Silene uniflora Extracts for Strawberry Postharvest Protection.

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.

A Coating Based on Bioactive Compounds from Streptomyces spp. and Chitosan Oligomers to Control Botrytis cinerea Preserves the Quality and Improves the Shelf Life of Table Grapes.

Botrytis cinerea is the most harmful postharvest disease of table grapes. Among the strategies that can be envisaged for its control, the use of coatings based on natural products is particularly promising. The study presented herein focuses on the assessment of the antagonistic capacity of two Streptomyces species and their culture filtrates against B. cinerea. Firstly, the secondary metabolites were characterized by gas chromatography-mass spectrometry, with N1-(4-hydroxybutyl)-N3-methylguanidine acetate and 2R,3S-9-[1,3,4-trihydroxy-2-butoxymethyl]guanine acetate as the main compounds produced by S. lavendofoliae DSM 40217; and cyclo(leucyloprolyl) and cyclo(phenylalanylprolyl) as the most abundant chemical species for S. rochei DSM 41729. Subsequently, the capacity of S. lavendofoliae DSM 40217 and S. rochei DSM 41729 to inhibit the growth of the pathogen was tested in dual culture plate assays, finding 85-90% inhibition. In agar dilution tests, their culture filtrates resulted in effective concentration values (EC90) in the 246-3013 µg·mL-1 range. Upon the formation of conjugate complexes with chitosan oligomers (COS) to improve solubility and bioavailability, a synergistic behavior was observed, resulting in lower EC90 values, ranging from 201 to 953 µg·mL-1. Ex situ tests carried out on 'Timpson' and 'Red Globe' table grapes using the conjugate complexes as coatings were found to maintain the turgor of the grapes and delay the appearance of the pathogen by 10-15 days at concentrations in the 750-1000 µg·mL-1 range. Hence, the conjugate complexes of COS and the selected Streptomyces spp. culture filtrates may be put forward as promising protection treatments for the sustainable control of gray mold.

Dwarf Pomegranate (Punica granatum L. var. nana): Source of 5-HMF and Bioactive Compounds with Applications in the Protection of Woody Crops.

While the properties of edible pomegranate varieties have been widely explored, there is little information on ornamental types. In this study, possible alternatives for the valorization of dwarf pomegranate fruits have been explored. The characterization of their hydromethanolic extract by gas chromatography-mass spectrometry evidenced the presence of high contents of 5-hydroxymethylfurfural (a carbon-neutral feedstock for the production of fuels and other chemicals) and ß- and γ-sitosterol stereoisomers. The microbicidal activity of the crude extract, both alone and in a conjugate complex with chitosan oligomers (COS), was investigated against three plant pathogenic microorganisms that cause significant losses in woody crops: Erwinia amylovora, E. vitivora, and Diplodia seriata. In in vitro assays, a strong synergistic behavior was found after conjugation of the bioactive constituents of the fruit extract with COS, resulting in minimum inhibitory concentration (MIC) values of 750 and 375 µg·mL-1 against E. amylovora and E. vitivora, respectively, and an EC90 value of 993 µg·mL-1 against D. seriata. Hence, extracts from the non-edible fruits of this Punicaceae may hold promise as a source of high value-added phytochemicals or as environmentally friendly agrochemicals.

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

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

Physicochemical Characterization and Antimicrobial Activity against Erwinia amylovora, Erwinia vitivora, and Diplodia seriata of a Light Purple Hibiscus syriacus L. Cultivar.

Phytochemicals are essential raw materials for the production of formulations that can be helpful in crop protection. In particular, Hibiscus spp., which are often used in traditional medicine, are rich in potential bioactive molecules. This study presents an analysis of the thermal, vibrational, and phytochemical characteristics of a light purple variety of Hibiscus syriacus, using thermal gravimetric and differential scanning calorimetry, Fourier-transform infrared spectroscopy, and gas chromatography-mass spectroscopy techniques. Further, with a view to its valorization, the antimicrobial activity of its extracts has been investigated in vitro against Erwinia amylovora (the phytopathogen responsible for fire blight in apples, pears, and some other members of the family Rosaceae), Erwinia vitivora (the causal agent of the "maladie d'Oléron" in grapevines), and Diplodia seriata (responsible for "Bot canker"). Higher heating values and thermal features showed similarities with kenaf biomass. The main compounds identified in the hydro-methanolic extracts were: in flowers, 1-heptacosanol, heptacosane, 1-tetracosanol, hexadecenoic acid, 9,12,15-octadecatrienoic acid, and 9,12-octadecadienoic acid; and in leaves, the coumarin derivative 4,4,6,8-tetramethyl-2-chromanone, vitamin E, phytol, and sitosterol. MIC values of 500 and 375 µg·mL-1 were obtained against E. amylovora for flower and leaf extracts, respectively, upon conjugation with chitosan oligomers (to improve solubility and bioavailability). In the case of E. vitivora, MIC values of 250 and 500 µg·mL-1, respectively, were registered. Regarding the antifungal activity, EC90 values of 975.8 and 603.5 µg·mL-1, respectively, were found. These findings suggest that H. syriacus (cv. 'Mathilde') may be a promising source of antimicrobials for agriculture.

Activity of Anthracenediones and Flavoring Phenols in Hydromethanolic Extracts of Rubia tinctorum against Grapevine Phytopathogenic Fungi.

In this work, the chemical composition of Rubia tinctorum root hydromethanolic extract was analyzed by GC-MS, and over 50 constituents were identified. The main phytochemicals were alizarin-related anthraquinones and flavoring phenol compounds. The antifungal activity of this extract, alone and in combination with chitosan oligomers (COS) or with stevioside, was evaluated against the pathogenic taxa Diplodia seriata, Dothiorella viticola and Neofusicoccum parvum, responsible for the so-called Botryosphaeria dieback of grapevine. In vitro mycelial growth inhibition tests showed remarkable activity for the pure extract, with EC50 and EC90 values as low as 66 and 88 µg·mL-1, respectively. Nonetheless, enhanced activity was attained upon the formation of conjugate complexes with COS or with stevioside, with synergy factors of up to 5.4 and 3.3, respectively, resulting in EC50 and EC90 values as low as 22 and 56 µg·mL-1, respectively. The conjugate with the best performance (COS-R. tinctorum extract) was then assayed ex situ on autoclaved grapevine wood against D. seriata, confirming its antifungal behavior on this plant material. Finally, the same conjugate was evaluated in greenhouse assays on grafted grapevine plants artificially inoculated with the three aforementioned fungal species, resulting in a significant reduction in the infection rate in all cases. This natural antifungal compound represents a promising alternative for developing sustainable control methods against grapevine trunk diseases.

Antifungal Activity against Botryosphaeriaceae Fungi of the Hydro-Methanolic Extract of Silybum marianum Capitula Conjugated with Stevioside.

Silybum marianum (L.) Gaertn, viz. milk thistle, has been the focus of research efforts in the past few years, albeit almost exclusively restricted to the medicinal properties of its fruits (achenes). Given that other milk thistle plant organs and tissues have been scarcely investigated for the presence of bioactive compounds, in this study, we present a phytochemical analysis of the extracts of S. marianum capitula during the flowering phenological stage (stage 67). Gas chromatography-mass spectroscopy results evidenced the presence of high contents of coniferyl alcohol (47.4%), and secondarily of ferulic acid ester, opening a new valorization strategy of this plant based on the former high-added-value component. Moreover, the application of the hydro-methanolic extracts as an antifungal agent has been also explored. Specifically, their activity against three fungal species responsible for the so-called Botryosphaeria dieback of grapevine (Neofusicoccum parvum, Dothiorella viticola and Diplodia seriata) has been assayed both in vitro and in vivo. From the mycelial growth inhibition assays, the best results (EC90 values of 303, 366, and 355 µg·mL-1 for N. parvum, D. viticola, and D. seriata, respectively) were not obtained for the hydroalcoholic extract alone, but after its conjugation with stevioside, which resulted in a strong synergistic behavior. Greenhouse experiments confirmed the efficacy of the conjugated complexes, pointing to the potential of the combination of milk thistle extracts with stevioside as a promising plant protection product in organic Viticulture.

Cyttaria hariotii E.Fisch. as a promising source of pullulan and Mn(II)-pullulan complexes for Mn-deficiency remediation in winter cereals.

Pullulan, a water-soluble polysaccharide consisting of maltotriose units used in the preparation of edible films and drug delivery, is generally produced from starch by Aureobasidium pullulans (de Bary & Löwenthal) G.Arnaud fungus. In this article, the characterisation of an alternative pullulan source - the stromata of Cyttaria hariotii E.Fisch. fungus - by elemental analysis, infrared spectroscopy and thermal analysis techniques is reported. With a view to a possible valorisation of this pullulan and its derivatives as bioactive formulations in agriculture, low-molecular-weight pullulan (<7 kDa) complexes with Mn(II), suitable for the remediation of Mn-deficiencies in winter cereal by foliar application, were synthesised and characterised by mass spectrometry.

Antifungal Activity against Fusarium culmorum of Stevioside, Silybum marianum Seed Extracts, and Their Conjugate Complexes.

Fusarium head blight (FHB) is a disease that poses a major challenge in cereal production that has important food and feed safety implications due to trichothecene contamination. In this study, the effect of stevioside-a glycoside found in the leaves of candyleaf (Stevia rebaudiana Bertoni)-was evaluated in vitro against Fusarium culmorum (W.G. Smith) Sacc., alone and in combination (in a 1:1 molar ratio) with polyphenols obtained from milk thistle seeds (Silybum marianum (L.) Gaertn). Different concentrations, ranging from 32 to 512 µg·mL-1, were assayed, finding EC50 and EC90 inhibitory concentrations of 156 and 221 µg·mL-1, respectively, for the treatment based only on stevioside, and EC50 and EC90 values of 123 and 160 µg·mL-1, respectively, for the treatment based on the stevioside-polyphenol conjugate complexes. Colony formation inhibition results were consistent, reaching full inhibition at 256 µg·mL-1. Given that synergistic behavior was observed for this latter formulation (SF = 1.43, according to Wadley's method), it was further assessed for grain protection at storage, mostly directed against mycotoxin contamination caused by the aforementioned phytopathogen, confirming that it could inhibit fungal growth and avoid trichothecene contamination. Moreover, seed tests showed that the treatment did not affect the percentage of germination, and it resulted in a lower incidence of root rot caused by the pathogen in Kamut and winter wheat seedlings. Hence, the application of these stevioside-S. marianum seed extract conjugate complexes may be put forward as a promising and environmentally friendly treatment for the protection of cereal crops and stored grain against FHB.

Effect of Sodium Hypochlorite and Benzalkonium Chloride on the Structural Parameters of the Biofilms Formed by Ten Salmonella enterica Serotypes.

The influence of the strain on the ability of Salmonella enterica to form biofilms on polystyrene was investigated by confocal laser scanning microscopy. The effects of sodium hypochlorite with 10% active chlorine (SHY; 25,000, 50,000, or 100,000 ppm), and benzalkonium chloride (BZK; 1000, 5000, or 10,000 ppm) on twenty-four-hour-old biofilms was also determined. The biofilms of ten Salmonella enterica isolates from poultry (S. Agona, S. Anatum, S. Enteritidis, S. Hadar, S. Infantis, S. Kentucky, S. Thompson, S. Typhimurium, monophasic variant of S. Typhimurium 1,4,(5),12:i:-, and S. Virchow) were studied. Biofilms produced by S. Anatum, S. Hadar, S. Kentucky, and S. Typhimurium showed a trend to have the largest biovolume and the greatest surface coverage and thickness. The smallest biofilms (P < 0.01) in the observation field (14.2 × 103 µm2) were produced by S. Enteritidis and S. 1,4,(5),12:i:- (average 12.9 × 103 ± 9.3 × 103 µm3) compared to the rest of the serotypes (44.4 × 103 ± 24.7 × 103 µm3). Biovolume and surface coverage decreased after exposure for ten minutes to SHY at 50,000 or 100,000 ppm and to BZK at 5000 or 10,000 ppm. However, the lowest concentrations of disinfectants increased biovolume and surface coverage in biofilms of several strains (markedly so in the case of BZK). The results from this study suggest that the use of biocides at low concentrations could represent a public health risk. Further research studies under practical field conditions should be appropriate to confirm these findings.

Antifungal Agents Based on Chitosan Oligomers, ε-polylysine and Streptomyces spp. Secondary Metabolites against Three Botryosphaeriaceae Species.

Grapevine trunk diseases (GTDs) are a major threat to the wine and grape industry. The aim of the study was to investigate the antifungal activity against Neofusicoccum parvum, Diplodia seriata, and Botryosphaeria dothidea of ε-polylysine, chitosan oligomers, their conjugates, Streptomyces rochei and S. lavendofoliae culture filtrates, and their binary mixtures with chitosan oligomers. In vitro mycelial growth inhibition tests suggest that the efficacy of these treatments, in particular those based on ε-polylysine and ε-polylysine:chitosan oligomers 1:1 w/w conjugate, against the three Botryosphaeriaceae species would be comparable to or higher than that of conventional synthetic fungicides. In the case of ε-polylysine, EC90 values as low as 227, 26.9, and 22.5 µg·mL-1 were obtained for N. parvum, D. seriata, and B. dothidea, respectively. Although the efficacy of the conjugate was slightly lower, with EC90 values of 507.5, 580.2, and 497.4 µg·mL-1, respectively, it may represent a more cost-effective option to the utilization of pure ε-polylysine. The proposed treatments may offer a viable and sustainable alternative for controlling GTDs.

Antibiotic susceptibility of methicillin-resistant staphylococci (MRS) of food origin: A comparison of agar disc diffusion method and a commercially available miniaturized test.

Methicillin-resistant staphylococci (MRS) are a major concern to public and animal health. Thirty MRS (Staphylococcus aureus, S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. lentus, S. lugdunensis, S. sciuri, and S. xylosus) isolates from meat and poultry preparations were tested for antimicrobial susceptibility to 11 antimicrobials (belonging to seven different categories) of clinical significance using both the standard agar disc diffusion method and a commercially available miniaturized system (Sensi Test Gram-positive). It is worth stressing that 16 isolates (53.33%) exhibited an extensively drug-resistant phenotype (XDR). The average number of resistances per strain was 4.67. These results suggest that retail meat and poultry preparations are a likely vehicle for the transmission of multi-drug resistant MRS. Resistance to erythromycin was the commonest finding (76.67% of strains), followed by tobramycin, ceftazidime (66.67%), ciprofloxacin (56.67%) and fosfomycin (53.33%). An agreement (kappa coefficient) of 0.64 was found between the two testing methods. Using the agar disc diffusion as the reference method, the sensitivity, specificity and accuracy of the miniaturized test were 98.44%, 69.44% and 83.33%, respectively. Most discrepancies between the two methods were due to isolates that were susceptible according to the disc diffusion method but resistant according to the miniaturized test (false positives).

Microbial loads and antibiotic resistance patterns of Staphylococcus aureus in different types of raw poultry-based meat preparations.

The hygiene status of raw chicken-meat preparations from retail outlets in North-Western Spain was investigated. Microbial counts (aerobic plate counts (APCs), psychrotrophs, Enterobacteriaceae, fecal coliforms, enterococci, pseudomonads, fluorescent pseudomonads, yeasts and molds, and Staphylococcus aureus) were determined for minced meat, hamburgers, nuggets, white sausages, red sausages, escalope, and roll-ups. S. aureus isolates were tested for susceptibility to twenty antimicrobials of veterinary and human clinical significance (disc diffusion method, CLSI). Average microbial loads (log10 cfu/g) ranged from 2.63 ± 0.80 (enterococci) to 6.66 ± 1.09 (psychrotrophs). Average APCs (6.44 ± 1.16 log10 cfu/g) were regarded as acceptable according to EU microbiological criteria. The type of product had an influence (P < 0.05) on microbial loads, samples of escalope showing the highest counts for most microbial groups. Two-thirds (66.7%) of the samples tested harbored S. aureus. All the S. aureus isolates were multi-resistant (to between three and fifteen antibiotics). The greatest prevalence of resistance was shown for ampicillin, oxacillin, penicillin G, ceftazidime, and nalidixic acid. The results of this study show that poultry-based meat preparations present high microbial loads and are a major reservoir of antibiotic-resistant S. aureus strains. This highlights the need for correct handling of such foodstuffs with a view to reducing risks to consumers.

Effect of Sub-Lethal Concentrations of Biocides on the Structural Parameters and Viability of the Biofilms Formed by Salmonella Typhimurium.

This study aimed to investigate the effect of sub-minimum inhibitory concentrations (sub-MICs) of three food-grade biocides (benzalkonium chloride -BZK-, trisodium phosphate -TSP-, and sodium hypochlorite -SHY-) on Salmonella biofilms. The structural parameters and bacterial viability of the biofilms formed by a S. Typhimurium isolate from poultry was investigated by means of confocal laser scanning microscopy after staining with SYTO9 and propidium iodide. The MIC values for Salmonella cells before exposure to subinhibitory concentrations of biocides were 8.0 µg/mL (BZK), 18.0 mg/mL (TSP), and 6.0 mg/mL (SHY). The cultures exhibited a stable acquired tolerance to BZK and SHY. The maximum concentrations of biocides that allowed bacterial growth after several passages through gradually higher concentrations of such compounds were 30.4 µg/mL (BZK) and 10.1 mg/mL (SHY). The architecture and viability of S. Typhimurium biofilms varied in response to sub-MICs of different biocides. Previous adaptation to SHY enhanced (p < 0.001) biofilm formation (average biovolume in the observed field -14,161 µm2-: 139,856.15 ± 155,213.27 µm3) relative to unexposed cells (53,779.05 ± 55,535.62 µm3) and cells previously exposed to BZK (58,216.97 ± 58,644.45 µm3) or TSP (30,052.13 ± 28,290.56 µm3). This was particularly marked when biofilm was grown in the absence of biocides or in the presence of sub-MICs of SHY. The highest percentage of dead cells was shown by biofilms formed by cultures previously exposed to TSP relative to the other conditions tested (34.08% ± 13.74% vs. 23.70% ± 16.16%; p < 0.001). The importance of maintaining higher than MICs of SHY during sanitizing procedures to fight foodborne infections by Salmonella biofilms is highlighted.

Effect of sub-inhibitory concentrations of biocides on the architecture and viability of MRSA biofilms.

The effect of sub-minimum inhibitory concentrations (sub-MICs) of three biocides (benzalkonium chloride [BZK], trisodium phosphate [TSP] and sodium hypochlorite [SHY]) upon the architecture and viability of the biofilms formed by a methicillin-resistant Staphylococcus aureus strain of food origin (MRSA 48a) was investigated. Images were examined through confocal laser scanning microscopy (CLSM) after staining with SYTO9 and propidium iodide. Sub-MICs of BZK or TSP reduced the ability of MRSA to produce biofilm. In contrast, the presence of sub-MICs of SHY enhanced the biofilm-forming ability of MRSA when cells had undergone previous adaptation to this compound (biovolume in the observation field was 137,785.31 ± 47,682.79 µm3 for biofilms formed in the presence of SHY, and 70,204.13 ± 31,603.98 µm3 in the absence of biocides; P < 0.05). The largest amount of live (green stained) cells (P < 0.05) was observed in biofilms grown in the presence of SHY relative to the other conditions tested (58,999.75 ± 55,312.37 µm3vs 31,976.29 ± 38,594.98 µm3). Findings from the present work constitute the first report of biofilm production by MRSA being induced by sub-inhibitory concentrations of SHY. The data suggest that repeated use of SHY at low concentrations could represent a public health risk.