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Article

Silene uniflora Extracts for Strawberry Postharvest Protection

by
Laura Buzón-Durán
1,2,
Eva Sánchez-Hernández
1,*,
Pablo Martín-Ramos
1,
Luis Manuel Navas-Gracia
1,
Mari Cruz García-González
2,
Rui Oliveira
3 and
Jesús Martín-Gil
1
1
Department of Agricultural and Forestry Engineering, ETSIIAA, Universidad de Valladolid, 34004 Palencia, Spain
2
Department of Agroforestry Sciences, ETSIIAA, Universidad de Valladolid, Avenida de Madrid 44, 34004 Palencia, Spain
3
Centre of Molecular and Environmental Biology (CBMA), Department of Biology, School of Sciences, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
*
Author to whom correspondence should be addressed.
Plants 2023, 12(9), 1846; https://doi.org/10.3390/plants12091846
Submission received: 23 March 2023 / Revised: 23 April 2023 / Accepted: 27 April 2023 / Published: 29 April 2023
(This article belongs to the Special Issue Plant Extracts as Biological Protective Agents)
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Abstract

:
Halophytes are gaining considerable attention due to their applications in saline agriculture, phytoremediation, medicine, and secondary metabolite production. This study investigated the bioactive components present in Silene uniflora (sea campion) hydromethanolic extract, and their antimicrobial activity was evaluated both in vitro and ex situ against two strawberry phytopathogens, namely Botrytis cinerea (grey mold) and Colletotrichum nymphaeae (anthracnose fruit rot). The main identified phytochemicals were mome inositol, saturated fatty acid esters, and cyclotetracosane. In vitro tests demonstrated complete inhibition of the growth of B. cinerea and C. nymphaeae at extract concentrations of 1000 and 1500 μg·mL−1, respectively, with an activity comparable to that of fosetyl-Al and substantially higher than that of azoxystrobin. This activity was improved upon conjugation with chitosan oligomers (COS), yielding inhibition values of 750 and 1000 μg·mL−1. The COS-S. uniflora conjugate complexes were then tested as protective treatments for postharvest storage of strawberry fruit, resulting in high protection against artificially inoculated B. cinerea and C. nymphaeae at doses of 3750 and 5000 μg·mL−1, respectively. The reported results open the door to the valorization of this halophyte as a source of biorationals for strawberry protection.

1. Introduction

Silene uniflora Roth is an herbaceous perennial plant of the Caryophyllaceae family, typically forming mats on cliffs. Its leaves are linear, gray-green, and glaucous, and the flowers are white with five sepals that form a bladder and five deeply notched petals (Figure 1a).
Limited research has been conducted on the chemical composition of Silene spp. Triterpene saponins and pectic polysaccharides have been isolated from Silene vulgaris (Moench) Garcke [1,2,3], and the oil compositions of various Silene species have also been investigated [4,5,6]. The phytochemical profiling of various extracts from species in the Silene genus has demonstrated the presence of sterols, alkaloids, tannins, flavonoids [7,8], and phytoecdysteroids (such as 2,22-dideoxyecdysone 25-O-β-d-glucopyranoside, Figure 1b) [9,10,11]. Coumaric acid derivatives and catechin were reported in the leaves and procyanidin B1 in the root extract of S. vulgaris subsp. macrocarpa [12]. Quinic, malic, protocatechuic, and p-coumaric acids, as well as hesperidin, were identified in six Silene species (i.e., Silene alba (Mill.) E.H.L.Krause, Silene conoidea L., Silene dichotoma Ehrh., Silene italica (L.) Pers., Silene supina M.Bieb., and S. vulgaris) [13]. However, the phytochemical profile of S. uniflora has yet to be reported.
Regarding antimicrobial activity, there have been promising reports on Silene parishii S.Watson [14], S. vulgaris [15,16], Silene cariensis Boiss. [17], S. alba, S. conoidea, S. dichotoma, S. italica, S. supina, and S. vulgaris [13], but there is a dearth of information on the activity of S. uniflora.
The aim of the study presented herein was two-fold: (i) to investigate the phytoconstituents of S. uniflora using vibrational spectroscopy (IR) and gas chromatography–mass spectrometry (GC-MS) and (ii) to evaluate S. uniflora antifungal activity, alone and in combination with chitosan oligomers (COS), and open new pathways for its valorization. In particular, two of the most important strawberry (Fragaria × ananassa Duch.) pathogens were selected: Botrytis cinerea Pers. and Colletotrichum nymphaeae (Pass.) Aa., which ranked second and eighth on a list of fungal pathogens of scientific and economic importance, respectively [18]. The former (grey mold) has a wide host range (over 200 plant species) and potential for causing severe damage, both pre- and post-harvest; the latter belongs to the Colletotrichum acutatum J.H.Simmonds clade and causes anthracnose [19]. The reported findings may contribute to the management of these diseases in agricultural ecosystems.

2. Results

2.1. Vibrational Characterization

Prior to extract preparation, infrared spectra of the different aerial plant organs of S. uniflora (Figure S1) were analyzed in an initial screening to identify functional groups and assess the presence/absence of significant differences among them. The fingerprint regions of the three spectra were very similar. Band assignments are summarized in Table 1. The band at 3366 cm−1 can be attributed to hydrogen bonding in pyranosides [20], and those at ca. 2916, 1443, 1371, 1244, and 1146 cm−1 are consistent with the presence of inositol, detected by GC-MS, as discussed below. Concerning the presence of saturated fatty acid vinyl esters, this is supported by the bands at 2916, 2848, 1636, 1472, 1417, 1378, 1307, 1243, 1146, 1101, and 719 cm−1.

2.2. GC-MS Characterization

The main phytochemicals identified in the extract prepared from a mixture of the aerial organs of the plant (Table 2, Figure S2) were: 4-O-methyl-myo-inositol or mome inositol (52.5%), saturated fatty acid vinyl esters (8.7%, Figure S3), and cyclotetracosane (3.7%), depicted in Figure 2.

2.3. In Vitro Growth Inhibition Tests

The results of the mycelial growth inhibition tests (Figure S4) are summarized in Figure 3. When tested separately, S. uniflora showed greater efficacy against B. cinerea than COS, as full inhibition was reached at 1000 and 1500 μg·mL−1, respectively. For C. nymphaeae, both S. uniflora and COS exhibited approximately the same efficacy (MIC = 1500 μg·mL−1). An enhancement in terms of efficacy was observed in both cases for the COS-S. uniflora extract conjugate complex, reaching full inhibition at 750 and 1000 μg·mL−1 for B. cinerea and C. nymphaeae, respectively.
Upon comparison of the effective concentrations (Table 3), it was possible to observe differences in the efficacy of the treatments more clearly. The highest efficacy (i.e., the lowest EC50 and EC90 values) was observed for the COS-S. uniflora conjugate complex against B. cinerea, followed by those of the same treatment against C. nymphaeae. Synergistic behavior was found between COS and S. uniflora extract (SF values ≥ 1), with the highest synergy factor being obtained for the EC90 of COS-S. uniflora against B. cinerea (SF = 1.56).
Table 4 summarizes the inhibition results for three synthetic fungicides. Mancozeb completely inhibited the growth of both fungal pathogens even at a tenth of the recommended dose (i.e., at 150 μg·mL−1), while Fosetyl-Al required a concentration of 2000 μg·mL−1, and azoxystrobin did not fully inhibit the growth of the two fungal taxa at 62,500 μg·mL−1.

2.4. Ex Situ Growth Inhibition Tests

Strawberry fruits of the variety “Calinda” were treated with the most active product according to the in vitro assays, the COS-S. uniflora conjugate complex, at MIC×5 (namely 3750 and 5000 μg·mL−1 for B. cinerea and C. nymphaeae, respectively). As depicted in Figure 4, the treatment noticeably reduced the incidence of both pathogens. Disease incidences were calculated on days 1, 7, and 10 of the experiment (Table 5). In the negative controls, the pathogens did not proliferate (thus ruling out the possibility of contamination), whereas in the strawberries that had been artificially inoculated with the pathogens but not treated (positive controls), B. cinerea and C. nymphaeae were able to invade more than 81% of the surface of all fruits on the tenth day, with an incidence of 5 at the end of the trial. Upon treatment with the COS-S. uniflora extract conjugate complex, an incidence of 1.3 was observed on the tenth day, with the most-affected fruits showing a colonization of less than 40% by B. cinerea, whereas the colonization of fruits artificially infected with C. nymphaeae was higher, with an incidence of 2.3 on the tenth day (i.e., most fruits showed a colonization equal to or higher than 40%).
Concerning fruit quality attributes, the COS-S. uniflora extract treatment exerted a beneficial effect on the firmness, with an average 24% decrease in flesh firmness values in the case of B. cinerea and a 33% decrease for C. nymphaeae vs. a 52% decrease in the untreated fruits (negative control) by the end of the experiment. As far as color is concerned, the COS-S. uniflora coating imparted a slightly paler shade of red on day 10, more evident in the fruits inoculated with B. cinerea than in those inoculated with C. nymphaeae, although quantitative color measurements would be needed to determine the actual impact on the hue degree and chroma. This should be taken into account, as it may influence consumer preferences.

3. Discussion

3.1. On the Phytochemical Profile

Given that only a small subset of the known organic compounds (amenable for GC-MS) is present in the largest mass spectral databases, limitations in the identification of some of the compounds present in the extracts were detected, with quality of resemblance (Qual) values below 80 (Table 2). Caution is advised as identification of such compounds may be unreliable. However, it is noteworthy that for the chemical species identified at RT = 26.8367 min, comprising 8.7% of the peak area with a “Qual” value of 41, its MS spectrum shows good agreement with those of myristic acid vinyl ester and palmitic acid vinyl ester (Figure S3), and the bands identified in the FTIR spectra of the dried aerial parts also support the presence of saturated fatty acid vinyl esters.
Concerning the presence of the main identified compounds in other plant extracts, mome inositol has previously been reported in high amounts in Corbichonia decumbens (Forssk.) Exell (49.5–75.5% depending on the plant organ) [21], Clitoria ternatea L. (38.7%) [22], Spergula arvensis L. (38.1%) [23], Nephelium lappaceum L. (36%) [24], and Macrotyloma uniflorum (Lam.) Verdc. (23.2%) [25]. This phytoconstituent is anti-alopecic, anti-cirrhotic, anti-neuropathic, cholesterolytic, lipotropic, and a sweetener [25].
With regard to vinyl palmitate and vinyl myristate, they have been documented in Simarouba glauca DC. [26], Eichhornia crassipes (Mart.) Solms [27], Phymatosorus scolopendria (Burm.fil.) Pic.Serm. [28], Cinnamomum javanicum Blume [29], and Petiveria alliaceae L. extracts [30].
In comparison to other salt-tolerant plants (namely Crithmum maritimum L. [31], Daucus carota subsp. gummifer (Syme) Hook. fil. [31], Tripleurospermum callosum (Boiss. and Heldr.) E.Hossain [32], Limonium binervosum (G.E.Sm.) C.E.Salmon [33] and Tamarix gallica L. [34]), it can be noted that the extract of a mixture of the aerial parts of S. uniflora shares with T. callosum and L. binervosum the presence of cyclotetracosane at a moderate concentration (3%). In those plant extracts in which cyclotetracosane is present in quantities greater than 10%, such as the essential oil of Valeriana officinalis L., the ethanol and methanol extracts of Cyclosorus dentatus (Forssk.) Ching [35] (an allelopathic plant), or the ethyl acetate root extract of Jatropha zeyheri Sond. [36], substantial antioxidant capacities have been reported. Cyclotetracosane has been demonstrated to possess α-amylase inhibitory activity [37,38].

3.2. On the Antimicrobial Activity

3.2.1. Activity of Other Silene spp. Extracts

In terms of activity against the two phytopathogens studied here, data are only available for methanolic Silene armeria L. leaf extract [39], for which a MIC value of 1000 μg·mL−1 was reported against B. cinerea and Colletotrichum capsici (Syd. and P.Syd.) E.J.Butler and Bisby, comparable to the MICs reported herein (1000 and 1500 μg·mL−1 against B. cinerea and C. nymphaeae, respectively). This same S. armeria extract achieved MIC values in the 500–2000 μg·mL−1 range against Rhizoctonia solani Kühn, Fusarium oxysporum Schlechtendal, Fusarium solani W.C.Snyder, Sclerotinia sclerotiorum (Lib.) de Bary, and Phytophthora capsici Leonian, suggesting that similar activity may be expected for the S. uniflora extract studied here.
Concerning other Silene spp. extracts, there are reports on their antimicrobial activity against other microorganisms. Back in 1993, Hoffmann et al. [14] showed that the ethanolic extract of S. parishii at a concentration of 1000 μg·mL−1 was effective against Bacillus subtilis (Ehrenberg, 1835) Cohn, 1872, partially effective against Candida albicans (C.P.Robin) Berkhout, and had no effect on Staphylococcus aureus Rosenbach, 1884 and Klebsiella pneumoniae (Schroeter, 1886) Trevisan, 1887. Subsequent studies by Boukhira et al. [15] and Thakur et al. [16] on S. vulgaris, Keskin et al. [17] on S. cariensis subsp. cariensis and S. pungens, and Zengin et al. [13] on S. alba, S. conoidea, S. dichotoma, S. italica, S. supina, and S. vulgaris have demonstrated that Silene spp. extracts have significant antibacterial and antifungal activities against S. aureus; Bacillus cereus Frankland and Frankland, 1887; Escherichia coli (Migula, 1895) Castellani and Chalmers, 1919; Pseudomonas aeruginosa (Schroeter, 1872) Migula, 1900; Aeromonas hydrophila (Chester, 1901) Stanier, 1943; Salmonella enterica enterica (ex Kauffmann and Edwards, 1952) Le Minor and Popoff, 1987; Listeria monocytogenes (Murray et al., 1926) Pirie, 1940; Enterococcus faecalis (Andrewes and Horder, 1906) Schleifer and Kilpper-Bälz, 1984; Micrococcus flavus Liu et al., 2007; C. albicans; Aspergillus brasiliensis Varga et al.; Aspergillus versicolor (Vuillemin) Tiraboschi; Aspergillus fumigatus Fresenius; Aspergillus ochraceus K.Wilhelm; Aspergillus niger van Tieghem; Penicillium ochrochloron Biourge; Penicillium funiculosum Thom; Penicillium verrucosum Dierckx; and Trichoderma viride Persoon.

3.2.2. Comparison with Synthetic Antimicrobials

The concentrations of S. uniflora extract required for full inhibition of B. cinerea and C. nymphaeae (1000 and 1500 μg·mL−1, respectively; see Table 3) were an order of magnitude higher than those of mancozeb (Table 4), demonstrating a substantially lower antimicrobial activity. The activity of S. uniflora extract was comparable to that of fosetyl-Al (MIC = 2000 μg·mL−1), but substantially higher than that of azoxystrobin (MIC > 62,500 μg·mL−1).

3.2.3. Comparison with Chitosan-Based Coatings for Postharvest Strawberry Protection

In order to compare the protective effect of the COS-S. uniflora extract conjugate complex with other chitosan-based coatings reported in the literature (on strawberries), the results of a brief bibliographical survey are presented in Table 6, itemized into those used for postharvest control of gray mold decay (B. cinerea) and those aimed at anthracnose (Colletotrichum spp.) control. In the case of B. cinerea, it may be observed that the efficacy of the COS-S. uniflora extract treatment (3750 and 5000 μg·mL−1 against B. cinerea and C. nymphaeae, respectively) was markedly superior to those reported for chitosan acetate, chitosan chloride, chitosan glutamate, and chitosan formate, in which a 1% w/v dose was applied, with higher disease severities at the end of the experiments [40]. However, it was not as effective as Zataria multiflora Boiss. essential oil encapsulated in chitosan nanoparticles (1500 µg·mL−1) [41] and COS-Uncaria tomentosa (Willd. ex Schult.) DC conjugate complexes (1000 µg·mL−1).
When it comes to protection against C. nymphaeae, to the best of our knowledge, no previous studies on postharvest protection using chitosan have been reported for strawberry fruits. Comparison with other treatments against Colletotrichum spp. reported in the literature reveals the efficacy of COS-S. uniflora would be similar to those reported by Arceo Martínez et al. [42] for chitosan at a concentration of 7500 µg·mL−1.
Table
Table 6. Summary of chitosan-based treatments used for postharvest control of gray mold (B. cinerea) and anthracnose (Colletotrichum spp.) on strawberry fruits reported in the literature and their associated disease severities.
Table 6. Summary of chitosan-based treatments used for postharvest control of gray mold (B. cinerea) and anthracnose (Colletotrichum spp.) on strawberry fruits reported in the literature and their associated disease severities.
PathogenChitosan ComplexStorage ConditionsDisease
Severity
(0–5)		Ref.
B. cinereaChitosan + Silene uniflora (3750 μg·mL−1)7 days at 4 °C, followed
by 3 days at 20 °C		1.3This work
Chitosan acetate (1% w/v)4 days at 20 ± 1 °C,
95–98% RH		3.1[40]
Chitosan chloride (1% w/v)3.2
Chitosan formate (1% w/v)3.4
Chitosan glutamate (1% w/v)3.4
Commercial chitosan (1% w/v)3.5
Chitosan (1% w/v)7 days at 0 ± 1 °C, 95–98% RH, followed by 3 days of shelf life at 20 ± 1 °C, 95–98% RH2.7
Chitosan NP (1500 μg·mL−1)7 days at 4 °C, followed
by 2 days at 20 °C		2.6[41]
Chitosan NP + Zataria multiflora (1500 μg·mL−1)1.5
Chitosan + Cinnamomum zeylanicum (1500 μg·mL−1)2.4[43]
Chitosan + Z. multiflora (1500 μg·mL−1)1.5
COS + Uncaria tomentosa (100 μg·mL−1)7 days at 4 °C, followed
by 3 days at 20 °C		3.5[44]
COS + U. tomentosa (500 μg·mL−1)1.7
COS + U. tomentosa (1000 μg·mL−1)0.5
Colletotrichum spp.C. nymphaeaeChitosan + S. uniflora (5000 μg·mL−1)7 days at 4 °C, followed
by 3 days at 20 °C		2.3This work
C. gloeosporioidesChitosan (7500 μg·mL−1)7 days at 2 ± 2.0 °C,
followed by 3 days at 25 ± 2.0 °C		2[42]
Chitosan (10,000 μg·mL−1)1.2
Chitosan (15,000 μg·mL−1)1
C. acutatumChitosan (7500 μg·mL−1)7 days at 2 ± 2.0 °C,
followed by 3 days at 25 ± 2.0 °C		2
Chitosan (10,000 μg·mL−1)1.8
Chitosan (15,000 μg·mL−1)1
C. fragariaeChitosan + cinnamon EO + aqueous extract of Roselle calycesStored at two different temperatures (5 and 20 °C) for 10 d1 at 5 °C[45]
5 at 20 °C

3.2.4. Mechanism of Action

Based on the activities referred to in the literature for the main constituents identified in S. uniflora extract, the observed antifungal activity should be mainly ascribed to the presence of 4-O-methyl-myo-inositol. Although there are no reports on the antimicrobial activity of pure 4-O-methyl-myo-inositol, the aforementioned extract of N. lappaceum showed antibacterial activity against food pathogenic and spoilage bacteria [24]. Furthermore, myo-inositol has demonstrated strong antifungal activity against Fusarium circinatum Nirenberg and O’Donnell, Cryphonectria parasitica (Murril) M.E. Barr, and Phytophthora cinnamomi Rands phytopathogens, with MIC values of 1000, 750, and 375 μg·mL−1, respectively [46]. Both vinyl palmitate and vinyl myristate have also shown antimicrobial activity [27,47]. However, the contributions from other minority constituents and synergistic behaviors among them cannot be ruled out.
Concerning COS, its antifungal activity is well-established [48] 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 permeability of the cell [49], resulting in a loss of intracellular components that disrupts the osmotic pressure and causes cell death [50]. COS can also alter chitin levels, leading to a weakened cell wall [51], and can generate ROS that damage biomolecules, triggering apoptosis and necrosis. Additionally, COS can interfere with DNA and RNA synthesis [52].
With regard to enhanced activity upon the formation of conjugate complexes, the observed synergism may stem from an enhanced additive fungicidal activity per se or by simultaneous action at multiple fungal metabolic sites [53], but it may also be due to the fact that chitosan oligomers can increase the solubility and bioavailability of the bioactive compounds present in the extract.

4. Materials and Methods

4.1. Plant Material and Chemicals

Samples of S. uniflora were collected in May 2021, during the flowering stage (Figure 1a), from Playa de Cué (Llanes, Asturias, Spain; 43°24′58.7″ N 4°43′53.3″ W). Specimens were identified and authenticated by Prof. Dr. Baudilio Herrero Villacorta (Departamento de Ciencias Agroforestales, ETSIIAA, Universidad de Valladolid) and voucher specimens are available from the herbarium of the ETSIIAA. Aerial parts from different specimens (n = 20) were mixed to obtain (separate) representative composite samples of flowers, fruits, and leaves. The composite samples were shade-dried (with a 72% weight loss), reduce to powder using a mechanical grinder, homogenized, and sieved (1 mm mesh).
Strawberry fruits (Fragaria × ananassa cv. “Calinda”) were supplied by Ideal Fruits (Chañe, Segovia, Spain). The fruits were produced without the addition of artificial pesticides in accordance with organic farming regulations. The fruits were collected and transported to the laboratory in refrigerated conditions, and ex situ tests began within 24 h of harvesting. Strawberries were chosen on the bases of uniform size, lack of physical damage and fungal infection, and a red coloration covering more than 75% of the surface, in agreement with Romanazzi et al. [40].
Potato dextrose agar (PDA) was provided by Becton Dickinson (Bergen County, NJ, USA), NeutraseTM 0.8 L enzyme was acquired from Novozymes A/S (Bagsværd, Denmark), and high-molecular-weight chitosan (CAS 9012-76-4) was purchased from Hangzhou Simit Chem. and Tech. Co. (Hangzhou, China).
For comparison purposes, the Plant Health and Certification Service of the Government of Aragon provided commercial fungicides, namely Ortiva® (azoxystrobin 25%; Syngenta), Vondozeb® (mancozeb 75%; UPL Iberia), and Fesil® (fosetyl-Al 80%; Bayer).

4.2. Fungal Isolates

The fungal isolates of B. cinerea (code not available, but details on its provenance are provided in [54]) and Colletotrichum nymphaeae were supplied as subcultures in PDA by Richerd Breia and Hernâni Gerós from the Centre of Molecular and Environmental Biology (CBMA) at the University of Minho and by Pedro Talhinhas, School of Agriculture, University of Lisbon, respectively. They were cultured in PDA at 25 °C in the dark.

4.3. Preparation of S. uniflora Extract, Chitosan Oligomers, and Their Conjugate Complex

The extract preparation procedure was similar to the one previously reported in [31]. Briefly, 20 g of dried S. uniflora flowering aerial parts were mixed with a 300 mL methanol/water solution (1:1 v/v) and heated in a water bath at 50 °C for 30 min. Then, the solution was subjected to sonication for 5 min in pulse mode with a 1 min stop every 2.5 min, using a model UIP1000hdT probe-type ultrasonicator (Hielscher Ultrasonics; Teltow, Germany). The solution was centrifuged at 9000 rpm for 15 min and the supernatant was filtered through Whatman No. 1 paper, followed by freeze-drying to obtain the solid residue. The extraction yield was 5%. 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 (COS) were prepared using the method described in [55] with the modifications described in [56], yielding oligomers of molecular weight < 2000 Da in a solution with a pH of 4.5. COS and S. uniflora extract solutions (150 mL of each solution, both at a concentration of 3000 μg·mL−1) were mixed in a 1:1 (v/v) ratio and sonicated for 15 min in five 3 min pulses to obtain the conjugate complexes.

4.4. Characterization Procedures

The infrared spectra of the S. uniflora dried plant organs were registered using a model Nicolet iS50 Fourier transform infrared spectrometer from Thermo Scientific (Waltham, MA, USA), equipped with a diamond attenuated total reflection (ATR) system. The spectra were acquired at 1 cm−1 spectral resolution over the 400–4000 cm−1 range by co-adding 64 scans.
The hydromethanolic extract was studied using GC-MS at the Research Support Services (STI) at Universidad de Alicante (Alicante, Spain), utilizing a model 7890A gas chromatograph coupled to a model 5975C quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The chromatographic conditions consisted of an injection volume of 1 µL; an injector temperature of 280 °C, in splitless mode; and an initial oven temperature of 60 °C for two minutes, followed by a 10 °C per minute ramp up to a final temperature of 300 °C, kept for 15 min. The chromatographic column used for the separation of the compounds was an HP-5MS UI of 30 m length, 0.250 mm diameter, and 0.25 µm film. The mass spectrometer conditions were set to a temperature of the electron impact source of 230 °C, and the quadrupole was set to 150 °C; the ionization energy was set to 70 eV. The identification of phytoconstituents was based on a comparison of their mass spectra and retention time with those of authentic compounds and by computer matching with the National Institute of Standards and Technology (NIST11) and Wiley databases.

4.5. Antifungal Activity Assessment

4.5.1. In Vitro Tests of Mycelial Growth Inhibition

The antimicrobial activities were evaluated using the agar dilution method [57]. Petri dishes with PDA incorporating ten different concentrations (ranging from 62.5 to 1500 μg·mL−1) of the various treatments—namely COS, S. uniflora extract, and COS-S. uniflora extract—were inoculated with 5 mm plugs and cultured at 25 °C for seven days. 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. Tests with commercial fungicides were performed in parallel and using the same source of inoculum. Growth inhibition was calculated by the following formula: ((dc − dt)/dc) × 100, where dc is the average colony diameter in the control colony and dt is the average colony diameter in the treated colony. The 50% and 90% effective concentrations, EC50 and EC90, were calculated using IBM (Armonk, NY, USA) SPSS Statistics v.25’s PROBIT analysis. The synergy factor [58], which measures the degree of interaction, was estimated using Wadley’s method [59].

4.5.2. Ex Situ Tests of Mycelial Growth Inhibition

Under controlled laboratory conditions, the efficacy of the COS-S. uniflora extract conjugate complex was evaluated on artificially inoculated strawberry fruits. The protocol was slightly modified from that proposed by Sánchez-Hernández et al. [44]. The strawberries were disinfected for 2 min with a NaOCl 3% solution, then washed three times with sterile distilled water and dried in a laminar flow hood on sterile absorbent paper. The strawberries were divided into three homogeneous groups of 45 fruits (three repetitions with 15 fruits per repetition, treatment, and pathogen), with all fruits measuring more than 22 mm in diameter. One group was treated with COS-S. uniflora extract conjugate complex (at a concentration of MIC×5, i.e., at 3750 μg·mL−1 for B. cinerea or 5000 μg·mL−1 for C. nymphaeae), while the other groups served as the negative (no treatment and no pathogen) and positive (pathogen and no treatment) controls. Superficial wounds (ø = 5 mm) were made in the equatorial zone of each fruit, and the strawberries were then immersed in the COS-S. uniflora conjugate complex treatment for five minutes and dried at room temperature in a laminar flow hood, using sterile absorbent paper. In the superficial wounds, a plug of a PDA culture from B. cinerea or C. nymphaeae was placed (with the mycelium facing the fruit wound). Following Hernández-Muñoz et al. [60], the fruits were placed in covered plastic boxes and stored for seven days at 4 °C and 95–98% RH, then exposed to a 3-day shelf life at 20 °C and 95–98% RH. In accordance with Romanazzi et al. [40], the percentage of rotten strawberries and the disease severity (according to an empirical scale with six degrees: 0, healthy fruit; 1, 1–20% of fruit surface infected; 2, 21–40% of fruit surface infected; 3, 41–60% of fruit surface infected; 4, 61–80% of fruit surface infected; 5, more than 81% of surface infected, with sporulation) were recorded during storage.
Concerning quality attributes, firmness was measured in the central zone of the strawberries (previously sliced into halves) using a TA-XT2 Texture Analyzer (Stable Micro Systems, Godalming, UK) with a 5 mm diameter flat probe. The penetration depth was 5 mm and the cross-head speed was 5 mm·s−1.

4.6. Statistical Analyses

In vitro and ex situ results were analyzed via one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test in IBM SPSS Statistics v.25.

5. Conclusions

The hydromethanolic extract of S. uniflora aerial parts (leaves, flower petals, and fruits) has 4-O-methyl-myo-inositol (52.5%), saturated fatty acid esters (8.7%), and cyclotetracosane (3.7%) as its main phytoconstituents, according to our GC-MS results. Upon testing of its in vitro activity against B. cinerea and C. nymphaeae strawberry pathogens, MIC values of 1000 and 1500 μg·mL−1 were obtained, which are comparable to those of the synthetic fungicide fosetyl-Al. A synergistic effect was observed upon conjugation of chitosan oligomers with the halophyte extract, resulting in MIC values of 750 and 1000 μg·mL−1. Concerning the use of the COS-S. uniflora conjugate complex in postharvest protection of strawberry fruits, a dose five times higher than the in vitro MIC was required to achieve high inhibition against the two phytopathogens after 10 days (with disease severities of 1.3 and 2.3 out of 5 for B. cinerea and C. nymphaeae, compared to 5 out of 5 for the non-treated fruits). This activity is one of the highest reported for chitosan-based coatings, suggesting that S. uniflora extract may be a suitable biorational for the protection of this crop.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants12091846/s1, Figure S1: Infrared spectra of S. uniflora aerial plant organs; Figure S2: GC-MS chromatogram of Silene uniflora extract; Figure S3: Comparison of MS spectra of two saturated fatty acid vinyl esters with that of the chemical species detected at RT = 26.837 min; Figure S4: Radial growth of B. cinerea and C. nymphaeae in the presence of the three treatments, namely S. uniflora extract, chitosan oligomers, and COS-S. uniflora extract conjugate complex, at different concentrations (expressed in μg·mL−1).

Author Contributions

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

Funding

This research was funded by the European Union through the Horizon Europe Program (HORIZON-CL6-2022-FARM2FORK-01) under the project “Agro-ecological strategies for resilient farming in West Africa (CIRAWA)”, with project ID 101084398. The authors also acknowledge the financial support by AgrifoodXXI (NORTE-01-0145-FEDER-000041).

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.

Acknowledgments

L.B.-D. gratefully acknowledges financial support from the Universidad de Valladolid through the Postdoctoral Program Call 2021 for Postdoctoral Contracts of the University of Valladolid.

Conflicts of Interest

The authors declare no conflict 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.

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Plants 12 01846 g001 550
Figure 1. (a) Photograph of Silene uniflora during its flowering stage growing on a cliff in Playa de Cué (Llanes, Asturias, Spain); (b) chemical structure of 2,22-dideoxyecdysone 25-O-β-d-glucopyranoside phytoecdysteroid reported in Silene spp. extracts.
Figure 1. (a) Photograph of Silene uniflora during its flowering stage growing on a cliff in Playa de Cué (Llanes, Asturias, Spain); (b) chemical structure of 2,22-dideoxyecdysone 25-O-β-d-glucopyranoside phytoecdysteroid reported in Silene spp. extracts.
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Figure 2. Chemical structures of the four most abundant phytochemicals identified using gas chromatography–mass spectrometry in the hydromethanolic extract of S. uniflora aerial parts.
Figure 2. Chemical structures of the four most abundant phytochemicals identified using gas chromatography–mass spectrometry in the hydromethanolic extract of S. uniflora aerial parts.
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Plants 12 01846 g003 550
Figure 3. Radial growth values of B. cinerea and C. nymphaeae in the presence of the different treatments under study—S. uniflora extract (S. uniflora), chitosan oligomers (COS), and COS-S. uniflora extract conjugate complex (COS-S. uniflora)—at different concentrations (in μg·mL−1). C represents the control. Concentrations of each treatment labeled with the same lowercase letters are not significantly different at p < 0.05 according to Tukey’s test. All values are presented as the average of three repetitions, with three replicates per repetition. Error bars represent the standard deviation.
Figure 3. Radial growth values of B. cinerea and C. nymphaeae in the presence of the different treatments under study—S. uniflora extract (S. uniflora), chitosan oligomers (COS), and COS-S. uniflora extract conjugate complex (COS-S. uniflora)—at different concentrations (in μg·mL−1). C represents the control. Concentrations of each treatment labeled with the same lowercase letters are not significantly different at p < 0.05 according to Tukey’s test. All values are presented as the average of three repetitions, with three replicates per repetition. Error bars represent the standard deviation.
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Figure 4. Evolution of the decay of strawberry fruits caused by B. cinerea and C. nymphaeae: (left) negative control, (center) positive control, (right) treated with COS-S. uniflora extract conjugate complex (COS-S. uniflora) at MIC×5 (3750 and 5000 μg·mL−1 for B. cinerea and C. nymphaeae, respectively).
Figure 4. Evolution of the decay of strawberry fruits caused by B. cinerea and C. nymphaeae: (left) negative control, (center) positive control, (right) treated with COS-S. uniflora extract conjugate complex (COS-S. uniflora) at MIC×5 (3750 and 5000 μg·mL−1 for B. cinerea and C. nymphaeae, respectively).
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Table
Table 1. Main absorption bands in the infrared spectra of Silene uniflora organs, expressed in cm−1.
Table 1. Main absorption bands in the infrared spectra of Silene uniflora organs, expressed in cm−1.
PetalsFruitLeavesAssignment
336633353366OH group in phenolic compounds/hydrogen bonding in pyranosides
2955 2954Symmetric C–H stretching (CH3 symmetric stretching)
291629182915O–H stretching/C–H stretching
2848 2848CH2 symmetric stretching
1733 1733C=O stretching, alkyl ester/carboxylic acid (monomeric form)
1706 1706C=O stretching of carboxylic acid (dimeric form)
163616361636Skeletal vibration due to aromatic C=C ring stretching
1472 1472CH2 scissors
146314431463Symmetric aromatic ring stretching vibration (C=C ring)
141714181417C–H vibration of the methyl group
137813711378C–H symmetric bending in CH3
130713161327CH2 wagging, C–O stretching
124312441243CH in-plane bending
114611461147C–O–C asymmetric stretching
11011101 In-plane =C–H bending/C=C stretching
1075 C–O stretching/O–H out plane bending
101810191020C–H bending
729 729CH2 rocking
719 719CH2 rocking
Table
Table 2. Main phytoconstituents identified in the GC-MS chromatogram of Silene uniflora extract.
Table 2. Main phytoconstituents identified in the GC-MS chromatogram of Silene uniflora extract.
RT (min)Area (%)AssignmentQual
4.68030.6939Pyrrolidine47
4.76330.95932-Cyclopenten-1-one, 2-hydroxy-87
5.72490.6416Succindialdehyde33
7.33330.85842(3H)-Furanone, dihydro-4-hydroxy-38
10.55621.15352-Methoxy-4-vinylphenol90
12.31310.76001-Butanol, 4-butoxy-32
12.77600.98633,4-Altrosan83
16.216452.53664-O-Methyl-myo-inositol (mome inositol)93
17.91600.5355n-Hexadecanoic acid99
25.09181.1509Squalene99
25.53103.6950Cyclotetracosane98
26.83678.6716Myristic acid vinyl ester/Palmitic acid vinyl ester41
26.96140.70221-Nonadecene/1-heptacosazol95/93
28.59360.9617Hexadecanoic acid, 4-nitrophenyl ester51
29.21680.67711H-Indole, 5-methyl-2-phenyl-41
Qual: quality of resemblance.
Table
Table 3. EC50 and EC90 effective concentrations of S. uniflora extract (S. uniflora), chitosan oligomers (COS), and COS-S. uniflora extract conjugate complex (COS-S. uniflora), expressed in µg·mL−1, and synergy factors (SF).
Table 3. EC50 and EC90 effective concentrations of S. uniflora extract (S. uniflora), chitosan oligomers (COS), and COS-S. uniflora extract conjugate complex (COS-S. uniflora), expressed in µg·mL−1, and synergy factors (SF).
Effective
Concentration		B. cinereaC. nymphaeae
COSS. unifloraCOS-S. unifloraSFCOSS. unifloraCOS-S. unifloraSF
EC502484382361.346746686441.04
EC9014269837461.5672114209911.46
EC50: effective concentration to reduce mycelial growth by 50%. EC90: effective concentration to reduce mycelial growth by 90%.
Table
Table 4. Radial growth of the mycelium of B. cinerea and C. nymphaeae in the in vitro assays performed on PDA medium loaded with two concentrations (the recommended dose and a tenth of the recommended dose) of three commercial synthetic fungicides.
Table 4. Radial growth of the mycelium of B. cinerea and C. nymphaeae in the in vitro assays performed on PDA medium loaded with two concentrations (the recommended dose and a tenth of the recommended dose) of three commercial synthetic fungicides.
Commercial
Fungicide		PathogenRadial Growth of Mycelium (mm)Inhibition (%)
ControlRd/10Rd *Rd/10Rd *
AzoxystrobinB. cinerea7551123284
C. nymphaeae7545404047
MancozebB. cinerea7500100100
C. nymphaeae7500100100
Fosetyl-AlB. cinerea7538049.3100
C. nymphaeae7563016100
* Rd stands for recommended dose, i.e., 62,500 μg·mL−1 of azoxystrobin (250,000 μg·mL−1 for Ortiva®, azoxystrobin 25%), 1500 μg·mL−1 of mancozeb (2000 μg·mL−1 for Vondozeb®, mancozeb 75%), and 2000 μg·mL−1 of fosetyl-Al (2500 μg·mL−1 for Fosbel®, fosetyl-Al 80%). All mycelial growth values (in mm) are average values (n = 3).
Table
Table 5. Disease severity of B. cinerea and C. nymphaeae attack on cv. “Calinda” strawberries after 1, 7, and 10 days.
Table 5. Disease severity of B. cinerea and C. nymphaeae attack on cv. “Calinda” strawberries after 1, 7, and 10 days.
Time
(Days)		B. cinereaC.nymphaeae
Negative
Control		Positive
Control		COS-S. unifloraNegative
Control		Positive
Control		COS-S. uniflora
10 ± 0 a0 ± 0 a0 ± 0 a0 ± 0 a0 ± 0 a0 ± 0 a
70 ± 0 a2.7 ± 0.9 b0.6 ± 0.2 b0 ± 0 a3.3 ± 0.7 b1.2 ± 0.3 b
100 ± 0 a5 ± 0 c1.3 ± 0.5 c0 ± 0 a5 ± 0 b2.3 ± 0.8 c
COS-S. uniflora: chitosan oligomers-S. uniflora extract conjugate complex. Different letters indicate that the disease severity is significantly different at p < 0.05.
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Buzón-Durán, L.; Sánchez-Hernández, E.; Martín-Ramos, P.; Navas-Gracia, L.M.; García-González, M.C.; Oliveira, R.; Martín-Gil, J. Silene uniflora Extracts for Strawberry Postharvest Protection. Plants 2023, 12, 1846. https://doi.org/10.3390/plants12091846

AMA Style

Buzón-Durán L, Sánchez-Hernández E, Martín-Ramos P, Navas-Gracia LM, García-González MC, Oliveira R, Martín-Gil J. Silene uniflora Extracts for Strawberry Postharvest Protection. Plants. 2023; 12(9):1846. https://doi.org/10.3390/plants12091846

Chicago/Turabian Style

Buzón-Durán, Laura, Eva Sánchez-Hernández, Pablo Martín-Ramos, Luis Manuel Navas-Gracia, Mari Cruz García-González, Rui Oliveira, and Jesús Martín-Gil. 2023. "Silene uniflora Extracts for Strawberry Postharvest Protection" Plants 12, no. 9: 1846. https://doi.org/10.3390/plants12091846

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