Armeria maritima (Mill.) Willd. Flower Hydromethanolic Extract for Cucurbitaceae Fungal Diseases Control

The cliff rose (Armeria maritima), like other halophytes, has a phenolics-based antioxidant system that allows it to grow in saline habitats. Provided that antioxidant properties are usually accompanied by antimicrobial activity, in this study we investigated the phytochemicals present in a hydromethanolic extract of A. maritima flowers and explored its antifungal potential. The main phytocompounds, identified by gas chromatography–mass spectrometry, were: hexadecanoic acid, octadecanoic acid, 9-octadecenoic acid, 3-(3,4-dihydroxy-phenyl)-acrylic acid ethyl ester, and benzeneacetaldehyde. The antifungal activity of the extract and its main constituents—alone and in combination with chitosan oligomers—was tested against six pathogenic taxa associated with soil-borne diseases of plant hosts in the family Cucurbitaceae: Fusarium equiseti, F. oxysporum f. sp. niveum, Macrophomina phaseolina, Neocosmospora falciformis, N. keratoplastica, and Sclerotinia sclerotiorum. In in vitro tests, EC90 effective concentrations in the 166−865 μg·mL−1 range were obtained for the chitosan oligomers–A. maritima extract conjugate complexes, lower than those obtained for fosetyl-Al and azoxystrobin synthetic fungicides tested for comparison purposes, and even outperforming mancozeb against F. equiseti. In ex situ tests against S. sclerotiorum conducted on artificially inoculated cucumber slices, full protection was achieved at a dose of 250 μg·mL−1. Thus, the reported results support the valorization of A. maritima as a source of biorationals for Cucurbitaceae pathogens protection, suitable for both organic and conventional agriculture.


Introduction
Armeria maritima (Mill.) Willd. (Plumbaginaceae), commonly known as sea thrift, sea rose, or cliff rose, is a compact, evergreen perennial plant that grows on cliffs and seashores in Iceland, the Atlantic coast of Europe, and the western region of the Baltic Sea [1].
Armeria maritima has been studied due to its potential for bioremediation, given its high tolerance to heavy metals [2]. Being a halophyte, A. maritima has a powerful antioxidant system based on phenolic acids and flavonoids [3,4]. Due to salinity, proline is the main amino acid [5]. Other bioactive compounds include β-alaninebetaine, glycinebetaine, and choline-O-sulphate [6]; gallic, caffeic, p-hydroxybenzoic as phenolic acids; and myricitrin, quercetin, and kaempferol glycoside flavonoids [3,4,7]. Table 1 provides a summary of the main absorption bands observed in the infrared spectra of flowers, stems, and roots of A. maritima. The identified functional groups are compatible with the presence of alkaloids, polyphenols, organic acid esters, and other phytoconstituents (elucidated by GC-MS).

Antifungal Activity of the Extract 2.3.1. In Vitro Antifungal Activity
The results of the antifungal susceptibility tests are summarized in Figure 3. For all the products assayed, higher concentrations led to lower radial growth of the fungal mycelium, resulting in statistically significant differences. In all cases, COS inhibited mycelial growth at 1500 µg·mL −1 ; meanwhile, the hydromethanolic extract of flowers achieved full inhibition at concentrations ranging from 375 to 1500 µg·mL −1 , depending on the fungal taxa tested. Comparatively, the main constituents of the extract, i.e., hexadecanoic acid, 9-octadecenoic acid, and octadecanoic acid, exhibited similar or better activity than the whole extract.
The formation of conjugate complexes further enhanced antifungal activity; COS-A. maritima extract led to complete inhibition at concentrations in the 250-1000 µg·mL −1 range, whereas full inhibition occurred at concentrations in the 78.12-375, 78.12-250, and 70.31-375 µg·mL −1 range for COS-hexadecanoic acid, COS-9-octadecenoic acid, and COSoctadecanoic acid conjugate complexes, respectively. To quantify this improved activity, effective concentration values were first calculated (Table 3), followed by synergy factors (Table 4) determined using the Wadley method. As a result, synergism (i.e., SFs > 1) was detected in all cases.     The results of the antifungal susceptibility tests are summarized in Fig the products assayed, higher concentrations led to lower radial growth of th celium, resulting in statistically significant differences. In all cases, COS inhib growth at 1500 μg·mL −1 ; meanwhile, the hydromethanolic extract of flowers inhibition at concentrations ranging from 375 to 1500 μg·mL −1 , depending o taxa tested. Comparatively, the main constituents of the extract, i.e., hexadec octadecenoic acid, and octadecanoic acid, exhibited similar or better activ whole extract. The formation of conjugate complexes further enhanced antifu The results of mycelial growth inhibition using three conventional synthetic fungicides chosen for comparison are presented in Table 5. The highest inhibition rates were observed for the dithiocarbamate fungicide (mancozeb), which fully inhibited the mycelial growth of all phytopathogens at one-tenth of the manufacturer's recommended dose (that is, 150 µg·mL −1 ), apart from F. equiseti, which was not completely inhibited at 1500 µg·mL −1 . The organophosphate fungicide (fosetyl-Al) led to full inhibition of all fungus taxa at the recommended dose (i.e., 2000 µg·mL −1 ), except for F. equiseti and S. sclerotiorum. The strobilurin fungicide (azoxystrobin) was the least effective, failing to fully inhibit the growth of all phytopathogens at the recommended dose (62,500 µg·mL −1 ), except for N. keratoplastica.

Ex Situ Antifungal Activity
Given that the COS−A. maritima conjugate complex was the most active product according to the previous in vitro tests, it was further tested as a protective treatment against white mold on cucumber fruits cv. "Urano". Three different concentrations, corresponding to the minimum inhibitory concentration (MIC), MIC×2, and MIC×4 (i.e., 250, 500, and 1000 µg·mL −1 , respectively), were assayed. Results are shown in Figure 4. In the positive control (i.e., S. sclerotiorum artificially inoculated on cucumber slices treated only with bi-distilled water), slices were fully colonized by the mold on the fifth day after inoculation and sclerotia were produced on the seventh day. In contrast, full protection was observed for the treated slices even at the lowest concentration (250 µg·mL −1 ), with an inhibition rate of 100%. Upon comparison of the slices' weight evolution (Table 6), significant differences (p < 0.0001) were detected for the between-subjects and within-subjects effects, i.e., both time and treatment had a significant impact on the slices' weight. A much more marked weight decrease, as a result of tissue maceration, was observed for the positive control, with no statistically significant differences between the negative control and the treated samples.

On the Phytochemical Profile Obtained by GC−MS
Considering that the chosen hydromethanolic extraction mixture also solubilizes polar compounds (non-volatile) that cannot be detected by GC−MS without derivatization of the extract, it is important to note that such prior derivatization was not done in the present work due to the associated drawbacks. These include making the procedural preparation steps longer and more expensive (which would decrease the economic viability of the crop protection treatments), increased complexity and length of the data acquisition process due to the potential for impurities and the uncertainty of conversion of compounds into derivatives, as well as the use of toxic reagents [31]. Additionally, the injection of non-volatile compounds may result in damage to the GC capillary column.

On the Phytochemical Profile Obtained by GC-MS
Considering that the chosen hydromethanolic extraction mixture also solubilizes polar compounds (non-volatile) that cannot be detected by GC-MS without derivatization of the extract, it is important to note that such prior derivatization was not done in the present work due to the associated drawbacks. These include making the procedural preparation steps longer and more expensive (which would decrease the economic viability of the crop protection treatments), increased complexity and length of the data acquisition process due to the potential for impurities and the uncertainty of conversion of compounds into derivatives, as well as the use of toxic reagents [31]. Additionally, the injection of non-volatile compounds may result in damage to the GC capillary column.
9-Octadecenoic acid (trans-oleic acid or elaidic acid) has been found in small amounts in pomegranates, peas, cabbage [42], Foeniculum vulgare Mill. [43], and Landolphia owariensis Beauv. [44]. In pot experiments conducted by Liu et al. [41], the mixture of palmitic and oleic acids was found to enhance the growth of tomato and cucumber seedlings.
The presence in the GC-MS chromatogram (at Rt = 12.18 min) of 2,1,3-benzothiadiazole (BTD) in a significant percentage (8.49%) but with a low Qual (<55) is a striking finding. BTD is a synthetic product used as an agrochemical, whose presence should be tentatively attributed to contamination. Nevertheless, it has previously been identified in a higher percentage (12.26%) in the ethanolic extract of Lawsonia inermis L. [50], so a possible natural origin cannot be completely ruled out. BTD is a plant defense inducer that has been used for the protection of various agronomically important crops, such as rice, wheat, potato, and tomato [51].
Methyl β-D-glucopyranoside, also known as β-methyl-D-glucoside (MeG) or methyl hexopyranoside (5.8%, Qual 58), is an O-glycosyl compound that has been found as a major compound in the leaves of the alpine herb Geum montanum L. and other plants of the Rosaceae family [52], as well as in Echinospartum horridum (Vahl) Rothm. [53]. It has been suggested that, like other methylated molecules (i.e., methyl-inositols), it might be involved in tolerance to osmotic stress [52].
The underlying mode of action of these fatty acids has been mainly studied in human pathogenic fungi, not specifically against phytopathogens [41]. Nevertheless, it has been suggested that it involves their insertion into fungal membrane lipid bilayers, compromising membrane integrity and leading to uncontrolled release of intracellular proteins and electrolytes, ultimately resulting in cytoplasmic disintegration of fungal cells [59]. Hydrostatic turgor pressure within the cell leading to disruption of the fungal membrane has also been suggested as a mechanism of fungicidal action [60]. Additionally, fatty acids have been found to inhibit topoisomerase I, an enzyme involved in DNA strand breakage and repair and topological changes necessary for cellular processes [61], as well as N-myristoyltransferase, resulting in inhibition of fungal growth [62].
Nonetheless, it is worth noting that other constituents not tested as individual compounds may also contribute to the antifungal activity (as discussed in Section 3.1, based on other studies reported in the literature) and that the presence of synergism between phytoconstituents cannot be discounted.
With regard to COS, its antifungal activity is well-established [63], and is thought to be due to its positive charge interacting with the negative charge of the fungal cell membrane. This interaction leads to increased cell permeability [64], resulting in a loss of intracellular components which disrupts the osmotic pressure and causes cell death [65]. COS can also alter chitin levels, leading to a weakened cell wall [66], and can generate ROS that damage biomolecules, triggering apoptosis and necrosis. Additionally, COS can interfere with DNA and RNA synthesis [67].
Concerning the enhanced activity upon the formation of conjugate complexes, without additional in-detail experiments on the mechanism of its action, only an educated guess can be made at this stage. The observed synergism may stem from an enhanced additive fungicidal activity per se or by simultaneous action on multiple fungal metabolic sites [68], but it may also be due to an increase in the solubility and bioavailability of the bioactive compounds present in the extract mediated by COS.
In line with the rationale behind the use of synthetic fungicides in pairs (not only to help prevent resistance development but also to benefit from the enhanced efficacy resulting from different modes of action), the better performance of the natural product versus the conventional fungicides may be tentatively attributed to the complex mixture of compounds found in the plant extract, given that these compounds may act synergistically to produce a more potent antifungal effect than synthetic fungicides based on one molecule. Table 7 presents a comparison of the efficacies reported for plant extracts and essential oils against five of the six studied phytopathogens. However, it should be noted that there are no data available for N. falciformis. It is important to exercise caution when interpreting these results, as the sensitivity may vary depending on the isolate. For instance, values for F. oxysporum spp. are presented due to the absence of specific data for F. oxysporum f. sp. niveum. Additionally, the results may be expressed in different forms (MIC values, inhibition rates, inhibition zones, etc.).    Aqueous extract A. sativum leaves MIC = 5000 µg·mL −1 [84] IR: inhibition rate; IZ: inhibition zone; MIC: minimum inhibitory concentration; MIC 50 : minimum inhibitory concentration that inhibited 50% of the radial growth; n.a.: no activity at the highest con-centration tested.

Comparison with Other Extracts Tested In Vitro against the Phytopathogens under Study
The non-conjugated A. maritima extract exhibited MIC values (1000, 750, 750, 1000, and 375 µg·mL −1 ) that are among the lowest for extracts. However, it is worth noting that some essential oils showed better performance. For F. equiseti, only Plumbago zeylanica L. root and Tamarix gallica L. bark extracts were more effective. Against F. oxysporum spp. and S. sclerotiorum, only Cestrum nocturnum L. flower extracts demonstrated activity comparable to that of the extract of A. maritima. Against M. phaseolina, it was only outperformed by Oxalis corniculata L., P. zeylanica, and Antigonon leptopus Hook. & Arn. extracts. Against N. keratoplastica, the efficacy of A. maritima extract was comparable to that of essential oils.

Comparison with Other Extracts Tested Ex Situ for Cucumber Protection
There is a limited amount of research that has investigated the use of natural extracts to inhibit white mold on cucumber ex situ. In particular, extracts of Cornus mas L. (fruits or leaves), Morus alba L. (immature fruits or leaves), and Prunus laurocerasus L. (leaves) at 1000 mg·mL −1 were shown to arrest the development of S. sclerotiorum on cucumber, with inhibition percentages in the 94 to 100% range [96]. Another study conducted by the same group [97] found that chitosan at 2000 µg·mL −1 was also effective in protecting cucumber fruits against S. sclerotiorum lesions. In comparison with the aforementioned treatments, the efficacy of the COS-A. maritima conjugate complex was notably higher.

Limitations of the Study and Further Research
While the preliminary in vitro and ex situ results suggest that the proposed COS-A. maritima conjugate complexes have potential as antifungal agents against Cucurbitaceae fungal pathogens, further research is needed to assess their practical applicability for crop protection. Tests with different fungal strains would be required to factor in differences in sensitivity, and field tests should be conducted on various Cucurbitaceae species. Furthermore, the impact of the treatment on other Cucurbitaceae bacterial and fungal pathogens not tested in this study should also be taken into consideration if traditional fungicides are to be replaced with this alternative based on natural products. Additionally, the timing of application, dosage, and other practical aspects such as cost, degradation tolerance, and efficacy of long-term protection should also be carefully evaluated in future studies.

Preparation of Armeria Extract, Chitosan Oligomers, and Conjugate Complexes
The flower samples were mixed (1:20 w/v) with a methanol/water solution (1:1 v/v) and heated in a water bath at 50 • C for 30 min, followed by sonication for 5 min in pulse mode with a 1-min stop every 2.5 min, using a model UIP1000 hdT probe-type ultrasonicator from Hielscher Ultrasonics (Teltow, Germany). The solution was then centrifuged at 9000 rpm for 15 min and the supernatant was filtered through Whatman No. 1 paper. For subsequent GC-MS analysis, 25 mg of the obtained freeze-dried extracts were dissolved in 5 mL of HPLC-grade MeOH to obtain a 5 mg·mL −1 solution, which was further filtered.
Chitosan oligomers were prepared according to the procedure previously described by our group [98], yielding a solution with a pH ranging from 4 to 6, containing oligomers of molecular weight less than 2 kDa.
The COS-A. maritima extract and COS−main bioactive compounds conjugate complexes were obtained by mixing the respective solutions in a 1:1 (v/v) ratio, followed by sonication for 15 min in 5 3-min pulses (so that the temperature did not exceed 60 • C). Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy of the freeze-dried products was used to confirm the formation of the conjugate complexes.

Physicochemical Characterization
A Nicolet iS50 Fourier-transform infrared spectrometer from Thermo Scientific (Waltham, MA, USA) with an in-built diamond attenuated total reflection (ATR) system was utilized to collect the infrared vibrational spectra of plant organs. The spectra were registered between 400 and 4000 cm −1 , with a spectral resolution of 1 cm −1 , co-adding 64 scans. A gas chromatograph model 7890A coupled to a quadrupole mass spectrometer model 5975C (both from Agilent Technologies, Santa Clara, CA, USA) was used to elucidate the constituents of A. maritima flowers hydromethanolic extract by gas chromatography-mass spectrometry (GC-MS). This characterization was outsourced to the research support services (STI) of the Universidad de Alicante (Alicante, Spain). The chromatographic conditions were: injection volume = 1 µL; injector temperature = 280 • C, in splitless mode; initial oven temperature = 60 • C, held for 2 min, followed by a ramp of 10 • C·min −1 up to a final temperature of 300 • C, held for 15 min. An HP-5MS UI chromatographic column (30 m length, 0.250 mm diameter, 0.25 µm film), also from Agilent Technologies, was employed for the separation of the compounds. The mass spectrometer conditions were: temperature of the electron impact source of the mass spectrometer = 230 • C and the quadrupole = 150 • C; ionization energy = 70 eV. The identification of components was based on a comparison of their mass spectra and retention times with those of authentic compounds and by computer matching with the database of the National Institute of Standards and Technology (NIST11).

In Vitro Antifungal Activity Assessment
The antifungal activity of the various treatments (including COS, the A. maritima flower extract, its main constituents (hexadecanoic acid, 9-octadecenoic acid, and octadecanoic acid), the conjugate complexes of all of them with COS, and certain commercial synthetic fungicides) was determined using the agar dilution method as per the EUCAST antifungal susceptibility testing standard procedures [99]. Stock solution aliquots were incorporated into the pouring PDA medium to produce final concentrations ranging from 15.62 to 1500 µg·mL −1 . Mycelial plugs (∅ = 5 mm), from the margin of 1-week-old PDA cultures of F. equiseti, F. oxysporum f. sp. niveum, M. phaseolina, N. falciformis, N. keratoplastica, and S. sclerotiorum were transferred to the center of PDA plates prepared with the aforementioned concentrations (3 plates per treatment and concentration, with 2 duplicates). The plates were incubated at 25 • C in the dark for 1 week. The control consisted in replacing the extract with the solvent used for extraction (i.e., methanol:water 1:1 v/v) in the PDA medium. Inhibition of mycelial growth was estimated according to the formula ((d c − d t )/d c ) × 100, where d c and d t represent the mean diameters of the control and treated fungal colonies, respectively. Given that the homogeneity and homoscedasticity requirements were met (according to Shapiro-Wilk and Levene tests, respectively), the results of mycelial growth inhibition were statistically analyzed in IBM SPSS Statistics (IBM, New York, NY, USA) v.25 software using one-way analysis of variance (ANOVA), followed by post hoc comparison of means using Tukey's test at p < 0.05.
Effective concentrations (EC 50 and EC 90 ) were determined via PROBIT analysis in IBM SPSS Statistics v.25. Interaction levels, i.e., synergy factors (SF), were estimated according to the Wadley method [100], which is based on the notion that one component of the mixture can substitute at a constant proportion for the other. Therefore, the anticipated efficacy of the mixture can be directly determined from the efficacy of the constituents when the relative proportions are known (as is the case here). SF = 1 indicates similar joint action (i.e., additivity), SF > 1 implies synergistic action, and SF < 1 implies antagonistic action between the two fungicide products.

Post-Harvest Protection Test in Cucumber
The cucumber fruits (C. sativus cv. "Urano") used to ascertain the ex situ protective effect of COS−A. maritima conjugate complex against S. sclerotiorum were sourced from the 'Huerta de Carabaña' orchard (Carabaña, Madrid, Spain) and previously grown under organic farming standards, without the use of synthetic pesticides. To begin the experiments within 24 h of harvest, the fruits were picked and sent by refrigerated express courier service. During selection, the fruits were chosen for their firmness, consistent size, caliber, lack of physical damage, and absence of signs of bacterial or fungal infection.
In controlled laboratory conditions, the efficacy of the treatment was determined by artificial inoculation of cucumber slices. The procedure was slightly modified from that proposed by Onaran and Yanar [96] and described in Sánchez-Hernández et al. [101]. The cucumber fruits were initially disinfected with a 3% NaOCl solution for 2 min, washed 3 times with sterile distilled water and dried in a laminar-flow hood on sterile absorbent paper. Then, under sterile conditions, cucumber fruits were cut into 8 mm-thick slices with a sterile knife. In each Petri plate containing sterile filter paper, one cucumber slice was placed, and a superficial wound (ø = 3 mm) was created in the equatorial zone of each slice. In these wounds, 100 µL of the COS−A. maritima conjugate complex at three concentrations (at the MIC obtained in previous in vitro assays, at MIC×2, and at MIC×4, i.e., 250, 500, and 1000 µg·mL −1 , respectively) were applied, followed by a two-hour waiting period for complete absorption. Then, a plug of S. sclerotiorum PDA culture was placed in each wound (with the mycelium facing the fruit wound). In the negative control, wounds were treated only with distilled water (without the pathogen), while positive controls were treated with distilled water and inoculated with the pathogen. All cucumber slices were incubated at 22 ± 2 • C and 75-90% RH for 7 days. Cucumber slices were weighed daily to study weight loss and disease progression, with the weight of each slice on day 0 being set as 100%. The experiment was conducted using three replicates, repeated three times. The results were statistically analyzed by repeated measures ANOVA with post hoc comparison of means by Tukey's test.

Conclusions
The application of GC-MS to an hydromethanolic extract of A. maritima flowers identified hexadecanoic acid (18%), 9-octadecenoic acid (14%), and octadecanoic acid (9%) as its main phytoconstituents. Subsequent antifungal tests against F. equiseti, F. oxysporum f. sp. niveum, N. falciformis, N. keratoplastica, M. phaseolina, and S. sclerotiorum revealed that the extract had strong inhibitory effects, with MIC values ranging from 375 to 1500 µg·mL −1 . This activity was even more prominent after conjugation with chitosan oligomers, resulting in MICs between 250 and 1000 µg·mL −1 depending on the fungal taxa. In comparison, these inhibitory effects were greater than those of conventional chemicals such as fosetyl-Al and azoxystrobin and, in the case of F. equiseti, exceeded those of mancozeb. The conjugate complex was also tested as a protective treatment in ex situ experiments on cucumber slices artificially inoculated with S. sclerotiorum, showing full inhibition at a concentration of 250 µg·mL −1 . The results suggest that the extracts of this halophyte could be valorized as biorationals for the protection of cucurbits against certain soil-borne diseases. However, further studies are needed to assess the impact of the proposed treatment on other Cucurbitaceae pathogens and long-term protection. Additionally, practical aspects for its field application need to be optimized.

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