of the flower and leaf composite samples), and the resulting hydromethanolic extracts were mixed to obtain the samples for GC-MS analysis.
Calorific values were calculated from elemental analysis data according to Talwalkar et al. [76], using the following equation: where HHV is the heating value for the dry material, expressed in kJ·g −1 , and %C, %H, %O and %N are the mass fractions, expressed in wt% of dry material. Thermal gravimetric (TGA) and differential scanning calorimetry (DSC) analyses were conducted with a simultaneous TG-DSC2 apparatus (Mettler Toledo; Columbus, OH, USA). Samples (10 mg/sample) were heated from 30 to 600 • C under N 2 :O 2 (4:1) flow (20 cm 3 ·min −1 ), at a 20 • C·min −1 heating rate.
The infrared spectra were obtained with a Nicolet iS50 Fourier-transform infrared spectrometer (Thermo Scientific; Waltham, MA, USA), equipped with an in-built diamond attenuated total reflection (ATR) system. A spectral resolution of 1 cm −1 over the 400-4000 cm −1 range was used, taking the interferograms that resulted from co-adding 64 scans.
The colorimetric quantification of the total polyphenol content (TPC), expressed in gallic acid equivalents (GAE), was conducted according to the procedure described in [77], using a UV-Vis Cary 100 spectrometer (Agilent Technologies; Santa Clara, CA, USA).
The gas chromatography-mass spectrometry (GC-MS) analyses of the hydroalcoholic plant extracts (obtained as a mixture of three extractions) were carried out at the Research Support Services (STI) at Universidad de Alicante (Alicante, Spain). A model 7890A gas chromatograph coupled to a model 5975C quadrupole mass spectrometer (Agilent Technologies). The chromatographic conditions were: 3 injections/vial; 1 µL injection volume; 280 • C injector temperature, in splitless mode; the 60 • C initial oven temperature was held for 2 min, followed by a 10 • C·min −1 ramp up to a 300 • C final temperature, kept for 15 min. The chromatographic column used for the separation of the compounds was an HP-5MS UI (Agilent Technologies) of 30 m length, 0.250 mm diameter and 0.25 µm film. The MS conditions were: temperature of the electron impact source of the mass spectrometer = 230 • C and of the quadrupole = 150 • C; 70 eV ionization energy. Equipment calibration was conducted using test mixture 2 for apolar capillary columns according to Grob (Supelco 86501) and PFTBA tuning standards. Compound identification was carried out using the NIST11 library [78].

In Vitro Antibacterial Activity Assessment
The antibacterial activity was assessed by determining the minimum inhibitory concentration (MIC). The agar dilution method was used, according to CLSI standard M07-11 [79]. An isolated colony of X. ampelinus was incubated in TSB liquid medium at 26 • C for 18 h. Starting from a 10 8 CFU·mL −1 concentration, serial dilutions were then conducted to obtain a final inoculum of~10 4 CFU·mL −1 . Subsequently, bacterial suspensions were delivered to the surface of TSA plates amended with the treatments at concentrations ranging from 62.5 to 1500 µg·mL −1 . The plates were incubated at 26 • C for 24 h. The procedure for E. amylovora was identical, except for the incubation temperature (30 • C). MICs were visually determined as the lowest concentrations at which no bacterial growth was visible in the agar dilutions. All experiments were run in triplicate, with each replicate consisting of 3 plates per treatment/concentration.

In Vitro Antifungal Activity Assessment
The antifungal activity of the different treatments was determined according to EU-CAST standard antifungal susceptibility testing procedures [80], using the agar dilution method. Aliquots of stock solutions were incorporated onto the PDA medium to obtain concentrations in the 62.5-1500 µg·mL −1 range. Mycelial plugs (5 mm in diameter), taken from the margin of 7-day-old D. seriata PDA cultures, were transferred to plates amended with aforementioned concentrations of each treatment (3 plates per treatment/concentration, with 2 replicates). Plates were incubated in the dark at 25 • C for 7 days. PDA medium without any amendment was used as the control. Mycelial growth inhibition was estimated according to the formula: where d c and d t represent the average diameters of the fungal colony of the control and of the treated fungal colony, respectively. EC 50 and EC 90 effective concentrations were estimated in IBM SPSS Statistics v.25 (IBM; Armonk, NY, USA) software using PROBIT analysis. The level of interaction was determined according to Wadley's method [81].

Statistical Analysis
Given that the homogeneity and homoscedasticity requirements were satisfied (according to Shapiro-Wilk and Levene tests, respectively), the mycelial growth inhibition results for D. seriata were statistically analyzed in IBM SPSS Statistics v.25 software using one-way analysis of variance (ANOVA), followed by post hoc comparison of means through Tukey's test at p < 0.05.

Conclusions
A halophyte from the cliffs of the Atlantic coasts of Europe, viz. Limonium binervosum (rock sea-lavender) was studied by elemental and thermal analysis, FTIR spectroscopy and GC-MS with a view to its valorization. The use of its biomass as a solid biofuel can be ruled out, given that its higher heating value (in the 16-18 kJ·g −1 range) and content of ashes (5.6% and 17% for flowers and leaves, respectively) do not meet the minimum legal requirements, but its high content in fatty acids open the door to potential exploitation as a biofuel feedstock. Another potential application would be related to the use of its hydrometanolic extracts as natural biocontrol products, given that phytochemicals with antimicrobial properties were found in significant amounts: both flower and leaf extracts contained eicosane (4-18%), β-sitosterol (9-19%) and tocopherols (7-13%), besides fatty acids and their esters (22% of tetradecanoic acid in the flower extract, and 30% of linolenic and linoleic acids in the leaf extract). The inhibitory activity of the extracts and their main constituents, alone or in combination with chitosan oligomers, was tested in vitro against X. ampelinus, E. amylovora and D. seriata phytopathogens. A remarkable antibacterial activity was observed against X. ampelinus (with a MIC value of 250 µg·mL −1 ) and E. amylovora (MIC = 500 µg·mL −1 ) for the conjugated complex of the flower extract with COS, which also resulted in an EC 90 of 914 µg·mL −1 against D. seriata. In view of these results, the conjugate complexes of this halophyte may be put forward as promising antimicrobial treatments for apple tree and grapevine diseases in organic agriculture.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

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 be part of an ongoing Ph.D. Thesis.