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Article

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

by
Eva Sánchez-Hernández
1,
Laura Buzón-Durán
1,
Natalia Langa-Lomba
2,
José Casanova-Gascón
2,
Belén Lorenzo-Vidal
3,
Jesús Martín-Gil
1 and
Pablo Martín-Ramos
2,*
1
Agriculture and Forestry Engineering Department, ETSIIAA, Universidad de Valladolid, Avenida de Madrid 44, 34004 Palencia, Spain
2
Instituto Universitario de Investigación en Ciencias Ambientales de Aragón (IUCA), EPS, Universidad de Zaragoza, Carretera de Cuarte, s/n, 22071 Huesca, Spain
3
Servicio de Microbiología, Hospital Universitario Rio Hortega, Calle Dulzaina 2, 47012 Valladolid, Spain
*
Author to whom correspondence should be addressed.
Plants 2021, 10(9), 1852; https://0-doi-org.brum.beds.ac.uk/10.3390/plants10091852
Submission received: 7 August 2021 / Revised: 3 September 2021 / Accepted: 4 September 2021 / Published: 7 September 2021
(This article belongs to the Special Issue Natural Resources of Medicinal and Cosmetic Plants)
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Versions Notes

Abstract

:
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.

Graphical Abstract

1. 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).
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-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].
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 activity of its extracts—alone and in combination with chitosan oligomers—against aforementioned phytopathogens.

2. Results

2.1. 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.

2.2. 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].

2.3. Vibrational Characterization

The main absorption bands in the FTIR spectra of the powdered dry samples of flowers and leaves are summarized in Table 2, together with their assignments. The bands at 2918, 2850, 1462 and 720 cm−1 are due to aliphatic features and are present in straight-chain alkanes (compatible with the presence of tetracosane, pentacosane, heptacosane, etc., identified by GC–MS in the extracts, as discussed below) [19]. The band at 2158 cm−1, ascribed to C-N stretching, may arise from the presence of carbonitrogenated compounds (e.g., n-methyl-1-adamantaneacetamide; 2-(4-fluoro-phenyl)-4-(3-methyl-benzylidene)-4h-oxazol-5-one, 2-ethylacridine, etc.) [20]. The bands at ca. 1730 and ca. 1165 cm−1, related to carbonyl (C=O) stretching and C-C(=O)-O stretching, respectively, illustrate the main spectral features of esters (e.g., 2-hydroxy-tetradecanoic acid methyl ester; hexadecanoic acid methyl ester; 9,12-octadecadienoic acid methyl ester; 9,12,15-octadecatrienoic acid methyl ester, etc.) [19]. The band at ca. 1640 cm−1, resulting from C=O and C=C stretching vibrations and asymmetric N–H bending vibrations, can be due to flavonoids and lipids [21,22]. The bands at 1513 and 1417 cm−1, related to aromatic C=C stretching, are compatible with the presence of flavonoids and aromatic rings. The band at 1235 cm−1 may be due to C–O group vibration in polyols, such as hydroxyflavonoids [23].
The FTIR spectrum of the lyophilized flower hydromethanolic extract (not included in Table 2) showed bands at 3362, 2917, 2849, 1733sh, 1636, 1462, 1340, 1228, 1067 and 957 cm−1, attributable to tetradecanoic (1727, 1448, 1310 cm−1) and eicosane (2914, 2847 and 1471 cm−1).

2.4. Hydromethanolic Extracts Characterization

2.4.1. Phenolic Contents

The total phenolic content of the flower and leaf extracts were 162 ± 7 and 58 ± 2 mg GAE/g DW, respectively.

2.4.2. Analysis of Hydromethanolic Extracts by GC–MS

The main constituents identified in the flower hydromethanolic extract (Table 3 and Table S1, and Figure S3) were: tetradecanoic acid and methyl 2-hydroxy tetradecanoate (22%); eicosane (18%); 1,2-tetradecanediol (15%); sitosterol (9%); tocopherols/vitamin E (7%); and n-alcanes (heneicosane, tetracosane, pentacosane, heptacosane, etc., which add up to 6%). Among the minority constituents, it is necessary to highlight the presence of 2-ethyl-acridine (1.6%) as the only carbonitrogenated compound.
Concerning the main phytoconstituents identified in the leaf extract (Table 4 and Table S2, and Figure S4), they were: octadecatrienoic acid (linolenic acid) and their esters (above 22%); sitosterol (19%); hexadecanoic acid and their esters (above 15%); octadecadienoic acid or linoleic acid (8%); vitamin E (8%) and other tocopherols (5%); trans-pinane (5%); eicosane (4%); and phytol (4%).

2.5. Antimicrobial Activity

2.5.1. 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 COS–tetradecanoic 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).

2.5.2. In Vitro Antifungal Activity

The results from the D. seriata mycelial growth inhibition tests are shown in Figure 2 and Figure 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 EC50 and EC90 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 EC90, respectively). In all the other cases, SFs > 1 (i.e., indicative of a synergistic behavior) were also obtained, albeit more moderate.

3. Discussion

3.1. 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].

3.2. Phytochemical Composition

The eicosane content in the flower extract (18%) is higher than the one reported in the aerial parts of L. leptophyllum (Schrenk) Kuntze (8%) [28]. Concerning β-sitosterol, its presence was reported in the rhizome of L. brasiliense [6], L. myrianthum (Schrenk) Kuntze [28], L. gmelinii (Willd.) Kuntze and L. popovii Kubansk. [29] and in the aerial parts of L. axillare (Forssk.) Kuntze [30]. Tetradecanoic, linolenic and linoleic acids were reported in the aerial parts and roots of L. gmelinii and L. popovii [29], with contents in the 1–4%, 11–27% and 15–32% range, respectively (vs. 22%, 22% and 8%, respectively, for L. binervosum).
Although flavonol myricetin (3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone), reported for L. aureum [7] and L. delicatulum [8], was not found among the phytochemicals identified by GC–MS in our experimental conditions, significant amounts (7–13%) of antioxidants alternative to myricetin, such as the α-, β-, γ- and δ-tocopherols (e.g., 2-[(4R,8R)-4,8,12-trimethyltridecyl]-3,4-dihydrochromen-6-ol natural vitamin E constituents) were identified. This feature is important because in the literature [31,32] some antimicrobial activity was advocated for myricetin analogs, and a synergistic antioxidant effect of α-tocopherol and myricetin was described [33].
Concerning the TPC of the flower extract (162 mg GAE/g DW), it was higher than those reported for L. sinuatum (L.) Mill. flowers (23–34 mg GAE/g DW) [34,35], but lower than those reported for L. algarvense flower methanol extract (228 mg GAE/g DW) [5]. In regards to the TPC in the leaf extract (58 mg GAE/g DW), it was similar to those reported for L. delicatulum shoot extracts (47 mg GAE/g DW) [36]: L. densiflorum (Guss.) Kuntze shoots (50–56 mg GAE/g DW) [37,38], L. algarvense leaves (54 mg GAE/g DW) [5], and L. morisianum Arrigoni aerial parts (59 mg GAE/g DW) [39]. These values are in the lower end of the range reported by Senizza et al. [40] and Ruiz-Riaguas et al. [8] for L. delicatulum, L. quesadense, L. bellidifolium (Gouan) Dumort., L. globuliferum (Boiss. and Heldr.) Kuntze, L. gmelinii, L. iconicum (Boiss. and Heldr.) Kuntze, L. lilacinum (Boiss. and Balansa) Wagenitz and L. sinuatum aerial parts extracts (44–172 mg GAE/g DW).

3.3. Antimicrobial Activity of Limonium spp. Extracts

The use of halophytes to obtain bioactive antimicrobial extracts is recent, and the effect of the natural products derived from them was generally evaluated against human pathogens (as in the case of the extracts from Pistacia atlantica Desf., Tamarix gallica L., T. articulata Vahl, Anabasis articulata (Forssk.) Moq. or Suaeda fructicosa (L.) Forssk. [41,42,43,44]), not against phytopathogens.
In the particular case of Limonium genus., antimicrobial studies were reported for other species, such as L. brasiliense [6], L. awei (De Not.) Brullo and Erben [45,46], L. morisianum [39], L. socotranum (Vierh.) Radcl.-Sm. [47], L. echioides [48], L. densiflorum [37], L. delicatulum [36], L. myrianthum, L. leptophyllum and L. gmelinii [49], but not for L. binervosum, so direct efficacy comparisons are not possible.
Regarding the antibacterial activity, Blainski et al. [6] reported a desirable inhibition of bacterial growth for the ethyl-acetate fraction of ternary extracts of L. brasiliense against vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus and Klebsiella pneumoniae, with MIC values of 19, 39 and 625 μg·mL−1, respectively. The activity of L. awei extracts was reported by Filocamo et al. [45], with MIC and minimum bactericidal concentration (MBC) values ranging from 15.6 to 500 μg·mL−1 and from 500 to 4000 μg·mL−1, respectively, against Gram-positive bacteria and >2000 μg·mL−1 for Gram-negative bacteria. For the same Limonium species, Nostro et al. [46] reported MIC and MBC values ranging from 7.8 to 62.5 μg·mL−1 and from 500 to 2000 μg·mL−1, respectively, against S. aureus (including methicillin-resistant strains). Recently, Mandrone et al. [39] found potent anti-staphylococcal properties for L. morisianum extract, with an average IC50 value of 9.2 [6.8–12.3] μg·mL−1. Moreover, recently, Al-Madhagi et al. [47] noted that methanol leaf and flower extracts from L. socotranum exhibited higher antibacterial activity against Micrococcus luteus (MIC 15.6 μg·mL−1), S. aureus (MIC 125 μg·mL−1) and Pseudomonas aeruginosa (MIC 125 μg·mL−1) than stem extracts.
Concerning the antifungal activity of Limonium spp., a low antifungal activity was reported for L. echioides (against Fusarium oxysporum and Penicillium sp. [48]), for L. avei (against Candida albicans [46]), and for L. densiflorum and L. delicatilum (against Candida spp. [36,37]). Nonetheless, a stronger antifungal activity against C. albicans and Aspergillus niger, with full inhibition at concentrations as low as 62 and 125 μg·mL−1, respectively, were found for L. socotranum leaf and flower extracts [47]. Significant antifungal activities against C. glabrata, with IC50 values in the 4.96–6.83 μg·mL−1, were also reported for secondary metabolites from L. myrianthum, L. leptophyllum and L. gmelinii by Gadetskaya et al. [49].

3.4. 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).
Eicosane is effective against bacteria such as Escherichia coli, Salmonella typhi and S. aureus [50], and against fungi such as Rhizoctonia solani [51]. Likewise, the antimicrobial activity of β-sitosterol against both bacteria (S. typhii, Corynebacterium diphtheriae, Bacillus subtilis, Shigella dysenteriae and Vibrio cholerae) and fungi (Fusarium spp. and Penicillium spp.) was reported by Kiprono et al. [52].
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].

3.5. 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 EC90 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.
For the same phytopathogens studied herein, and also for extracts from halophytes, MIC values of 375 and 500 μg·mL−1 against X. ampelinus and 187.5 and 500 μg·mL−1 against E. amylovora were found for the conjugate complexes formed between COS and rock samphire (Crithmum maritimum L.) and sea carrot (Daucus carota subsp. gummifer (Syme) Hook.fil.) hydromethanolic extracts, respectively. Such inhibition values are worse than the one reported herein against X. ampelinus for the COS–flower extract conjugate complex (MIC = 250 μg·mL−1), but slightly better than/comparable to that obtained against E. amylovora (MIC = 500 μg·mL−1) [69].
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].

4. Material and Methods

4.1. Reagents

High-molecular weight (310,000–375,000 Da) chitosan (CAS 9012-76-4) was purchased from Hangzhou Simit Chem. and Tech. Co. (Hangzhou, China). NeutraseTM 0.8 L enzyme was obtained from Novozymes A/S (Bagsværd, Denmark). The preparation of chitosan oligomers (COS) was carried out according to the procedure reported by Santos-Moriano et al. [74], with the modifications indicated in [73].
Eicosane (CAS 112-95-8, 99%), 1,2-tetradecanoic acid (CAS 544-63-8, Sigma Grade, ≥99%), linolenic acid (CAS 463-40-1, ≥99%), β-sitosterol (CAS 83-46-5, analytical standard), vitamin E (α-tocopherol, CAS 10191-41-0, analytical standard), methanol (CAS 67-56-1, UHPLC, suitable for MS), tryptic soy broth (TSB, CAS 8013-01-2) and tryptic soy agar (TSA, CAS 91079-40-2) were supplied by Sigma-Aldrich (Madrid, Spain). Potato dextrose agar (PDA) was acquired from Becton Dickinson (Bergen County, NJ, USA). All reagents were used as supplied without further purification.

4.2. Plant Material and Extraction Procedure

L. binervosum was collected in sea cliffs in Llanes (Asturias, Spain; 43°26′10.7″ N 4°49′25.1″ W) in early September 2020. Separate composite samples of flowers and leaves were obtained by thoroughly mixing the aerial parts from different specimens (n = 15). The composite samples were shade-dried, pulverized in a mechanical grinder, homogenized and sieved (1 mm mesh).
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.

4.3. Bacterial and Fungal Isolates

The X. ampelinus and E. amylovora bacterial isolates were supplied by CECT (Valencia, Spain), with CCUG 21976 and NCPPB 595 strain designations, respectively. D. seriata (isolate Y-084-01-01a, code ITACYL_F098), obtained from “Tempranillo” grapevine plants from P.D.O. Toro (Spain), was supplied by ITACYL (Valladolid, Spain) [75] as a lyophilized vial, which was reconstituted and refreshed as a PDA subculture.

4.4. Physicochemical Characterization

Elemental analyses of dry ground samples (3 mg/sample) were performed with a CHNS-932 apparatus (LECO, St. Joseph, MI, USA).
Calorific values were calculated from elemental analysis data according to Talwalkar et al. [76], using the following equation:
HHV = ( 0.341   ×   % C ) + ( 1.322   ×   % H )   −   0.12   ( % O + % N ) ,
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 N2:O2 (4:1) flow (20 cm3·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].

4.5. 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 108 CFU·mL−1 concentration, serial dilutions were then conducted to obtain a final inoculum of ~104 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.

4.6. In Vitro Antifungal Activity Assessment

The antifungal activity of the different treatments was determined according to EUCAST 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:
( ( d c − d t ) / d c ) × 100 ,
where dc and dt represent the average diameters of the fungal colony of the control and of the treated fungal colony, respectively. EC50 and EC90 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].

4.7. 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.

5. 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 EC90 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.

Supplementary Materials

The following are available online at https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/plants10091852/s1, Figure S1: TG, DTG and DSC curves for L. binervosum flowers, Figure S2: TG, DTG and DSC curves for L. binervosum leaves, Figure S3: GC–MS spectrum of L. binervosum flower hydromethanolic extract, Figure S4: GC–MS spectrum of L. binervosum leaf hydromethanolic extract, Figure S5: Growth inhibition of D. seriata for the conjugate complexes under study, Table S1: GC‒MS results for the L. binervosum flower hydromethanolic extract, Table S2: GC‒MS results for the L. binervosum leaf hydromethanolic extract.

Author Contributions

Conceptualization, J.M.-G. and P.M.-R.; methodology, B.L.-V. and J.C.-G.; validation, B.L.-V., J.C.-G. and J.M.-G.; formal analysis, J.C.-G. and P.M.-R.; investigation, E.S.-H., L.B.-D., N.L.-L., J.C.-G., B.L.-V., J.M.-G. and P.M.-R.; resources, J.M.-G. and P.M.-R.; writing—original draft preparation, E.S.-H., L.B.-D., N.L.-L., J.C.-G., B.L.-V., J.M.-G. and P.M.-R.; writing—review and editing, P.M.-R.; visualization, E.S.-H. and L.B.-D.; supervision, P.M.-R.; project administration, J.M.-G. and P.M.-R.; funding acquisition, J.M.-G. and P.M.-R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Junta de Castilla y León under project VA258P18, with FEDER co-funding, by Cátedra Agrobank under “IV Convocatoria de Ayudas de la Cátedra AgroBank para la transferencia del conocimiento al sector agroalimentario” program; by Fundación Ibercaja-Universidad de Zaragoza under the “Convocatoria Fundación Ibercaja-Universidad de Zaragoza de proyectos de investigación, desarrollo e innovación para jóvenes investigadores” program.

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.

Acknowledgments

The authors gratefully acknowledge the support of Pilar Blasco and Pablo Candela at the Servicios Técnicos de Investigación, Universidad de Alicante, for conducting the GC–MS analyses.

Conflicts of Interest

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Caperta, A.D.; Castro, S.; Loureiro, J.; Róis, A.; Conceição, S.; Costa, J.; Rhazi, L.; Santo, D.E.; Arsénio, P. Biogeographical, ecological and ploidy variation in related asexual and sexual Limoniumtaxa (Plumbaginaceae). Bot. J. Linn. Soc. 2016, 183, 75–93. [Google Scholar] [CrossRef]
  2. Lledó, M.D.; Crespo, M.B.; Fay, M.F.; Chase, M.W. Molecular phylogenetics of Limonium and related genera (Plumbaginaceae): Biogeographical and systematic implications. Am. J. Bot. 2005, 92, 1189–1198. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Salmon, C.E. Limonium binervosum Salm. In Reports of the Botanical Society and Exchange Club; Report for 1922; Druce, G.C., Ed.; T. Buncle & Co.: Arbroath, UK, 1923; Volume 7, p. 1054. [Google Scholar]
  4. Ingrouille, M.J.; Stace, C.A. The Limonium binervosum aggregate (Plumbaginaceae) in the British Isles. Bot. J. Linn. Soc. 1986, 92, 177–217. [Google Scholar] [CrossRef]
  5. Rodrigues, M.J.; Soszynski, A.; Martins, A.; Rauter, A.; Neng, N.R.; Nogueira, J.M.; Varela, J.; Barreira, L.; Custódio, L. Unravelling the antioxidant potential and the phenolic composition of different anatomical organs of the marine halophyte Limonium algarvense. Ind. Crop. Prod. 2015, 77, 315–322. [Google Scholar] [CrossRef]
  6. Blainski, A.; Gionco, B.; Oliveira, A.G.; Andrade, G.; Scarminio, I.S.; Silva, D.B.; Lopes, N.P.; Mello, J.C. Antibacterial activity of Limonium brasiliense (Baicuru) against multidrug-resistant bacteria using a statistical mixture design. J. Ethnopharmacol. 2017, 198, 313–323. [Google Scholar] [CrossRef] [PubMed]
  7. Geng, D.; Chi, X.; Dong, Q.; Hu, F. Antioxidants screening in Limonium aureum by optimized on-line HPLC–DPPH assay. Ind. Crop. Prod. 2015, 67, 492–497. [Google Scholar] [CrossRef]
  8. Ruiz-Riaguas, A.; Zengin, G.; Sinan, K.; Salazar-Mendías, C.; Llorent-Martínez, E. Phenolic Profile, Antioxidant Activity, and Enzyme Inhibitory Properties of Limonium delicatulum (Girard) Kuntze and Limonium quesadense Erben. J. Chem. 2020, 2020, 1–10. [Google Scholar] [CrossRef] [Green Version]
  9. Mutlu-Ingok, A.; Devecioglu, D.; Dikmetas, D.N.; Karbancioglu-Guler, F.; Capanoglu, E. Antibacterial, Antifungal, Antimycotoxigenic, and Antioxidant Activities of Essential Oils: An Updated Review. Molecules 2020, 25, 4711. [Google Scholar] [CrossRef]
  10. Willems, A.; Gillis, M.; Kersters, K.; Broecke, L.V.D.; De Ley, J. Transfer of Xanthomonas ampelina Panagopoulos 1969 to a New Genus, Xylophilus gen. nov., as Xylophilus ampelinus (Panagopoulos 1969) comb. nov. Int. J. Syst. Bacteriol. 1987, 37, 422–430. [Google Scholar] [CrossRef]
  11. Szegedi, E.; Civerolo, E.L. Bacterial diseases of grapevine. Int. J. Hortic. Sci. 2011, 17, 45–49. [Google Scholar] [CrossRef] [Green Version]
  12. Donat, V.; Biosca, E.; Peñalver, J.; Lopez, M. Exploring diversity among Spanish strains of Erwinia amylovora and possible infection sources. J. Appl. Microbiol. 2007, 103, 1639–1649. [Google Scholar] [CrossRef] [PubMed]
  13. Larignon, P.; Fulchic, R.; Cere, L.; Dubos, B. Observation on Black Dead Arm in French Vineyards. Phytopathol. Mediterr. 2001, 40, 336–342. [Google Scholar] [CrossRef]
  14. Mondello, V.; Songy, A.; Battiston, E.; Pinto, C.; Coppin, C.; Trotel-Aziz, P.; Clément, C.; Mugnai, L.; Fontaine, F. Grapevine Trunk Diseases: A Review of Fifteen Years of Trials for Their Control with Chemicals and Biocontrol Agents. Plant Dis. 2018, 102, 1189–1217. [Google Scholar] [CrossRef] [Green Version]
  15. Stevens, N.E. Two Apple Black Rot Fungi in the United States. Mycologia 1933, 25, 536–548. [Google Scholar] [CrossRef]
  16. Brown-Rytlewski, D.E.; McManus, P.S. Virulence of Botryosphaeria dothidea and Botryosphaeria obtusa on Apple and Management of Stem Cankers with Fungicides. Plant Dis. 2000, 84, 1031–1037. [Google Scholar] [CrossRef] [Green Version]
  17. Ii, E.A.B. BotryosphaeriaDiseases of Apple and Peach in the Southeastern Unites Stated. Plant Dis. 1986, 70, 480. [Google Scholar] [CrossRef]
  18. Kanakiya, A.; Padalia, H.; Pande, J.; Chanda, S. Physicochemical, phytochemical and pharmacognostic study of Limonium stocksii. A halophyte from Gujarat. J. Phytopharm. 2018, 7, 312–318. [Google Scholar]
  19. da Luz, B.R. Attenuated total reflectance spectroscopy of plant leaves: A tool for ecological and botanical studies. New Phytol. 2006, 172, 305–318. [Google Scholar] [CrossRef] [PubMed]
  20. Socrates, G. Infrared and Raman Characteristic Group Frequencies: Tables and Charts, 3rd ed.; Wiley: Chichester, NY, USA, 2001; p. 347. [Google Scholar]
  21. Mot, A.; Silaghi-Dumitrescu, R.; Sarbu, C. Rapid and effective evaluation of the antioxidant capacity of propolis extracts using DPPH bleaching kinetic profiles, FT-IR and UV–vis spectroscopic data. J. Food Compos. Anal. 2011, 24, 516–522. [Google Scholar] [CrossRef]
  22. Junior, V.; Arruda, I.; Bemme, L. Caracterização térmica e espectroscópica de microcápsulas de quitosana incorporada de própolis. Rev. Eletrônica da Univar 2013, 2, 161–165. [Google Scholar]
  23. Silva, A.J.; Silva, J.R.; de Souza, N.; Souto, P.C.D.S. Membranes from latex with propolis for biomedical applications. Mater. Lett. 2014, 116, 235–238. [Google Scholar] [CrossRef]
  24. Park, S.-I.; Hwang, Y.-S.; Um, J.-S. Estimating blue carbon accumulated in a halophyte community using UAV imagery: A case study of the southern coastal wetlands in South Korea. J. Coast. Conserv. 2021, 25, 1–9. [Google Scholar] [CrossRef]
  25. Ochoa-Hueso, R.; Corona, M.E.P.; Manrique, E. Impacts of Simulated N Deposition on Plants and Mycorrhizae from Spanish Semiarid Mediterranean Shrublands. Ecosystems 2013, 16, 838–851. [Google Scholar] [CrossRef]
  26. Duca, D.; Riva, G.; Pedretti, E.F.; Toscano, G. Wood pellet quality with respect to EN 14961-2 standard and certifications. Fuel 2014, 135, 9–14. [Google Scholar] [CrossRef]
  27. Patel, M.K.; Pandey, S.; Brahmbhatt, H.R.; Mishra, A.; Jha, B. Lipid content and fatty acid profile of selected halophytic plants reveal a promising source of renewable energy. Biomass Bioenergy 2019, 124, 25–32. [Google Scholar] [CrossRef]
  28. Gadetskaya, A.; Zhusupova, G.; Ross, S. Composition of the volatile oil from aerial parts of Limonium genus. Chem. J. Kazakhstan 2015, 2, 274–279. [Google Scholar]
  29. Korul’Kina, L.M.; Zhusupova, G.E.; Shul’Ts, E.E.; Erzhanov, K.B. Fatty-acid composition of two Limonium plant species. Chem. Nat. Compd. 2004, 40, 417–419. [Google Scholar] [CrossRef]
  30. Bashir, A.; Abdalla, A.A.; Wasfi, I.A.; Hassan, E.S.; Amiri, M.H.; Crabb, T.A. Flavonoids of Limonium axillare. Int. J. Pharmacogn. 1994, 32, 366–372. [Google Scholar] [CrossRef]
  31. Yurchyshyn, O.; Rusko, G.; Kutsyk, R. Antimicrobial effect of south Ukrainian sea lavender Limonium meyeri (Boiss.) O. Kuntze and Limonium hypanicum Klok. with especial emphasizing against staphylococci and Propionibacteria. Pharma Innov. J. 2017, 6, 380–384. [Google Scholar]
  32. Taheri, Y.; Suleria, H.A.R.; Martins, N.; Sytar, O.; Beyatli, A.; Yeskaliyeva, B.; Seitimova, G.; Salehi, B.; Semwal, P.; Painuli, S.; et al. Myricetin bioactive effects: Moving from preclinical evidence to potential clinical applications. BMC Complement. Med. Ther. 2020, 20, 1–14. [Google Scholar] [CrossRef]
  33. Marinova, E.; Toneva, A.; Yanishlieva, N. Synergistic antioxidant effect of α-tocopherol and myricetin on the autoxidation of triacylglycerols of sunflower oil. Food Chem. 2008, 106, 628–633. [Google Scholar] [CrossRef]
  34. Li, A.-N.; Li, S.; Li, H.-B.; Xu, D.-P.; Xu, X.-R.; Chen, F. Total phenolic contents and antioxidant capacities of 51 edible and wild flowers. J. Funct. Foods 2014, 6, 319–330. [Google Scholar] [CrossRef]
  35. Skrajda, M.N. Phenolic compounds and antioxidant activity of edible flowers. J. Educ. Health Sport 2017, 7, 946–956. [Google Scholar]
  36. Medini, F.; Fellah, H.; Ksouri, R.; Abdelly, C. Total phenolic, flavonoid and tannin contents and antioxidant and antimicrobial activities of organic extracts of shoots of the plant Limonium delicatulum. J. Taibah Univ. Sci. 2014, 8, 216–224. [Google Scholar] [CrossRef] [Green Version]
  37. Medini, F. Effects of physiological stage and solvent on polyphenol composition, antioxidant and antimicrobial activities of Limonium densiflorum. J. Med. Plants Res. 2011, 5. [Google Scholar] [CrossRef]
  38. Medini, F.; Bourgou, S.; Lalancette, K.; Snoussi, M.; Mkadmini, K.; Coté, I.; Abdelly, C.; Legault, J.; Ksouri, R. Phytochemical analysis, antioxidant, anti-inflammatory, and anticancer activities of the halophyte Limonium densiflorum extracts on human cell lines and murine macrophages. S. Afr. J. Bot. 2015, 99, 158–164. [Google Scholar] [CrossRef]
  39. Mandrone, M.; Bonvicini, F.; Lianza, M.; Sanna, C.; Maxia, A.; Gentilomi, G.; Poli, F. Sardinian plants with antimicrobial potential. Biological screening with multivariate data treatment of thirty-six extracts. Ind. Crop. Prod. 2019, 137, 557–565. [Google Scholar] [CrossRef]
  40. Senizza, B.; Zhang, L.; Rocchetti, G.; Zengin, G.; Ak, G.; Yıldıztugay, E.; Elbasan, F.; Jugreet, S.; Mahomoodally, M.F.; Lucini, L. Metabolomic profiling and biological properties of six Limonium species: Novel perspectives for nutraceutical purposes. Food Funct. 2021, 12, 3443–3454. [Google Scholar] [CrossRef]
  41. Belyagoubi-Benhammou, N.; Belyagoubi, L.; Gismondi, A.; DI Marco, G.; Canini, A.; Bekkara, F.A. GC/MS analysis, and antioxidant and antimicrobial activities of alkaloids extracted by polar and apolar solvents from the stems of Anabasis articulata. Med. Chem. Res. 2019, 28, 754–767. [Google Scholar] [CrossRef]
  42. Benmahieddine, A.; Belyagoubi-Benhammou, N.; Belyagoubi, L.; El Zerey-Belaskri, A.; Gismondi, A.; Di Marco, G.; Canini, A.; Bechlaghem, N.; Bekkara, F.A.; Djebli, N. Influence of plant and environment parameters on phytochemical composition and biological properties of Pistacia atlantica Desf. Biochem. Syst. Ecol. 2021, 95, 104231. [Google Scholar] [CrossRef]
  43. Saïdana, D.; Mahjoub, S.; Boussaada, O.; Chriaa, J.; Mahjoub, M.A.; Chéraif, I.; Daami, M.; Mighri, Z.; Helal, A.N. Antibac-terial and antifungal activities of the essential oils of two saltcedar species from Tunisia. J. Am. Oil Chem. Soc. 2008, 85, 817–826. [Google Scholar] [CrossRef]
  44. Tabet, A.; Boukhari, A. Antioxidant and antibacterial activities of two Algerian halophytes. Int. J. Pharm. Sci. Rev. Res. 2018, 50, 114–121. [Google Scholar]
  45. Filocamo, A.; Nostro, A.; Giovannini, A.; Catania, S.; Costa, C.; Marino, A.; Bisignano, G. Antimicrobial activity of Limonium avei (De Not.) Brullo & Erben extracts. Planta Med. 2010, 76, P465. [Google Scholar] [CrossRef]
  46. Nostro, A.; Filocamo, A.; Giovannini, A.; Catania, S.; Costa, C.; Marino, A.; Bisignano, G. Antimicrobial activity and phenolic content of natural site and micropropagated Limonium avei (De Not.) Brullo & Erben plant extracts. Nat. Prod. Res. 2011, 26, 1–5. [Google Scholar] [CrossRef]
  47. Al-Madhagi, W.M.; Hashim, N.M.; Ali, N.A.A.; Othman, R. Phytochemical screening, cytotoxic and antimicrobial activities of Limonium socotranum and Peperomia blanda extracts. Trop. Biomed. 2019, 36, 11–21. [Google Scholar]
  48. Saidana, D.; Boussaada, O.; Ayed, F.; Mahjoub, M.A.; Mighri, Z.; Helal, A.N. Thein vitrofree radical-scavenging and antifungal activities of the medicinal Herblimonium Echioides L. growing wild in Tunisia. J. Food Process. Preserv. 2012, 37, 533–540. [Google Scholar] [CrossRef]
  49. Gadetskaya, A.; Mohamed, S.; Tarawneh, A.; Ma, G.; Ponomarev, B.; Zhussupova, G.; Cantrell, C.; Cutler, S.; Ross, S. Biologically potent metabolites from Limonium species. Planta Med. 2016, 81, S1–S381. [Google Scholar] [CrossRef]
  50. Firdaus, M.; Kartikaningsih, H.; Sulifah, U. Sargassum spp extract inhibits the growth of foodborne illness bacteria. AIP Conf. Proc. 2019, 2202, 020083. [Google Scholar] [CrossRef]
  51. Ahsan, T.; Chen, J.; Zhao, X.; Irfan, M.; Wu, Y. Extraction and identification of bioactive compounds (eicosane and dibutyl phthalate) produced by Streptomyces strain KX852460 for the biological control of Rhizoctonia solani AG-3 strain KX852461 to control target spot disease in tobacco leaf. AMB Express 2017, 7, 54. [Google Scholar] [CrossRef] [Green Version]
  52. Kiprono, P.C.; Kaberia, F.; Keriko, J.M.; Karanja, J.N. The in vitro Anti-Fungal and Anti-Bacterial Activities of β-Sitosterol from Senecio lyratus (Asteraceae). Z. Naturforsch. C. J. Biosci. 2000, 55, 485–488. [Google Scholar] [CrossRef]
  53. Zheng, C.J.; Yoo, J.-S.; Lee, T.-G.; Cho, H.-Y.; Kim, Y.-H.; Kim, W.-G. Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids. FEBS Lett. 2005, 579, 5157–5162. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Liu, S.; Ruan, W.; Li, J.; Xu, H.; Wang, J.; Gao, Y.; Wang, J. Biological Control of Phytopathogenic Fungi by Fatty Acids. Mycopathologia 2008, 166, 93–102. [Google Scholar] [CrossRef] [PubMed]
  55. Chen, X.; Zhao, X.; Deng, Y.; Bu, X.; Ye, H.; Guo, N. Antimicrobial potential of myristic acid against Listeria monocytogenes in milk. J. Antibiot. 2019, 72, 298–305. [Google Scholar] [CrossRef] [PubMed]
  56. Prasath, K.G.; Sethupathy, S.; Pandian, S.K. Proteomic analysis uncovers the modulation of ergosterol, sphingolipid and oxidative stress pathway by myristic acid impeding biofilm and virulence in Candida albicans. J. Proteom. 2019, 208, 103503. [Google Scholar] [CrossRef] [PubMed]
  57. Carballeira, N.M.; O’Neill, R.; Parang, K. Synthesis and antifungal properties of α-methoxy and α-hydroxyl substituted 4-thiatetradecanoic acids. Chem. Phys. Lipids 2007, 150, 82–88. [Google Scholar] [CrossRef] [Green Version]
  58. Lee, J.-Y.; Kim, Y.-S.; Shin, D.-H. Antimicrobial Synergistic Effect of Linolenic Acid and Monoglyceride againstBacillus cereusandStaphylococcus aureus. J. Agric. Food Chem. 2002, 50, 2193–2199. [Google Scholar] [CrossRef]
  59. Raynor, L.; Mitchell, A.; Walker, R. Antifungal Activities of Four Fatty Acids against Plant Pathogenic Fungi. Mycopathologia 2004, 157, 87–90. [Google Scholar] [CrossRef] [PubMed]
  60. Al-Salih, D.A.A.K.; Aziz, F.M.; Mshimesh, B.A.R.; Jehad, M.T. Antibacterial effects of vitamin E: In vitro study. J. Biotechnol. Res. Cent. 2013, 7, 17–23. [Google Scholar]
  61. Baran, R.; Thomas, L. Combination of fluconazole and alpha-tocopherol in the treatment of yellow nail syndrome. J. Drugs Dermatol. JDD 2009, 8, 276–278. [Google Scholar]
  62. Yeamsuksawat, T.; Liang, J. Characterization and release kinetic of crosslinked chitosan film incorporated with α-tocopherol. Food Packag. Shelf Life 2019, 22, 100415. [Google Scholar] [CrossRef]
  63. Martins, J.T.; Cerqueira, M.; Vicente, A.A. Influence of α-tocopherol on physicochemical properties of chitosan-based films. Food Hydrocoll. 2012, 27, 220–227. [Google Scholar] [CrossRef] [Green Version]
  64. Raza, Z.A.; Abid, S.; Azam, A.; Rehman, A. Synthesis of alpha-tocopherol encapsulated chitosan nano-assemblies and their impregnation on cellulosic fabric for potential antibacterial and antioxidant cosmetotextiles. Cellulose 2019, 27, 1717–1731. [Google Scholar] [CrossRef]
  65. Liu, C.-G.; Desai, K.G.H.; Chen, A.X.-G.; Park, H.-J. Linolenic Acid-Modified Chitosan for Formation of Self-Assembled Nanoparticles. J. Agric. Food Chem. 2004, 53, 437–441. [Google Scholar] [CrossRef] [PubMed]
  66. Kurniasih, M.; Purwati; Dewi, R.S. Toxicity tests, antioxidant activity, and antimicrobial activity of chitosan. IOP Conf. Ser. Mater. Sci. Eng. 2018, 349, 012037. [Google Scholar] [CrossRef] [Green Version]
  67. Rajaei, A.; Hadian, M.; Mohsenifar, A.; Rahmani-Cherati, T.; Tabatabaei, M. A coating based on clove essential oils encapsulated by chitosan-myristic acid nanogel efficiently enhanced the shelf-life of beef cutlets. Food Packag. Shelf Life 2017, 14, 137–145. [Google Scholar] [CrossRef]
  68. Langa-Lomba, N.; Buzón-Durán, L.; Martín-Ramos, P.; Casanova-Gascón, J.; Martín-Gil, J.; Sánchez-Hernández, E.; González-García, V. Assessment of Conjugate Complexes of Chitosan and Urtica dioica or Equisetum arvense Extracts for the Control of Grapevine Trunk Pathogens. Agronomy 2021, 11, 976. [Google Scholar] [CrossRef]
  69. Sánchez-Hernández, E.; Buzón-Durán, L.; Andrés-Juan, C.; Lorenzo-Vidal, B.; Martín-Gil, J.; Martín-Ramos, P. Physicochemical Characterization of Crithmum maritimum L. and Daucus carota subsp. gummifer (Syme) Hook.fil. and Their Antimicrobial Activity against Apple Tree and Grapevine Phytopathogens. Agronomy 2021, 11, 886. [Google Scholar] [CrossRef]
  70. Liang, C.; Yuan, F.; Liu, F.; Wang, Y.; Gao, Y. Structure and antimicrobial mechanism of ɛ-polylysine–chitosan conjugates through Maillard reaction. Int. J. Biol. Macromol. 2014, 70, 427–434. [Google Scholar] [CrossRef] [PubMed]
  71. Buzón-Durán, L.; Langa-Lomba, N.; González-García, V.; Casanova-Gascón, J.; Martín-Gil, J.; Pérez-Lebeña, E.; Martín-Ramos, P. On the Applicability of Chitosan Oligomers-Amino Acid Conjugate Complexes as Eco-Friendly Fungicides against Grapevine Trunk Pathogens. Agronomy 2021, 11, 324. [Google Scholar] [CrossRef]
  72. Buzón-Durán, L.; Martín-Gil, J.; Marcos-Robles, J.L.; Fombellida-Villafruela, A.; Pérez-Lebeña, E.; Martín-Ramos, P. Antifungal Activity of Chitosan Oligomers–Amino Acid Conjugate Complexes against Fusarium culmorum in Spelt (Triticum spelta L.). Agronomy 2020, 10, 1427. [Google Scholar] [CrossRef]
  73. Buzón-Durán, L.; Martín-Gil, J.; Pérez-Lebeña, E.; Ruano-Rosa, D.; Revuelta, J.L.; Casanova-Gascón, J.; Ramos-Sánchez, M.C.; Martín-Ramos, P. Antifungal Agents Based on Chitosan Oligomers, ε-polylysine and Streptomyces spp. Secondary Metabolites against Three Botryosphaeriaceae Species. Antibiotics 2019, 8, 99. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  74. Santos-Moriano, P.; Fernandez-Arrojo, L.; Mengíbar, M.; Belmonte-Reche, E.; Peñalver, P.; Acosta, F.N.; Ballesteros, A.O.; Morales, J.C.; Kidibule, P.; Fernandez-Lobato, M.; et al. Enzymatic production of fully deacetylated chitooligosaccharides and their neuroprotective and anti-inflammatory properties. Biocatal. Biotransform. 2017, 36, 57–67. [Google Scholar] [CrossRef] [Green Version]
  75. Martin, M.T.; Cobos, R. Identification of Fungi Associated with Grapevine Decline in Castilla y León (Spain). Phytopathol. Mediterr. 2007, 46, 18–25. [Google Scholar] [CrossRef]
  76. Talwalkar, A.T. IGT/DOE Coal-Conversion Systems Technical Data Book; Institute of Gas Technology: Chicago, IL, USA, 1981; p. 23. [Google Scholar]
  77. Jallali, I.; Zaouali, Y.; Missaoui, I.; Smeoui, A.; Abdelly, C.; Ksouri, R. Variability of antioxidant and antibacterial effects of essential oils and acetonic extracts of two edible halophytes: Crithmum Maritimum L. and Inula Crithmoïdes L. Food Chem. 2014, 145, 1031–1038. [Google Scholar] [CrossRef] [PubMed]
  78. Langa-Lomba, N.; Sánchez-Hernández, E.; Buzón-Durán, L.; González-García, V.; Casanova-Gascón, J.; Martín-Gil, J.; Martín-Ramos, P. Activity of Anthracenediones and Flavoring Phenols in Hydromethanolic Extracts of Rubia tinctorum against Grapevine Phytopathogenic Fungi. Plants 2021, 10, 1527. [Google Scholar] [CrossRef]
  79. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, 11th ed.; CLSI standard M07; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2018. [Google Scholar]
  80. Arendrup, M.C.; Cuenca-Estrella, M.; Lass-Flörl, C.; Hope, W. EUCAST technical note on the EUCAST definitive document EDef 7.2: Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST). Clin. Microbiol. Infect. 2012, 18, E246–E247. [Google Scholar] [CrossRef] [Green Version]
  81. Levy, Y.; Benderly, M.; Cohen, Y.; Gisi, U.; Bassand, D. The joint action of fungicides in mixtures: Comparison of two methods for synergy calculation. EPPO Bull. 1986, 16, 651–657. [Google Scholar] [CrossRef]
Plants 10 01852 g001 550
Figure 1. (a) habitat of Limonium binervosum (G.E.Sm.) C.E.Salmon; L. binervosum in cliffs in Llanes (Asturias, Spain): (b) whole plant and (c) flowers. Credit: habitat map generated with OpenStreetMap using GBIF data, under CC BY-NC license.
Figure 1. (a) habitat of Limonium binervosum (G.E.Sm.) C.E.Salmon; L. binervosum in cliffs in Llanes (Asturias, Spain): (b) whole plant and (c) flowers. Credit: habitat map generated with OpenStreetMap using GBIF data, under CC BY-NC license.
Plants 10 01852 g001
Plants 10 01852 g002 550
Figure 2. Radial growth of the mycelium for D. seriata in in vitro tests conducted in PDA medium with different concentrations (62.5, 93.75, 125, 187.5, 250, 375, 500, 750, 1000 and 1500 µg·mL−1) of chitosan oligomers (COS), L. binervosum flower and leaf extracts, and their main phytochemical constituents (a), and their respective conjugate complexes (b). The same letters above concentrations mean that they are not significantly different at p < 0.05. Error bars represent standard deviations.
Figure 2. Radial growth of the mycelium for D. seriata in in vitro tests conducted in PDA medium with different concentrations (62.5, 93.75, 125, 187.5, 250, 375, 500, 750, 1000 and 1500 µg·mL−1) of chitosan oligomers (COS), L. binervosum flower and leaf extracts, and their main phytochemical constituents (a), and their respective conjugate complexes (b). The same letters above concentrations mean that they are not significantly different at p < 0.05. Error bars represent standard deviations.
Plants 10 01852 g002
Table
Table 1. Elemental composition (wt%) of L. binervosum fractions.
Table 1. Elemental composition (wt%) of L. binervosum fractions.
FractionCHNSOC/N Ratio
Flowers44.7%6.5%1.3%0.3%47.2%34.9
Leaves40.5%6.4%2.6%0.9%49.6%15.7
Table
Table 2. Main bands in the FTIR spectra of L. binervosum flowers and leaves and their assignments. Peak positions are expressed in cm−1.
Table 2. Main bands in the FTIR spectra of L. binervosum flowers and leaves and their assignments. Peak positions are expressed in cm−1.
FractionAssignment
FlowersLeaves
3382Bonded O–H stretching (cellulose)
29212918–CH2 asymmetric stretching of alkyls
28522850–CH2 symmetric stretching; CH2–(C6)– bending (cellulose)
2158 CN stretching
17311728C=O stretching of alkyl ester
16531636Amide I; C=C stretching; C=O stretching
16051617Aromatic C=C skeletal stretching; COO− antisymmetric stretching (polygalacturonic, pectin ester)
1558 Amide II; COO− symmetric stretching; polynuclear aromatics
15151517C=C stretching vibrations of aromatic structures
1457
1441		1462O–CH3 stretching; C–H bending of CH2 or CH3
1417CH2 symmetric bending; aromatic C=C; COO symmetric stretching
13621372C–H (cellulose)
12361236Amide III; C–C–O asymmetric stretching acetylated glucomannan; C–O stretching of aryl ether; C–O and OH of COOH groups
11621168C–O–C in bridge asymmetric; C–C in plane
11001104C–O–C symmetric stretching
10171021C–H bending (typical of carotenes); polygalacturonic acid (a variety of pectin in plant cuticles)
874β-glycosidic linkages (glucose units of cellulose chains)
832830O–C=O in-plane deformation or a CH2 rocking deformation
720 In-plane bending or rocking of the methylenes (–CH2–)
668 C–C out-of-plane bending
Table
Table 3. Main compounds identified in L. binervosum flower hydromethanolic extract by GC‒MS.
Table 3. Main compounds identified in L. binervosum flower hydromethanolic extract by GC‒MS.
PeakRt (min)Area (%)AssignmentMW (Da)Qual
211.8420.92geranyl acetate or 2,6-octadien-1-ol, 3,7-dimethyl-, acetate (stereoisomers)196.390; 86
317.1541.03bicyclo[3.1.1]heptane, 2,6,6-trimethyl-, (1α,2β,5α) (also named trans-pinane)138.390
618.4054.94tetradecanoic acid228.493
719.6661.07heneicosane; hexacosane296.6; 366.798; 92
1121.45817.61eicosane; hexadecane, 2,6,10,14-tetramethyl-; heptadecane282.5; 282.5; 240.597; 97; 96
1323.0603.36heneicosane; pentacosane296.6; 352.796; 93
1724.60816.87tetradecanoic acid, 2-hydroxy-, methyl ester (or methyl 2-hydroxy tetradecanoate)258.493
1825.0951.66tetracosane; heptadecane, 9-octyl-; tricosane, 2-methyl-338.7; 352.7; 338.7 93; 93; 86
1925.3091.261,2-tetradecanediol230.464
2025.5382.35squalene410.798
2125.5921.21pentacosane, 13-undecyl-; heneicosane, 3-methyl-507; 310.652; 38
2225.7080.90octacosane; hexacosane394.8; 366.799; 98
2326.02514.371,2-tetradecanediol230.490
2827.2521.13γ-tocopherol416.798
2927.5541.23fumaric acid, 3,5-difluorophenyl dodecyl ester; Z-14-nonacosane396.5; 406.868; 64
3027.6073.19octacosyl trifluoroacetate; tetratriacontyl pentafluoropropionate506.8; 640.938; 38
3127.9925.56vitamin E; dl-α-tocopherol430.7; 430.799; 99
3329.1121.74campesterol400.762
3430.1738.83γ-sitosterol; β-sitosterol414.7; 414.799; 95
3531.1661.592-ethylacridine 207.390
Rt: retention time; MW: molecular weight; Qual: percentage of similarity between the molecules present in the sample and those registered in the NIST11 library. When more than one possible assignment is indicated, MW and Qual values for each of the compounds are separated by a semicolon.
Table
Table 4. Main compounds identified in L. binervosum leaf hydromethanolic extract by GC‒MS.
Table 4. Main compounds identified in L. binervosum leaf hydromethanolic extract by GC‒MS.
PeakRt (min)Area (%)AssignmentMW (Da)Qual
117.1545.41bicyclo[3.1.1]heptane, 2,6,6-trimethyl-, (1α,2β,5α) (also named (-)-trans-pinane); 3-octadecyne138.3; 250.564;58
417.5932.20cyclohexanol, 1-ethynyl-; phytol, acetate; 1-hexadecyne124.2; 338.6; 222.438; 38; 38
518.0269.83hexadecanoic acid, methyl ester270.599
618.3864.25n-hexadecanoic acid; n-decanoic acid256.4; 172.399; 90
719.6677.639,12-octadecadienoic acid (Z,Z)-, methyl ester294.599
819.74022.269,12,15-octadecatrienoic acid, methyl ester, (Z,Z,Z)-; 9,12,15-octadecatrienoic acid, (Z,Z,Z)-292.5; 278.499; 95
919.8323.80phytol296.598
1225.5381.08squalene410.799
1325.9622.82nonacosane; eicosane; docosane408.8; 282.5; 310.699; 98; 96
1426.4151.77δ-tocotrienol (or 2H-1-benzopyran-6-ol, 3,4-dihydro-2,8-dimethyl-2-(4,8,12-trimethyltridecyl)-, [2R-[2*(4R*,8R*)]]-)396.698
1527.1251.14β-tocopherol416.799
1627.2521.84γ-tocopherol; β-tocopherol; δ-tocopherol, o-methyl-416.7; 416.7; 416.797; 94; 94
1727.4761.21eicosane; octadecane282.5; 254.596; 96
1827.6071.571H-indole-2-carboxylic acid, 6-(4-ethoxyphenyl)-3-methyl-4-oxo-4,5,6,7-tetrahydro-, isopropyl ester; n-methyl-1-adamantaneacetamide355.4; 207.3140; 38
1927.9878.08α-tocopherol416.799
2028.0701.35phytol, acetate; 2-(4-fluoro-phenyl)-4-(3-methyl-benzylidene)-4h-oxazol-5-one338.6; 281.349; 43
2130.16319.15γ-sitosterol; β-sitosterol414.7; 414.799; 99
Rt: retention time; MW: molecular weight; Qual: percentage of similarity between the molecules present in the sample and those registered in the NIST11 library. When more than one possible assignment is indicated, MW and Qual values for each of the compounds are separated by a semicolon.
Table
Table 5. Antibacterial activity of chitosan oligomers (COS), L. binervosum flower and leaf hydromethanolic extracts, their main constituents (eicosane, tetradecanoic acid, linolenic acid, β-sitosterol and vitamin E), and their corresponding conjugate complexes (COS–flower extract, COS–leaf extract, COS–eicosane, COS–tetradecanoic acid, COS–linolenic acid, COS–β-sitosterol and COS–vitamin E) against the two phytopathogenic bacteria under study at different concentrations (expressed in μg·mL−1).
Table 5. Antibacterial activity of chitosan oligomers (COS), L. binervosum flower and leaf hydromethanolic extracts, their main constituents (eicosane, tetradecanoic acid, linolenic acid, β-sitosterol and vitamin E), and their corresponding conjugate complexes (COS–flower extract, COS–leaf extract, COS–eicosane, COS–tetradecanoic acid, COS–linolenic acid, COS–β-sitosterol and COS–vitamin E) against the two phytopathogenic bacteria under study at different concentrations (expressed in μg·mL−1).
PathogenCompoundConcentration (μg·mL−1)
62.593.75125187.525037550075010001500
X. ampelinusCOS+++++++++−
Flower extract+++++++++−
Leaf extract+++++++++−
Eicosane++++++++−−
β-sitosterol++++++++−−
Tetradecanoic acid++++++−−−−
Linolenic acid++++++−−−−
Vitamin E++++++−−−−
COS–flower extract++++−−−−−−
COS–leaf extract+++++++−−−
COS–eicosane+++++++−−−
COS–β-sitosterol++++++−−−−
COS–tetradecanoic acid++++−−−−−−
COS–linolenic acid++++−−−−−−
COS–vitamin E++++−−−−−−
E. amylovoraCOS+++++++++−
Flower extract+++++++++−
Leaf extract+++++++++−
Eicosane++++++++++
β-sitosterol+++++++++−
Tetradecanoic acid++++++−−−−
Linolenic acid++++++−−−−
Vitamin E+++++++−−−
COS–flower extract++++++−−−−
COS–leaf extract+++++++−−−
COS–eicosane+++++++++−
COS–β-sitosterol+++++−−−−−
COS–tetradecanoic acid++++−−−−−−
COS–linolenic acid++++−−−−−−
COS–vitamin E++++−−−−−−
“+” and “−“ indicate presence and absence of bacterial growth, respectively.
Table
Table 6. EC50 and EC90 effective concentrations for the different treatments, expressed in µg·mL−1.
Table 6. EC50 and EC90 effective concentrations for the different treatments, expressed in µg·mL−1.
ECCOSFlower ExtractLeaf ExtractEicosaneβ-SitosterolTetradecanoicLinolenicVitamin E
EC50744 ± 42845 ± 191033 ± 107154 ± 2982 ± 11153 ± 17227 ± 17212 ± 13
EC901180 ± 461555 ± 712167 ± 2151023 ± 96151 ± 26394 ± 49538 ± 73434 ± 57
EC COS–Flower ExtractCOS–Leaf ExtractCOS–EicosaneCOS–β-SitosterolCOS–TetradecanoicCOS–LinolenicCOS–Vitamin E
EC50 611 ± 33625 ± 20234 ± 1351 ± 2109 ± 239 ± 1217 ± 7
EC90 914 ± 75966 ± 64678 ± 54124 ± 4130 ± 4129 ± 8406 ± 10
Table
Table 7. Synergy factors, estimated according to Wadley’s method, for the conjugate complexes under study.
Table 7. Synergy factors, estimated according to Wadley’s method, for the conjugate complexes under study.
ECCOS–Flower ExtractCOS–Leaf ExtractCOS–EicosaneCOS–β-SitosterolCOS–TetradecanoicCOS–LinolenicCOS–Vitamin E
EC501.301.381.092.902.338.981.52
EC901.471.581.632.154.555.751.56
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Sánchez-Hernández, E.; Buzón-Durán, L.; Langa-Lomba, N.; Casanova-Gascón, J.; Lorenzo-Vidal, B.; Martín-Gil, J.; Martín-Ramos, P. Characterization and Antimicrobial Activity of a Halophyte from the Asturian Coast (Spain): Limonium binervosum (G.E.Sm.) C.E.Salmon. Plants 2021, 10, 1852. https://0-doi-org.brum.beds.ac.uk/10.3390/plants10091852

AMA Style

Sánchez-Hernández E, Buzón-Durán L, Langa-Lomba N, Casanova-Gascón J, Lorenzo-Vidal B, Martín-Gil J, Martín-Ramos P. Characterization and Antimicrobial Activity of a Halophyte from the Asturian Coast (Spain): Limonium binervosum (G.E.Sm.) C.E.Salmon. Plants. 2021; 10(9):1852. https://0-doi-org.brum.beds.ac.uk/10.3390/plants10091852

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Sánchez-Hernández, Eva, Laura Buzón-Durán, Natalia Langa-Lomba, José Casanova-Gascón, Belén Lorenzo-Vidal, Jesús Martín-Gil, and Pablo Martín-Ramos. 2021. "Characterization and Antimicrobial Activity of a Halophyte from the Asturian Coast (Spain): Limonium binervosum (G.E.Sm.) C.E.Salmon" Plants 10, no. 9: 1852. https://0-doi-org.brum.beds.ac.uk/10.3390/plants10091852

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