Helichrysum stoechas (L.) Moench Inflorescence Extract for Tomato Disease Management.

Helichrysum stoechas is a singular halophyte that has been shown to have anti-inflammatory, antioxidant, and allelopathic properties. In the work presented herein, we have characterized its inflorescences hydromethanolic extract and assessed its antifungal activity for the pre- and postharvest management of tomato crop diseases. Gas chromatography-mass spectrometry characterization of the extract showed that 4-ethenyl-1,3-benzenediol, 2,3-dihydro-benzofuran, quinic acid, 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-1-benzopyran-4-one, 1,6-anhydro-ß-D-glucopyranose, catechol, scopoletin, and maltol were the main constituents. The co-occurrence of pyranones, benzenediols, and quinic acids as phytoconstituents of H. stoechas extract resulted in promising in vitro minimum inhibitory concentrations of 500, 375, 500, 187.5, 187.5, and 375 µg·mL-1 against mycelia of Alternaria alternata, Colletotrichum coccodes, Fusarium oxysporum f. sp. lycopersici, Rhizoctonia solani, Sclerotinia sclerotiorum, and Verticillium dahliae, respectively. Further, to assess the potential of H. stoechas inflorescence extract for postharvest tomato crop protection, ex situ tests were conducted against C. coccodes, obtaining high protection at a dose of 750 µg·mL-1. Taking into consideration that the demonstrated activity is among the highest reported to date for plant extracts and comparable to that of the synthetic fungicides tested as positive controls, H. stoechas inflorescence extract may be put forward as a promising biorational and may deserve further testing in field-scale studies.

Metagenomic Study of Fungal Microbial Communities in Two PDO Somontano Vineyards (Huesca, Spain): Effects of Age, Plant Genotype, and Initial Phytosanitary Status on the Priming and Selection of their Associated Microorganisms.

The study of microbial communities associated with different plants of agronomic interest has allowed, in recent years, to answer a number of questions related to the role and influence of certain microbes in key aspects of their autoecology, such as improving the adaptability of the plant host to different abiotic or biotic stresses. In this study, we present the results of the characterization, through both high-throughput sequencing and classical microbiological methods, of the fungal microbial communities associated with grapevine plants in two vineyards of different ages and plant genotypes located in the same biogeographical unit. The study is configured as an approximation to the empirical demonstration of the concept of "microbial priming" by analyzing the alpha- and beta-diversity present in plants from two plots subjected to the same bioclimatic regime to detect differences in the structure and taxonomic composition of the populations. The results were compared with the inventories of fungal diversity obtained by culture-dependent methods to establish, where appropriate, correlations between both microbial communities. Metagenomic data showed a differential enrichment of the microbial communities in the two vineyards studied, including the populations of plant pathogens. This is tentatively explained due to factors such as the different time of exposure to microbial infection, different plant genotype, and different starting phytosanitary situation. Thus, results suggest that each plant genotype recruits differential fungal communities and presents different profiles of associated potential microbial antagonists or communities of pathogenic species.

Phytochemical Profiling of Sambucus nigra L. Flower and Leaf Extracts and Their Antimicrobial Potential against Almond Tree Pathogens.

Despite extensive research on the chemical composition of elderberries and their numerous uses in pharmaceutical, beverage, and food production, there is still a lack of knowledge about Sambucus nigra leaves and flowers' antimicrobial activity against plant pathogens. In this study, the phytoconstituents of their aqueous ammonia extracts were first characterized by infrared spectroscopy and gas chromatography-mass spectrometry. The major phytocompounds identified in the flower extract were octyl 2-methylpropanoate; 3,5-dihydroxy-6-methyl-2,3-dihydropyran-4-one; propyl malonic acid; adenine; and 1-methyl-2-piperidinemethanol. Concerning the leaf extract, 1,6-anhydro-ß-D-glucopyranose; oleic acid; 2,1,3-benzothiadiazole; 2,3-dihydro-benzofuran; and 4-((1E)-3-hydroxy-1-propenyl)-2-methoxyphenol and other phenol derivatives were the main constituents. The potential of the extracts to act as bioprotectants was then investigated against three almond tree pathogens: Diaporthe amygdali, Phytophthora megasperma, and Verticillium dahliae. In vitro tests showed higher activity of the flower extract, with EC90 values in the 241-984 µg·mL-1 range (depending on the pathogen) vs. 354-1322 µg·mL-1 for the leaf extract. In addition, the flower extract led to full protection against P. megasperma at a dose of 1875 µg·mL-1 in ex situ tests on artificially-infected excised almond stems. These inhibitory concentrations were lower than those of commercial fungicides. These findings suggest that S. nigra aerial organs may be susceptible to valorization as an alternative to synthetic fungicides for the protection of this important crop.

New insights in the Spanish gene pool of olive (Olea europaea L.) preserved ex situ and in situ based on high-throughput molecular markers.

In Spain, several local studies have highlighted the likely presence of unknown olive cultivars distinct from the approximately 260 ones previously described in the literature. Furthermore, recent advancements in identification techniques have significantly enhanced in terms of efficacy and precision. This scenario motivated a new nationwide prospecting effort aimed at recovering and characterizing new cultivated germplasm using high-throughput molecular markers. In the present study, the use of 96 EST-SNP markers allowed the identification of a considerable amount of new material (173 new genotypes) coming from areas with low intensification of production in different regions of Spain. As a result, the number of distinct national genotypes documented in the World Olive Germplasm Bank of IFAPA, Córdoba (WOGBC-ESP046) increased to 427. Likewise, 65 and 24 new synonymy and homonymy cases were identified, respectively. This rise in the number of different national cultivars allowed to deepen the knowledge about the underlying genetic structure. The great genetic variability of Spanish germplasm was confirmed, and a new hot spot of diversity was identified in the northern regions of La Rioja and Aragon. Analysis of the genetic structure showed a clear separation between the germplasm of southern and northern-northeastern Spain and indicated a significantly higher level of admixture in the latter. Given the expansion of modern olive cultivation with only a few cultivars, this cryptic germplasm is in great danger of disappearing. This underlines the fact that maintaining as many cultivars as possible will increase the genetic variability of the olive gene pool to meet the future challenges of olive cultivation.

Valorization of Quercus suber L. Bark as a Source of Phytochemicals with Antimicrobial Activity against Apple Tree Diseases.

Cork, an anatomic adaptation of the bark of Quercus suber L. through its suberization process, finds its main application in the production of bottle stoppers. Its processing results in a large waste stream of cork fragments, granulates, and dust, which may be susceptible to valorization. The work presented here explored the use of its extracts to inhibit the growth of phytopathogenic microorganisms associated with apple tree diseases. The in vitro antimicrobial activity of cork aqueous ammonia extract was assayed against four fungi, viz. Monilinia fructigena and M. laxa (brown rot), Neofussicoccum parvum (dieback), and Phytophthora cactorum (collar and root rot), and two bacteria, viz. Erwinia amylovora and Pseudomonas syringae pv. syringae, either alone or in combination with chitosan oligomers (COS). Effective concentration values of EC90 in the 675-3450 µg·mL-1 range, depending on the fungal pathogen, were obtained in growth inhibition tests, which were substantially improved for the conjugate complexes (340-801 µg·mL-1) as a result of strong synergism with COS. Similar enhanced behavior was also observed in antibacterial activity assays, with MIC values of 375 and 750 µg·mL-1 for the conjugate complexes against P. syringae pv. syringae and E. amylovora, respectively. This in vitro inhibitory activity was substantially higher than those exhibited by azoxystrobin and fosetyl-Al, which were tested for comparison purposes, and stood out among those reported for other natural compounds in the literature. The observed antimicrobial activity may be mainly attributed to the presence of glycerin and vanillic acid, identified by gas chromatography-mass spectroscopy. In the first step towards in-field application, the COS-Q. suber bark extract conjugate complex was further tested ex situ against P. cactorum on artificially inoculated excised stems of the 'Garnem' almond rootstock, achieving high protection at a dose of 3750 µg·mL-1. These results suggest that cork industrial leftovers may, thus, be a promising source of bioactive compounds for integrated pest management.

Multifunctional Nanocarriers Based on Chitosan Oligomers and Graphitic Carbon Nitride Assembly.

In this study, a graphitic carbon nitride and chitosan oligomers (g-C3N4−COS) nanocarrier assembly, which was obtained by cross-linking with methacrylic anhydride (MA), was synthesized and characterized. Its characterization was carried out using infrared spectroscopy, elemental and thermal analyses, and transmission electron microscopy. The new nanocarriers (NCs), with an average particle size of 85 nm in diameter and a 0.25 dispersity index, showed photocatalytic activity (associated with the g-C3N4 moiety), susceptibility to enzymatic degradation (due to the presence of the COS moiety), and high encapsulation and moderate-high release efficiencies (>95% and >74%, respectively). As a proof of concept, the visible-light-driven photocatalytic activity of the NCs was tested for rhodamine B degradation and the reduction of uranium(VI) to uranium(IV). Regarding the potential of the nanocarriers for the encapsulation and delivery of bioactive products for crop protection, NCs loaded with Rubia tinctorum extracts were investigated in vitro against three Vitis vinifera phytopathogens (viz. Neofusicoccum parvum, Diplodia seriata, and Xylophilus ampelinus), obtaining minimum inhibitory concentration values of 750, 250, and 187.5 µg·mL−1, respectively. Their antifungal activity was further tested in vivo as a pruning wound protection product in young 'Tempranillo' grapevine plants that were artificially infected with the two aforementioned species of the family Botryosphaeriaceae, finding a significant reduction of the necrosis lengths in the inner woody tissues. Therefore, g-C3N4-MA-COS NCs may be put forward as a multifunctional platform for environmental and agrochemical delivery applications.

Holm Oak (Quercus ilex subsp. ballota (Desf.) Samp.) Bark Aqueous Ammonia Extract for the Control of Invasive Forest Pathogens.

Holm oak (Quercus ilex subsp. ballota (Desf.) Samp.) bark is a commonly used remedy to treat gastrointestinal disorders, throat and skin infections, hemorrhages, and dysentery. It has also been previously reported that its methanol extracts possess antibacterial activity, which can be related to the richness of Quercus spp. extracts in phenolic compounds, such as flavonoids and tannins. However, there is no information on the antifungal (including oomycete) properties of the bark from Q. ilex or its subspecies (ilex and ballota). In this work, we report the characterization of the aqueous ammonia extract of its bark by FTIR and GC-MS and the results of in vitro and ex situ inhibition tests against three phytopathogens. The main phytochemical components identified were inositols (19.5%), trans-squalene (13%), 4-butoxy-1-butanol (11.4%), gulopyranose (9.6%), lyxose (6.5%), 2,4-dimethyl-benzo[H]quinoline (5.1%), catechol (4.5%), and methoxyphenols (4.2%). The efficacy of the extract in controlling forest phytopathogens was tested in vitro against Fusarium circinatum (responsible for pitch canker of Pinus spp.), Cryphonectria parasitica (which causes chestnut blight), and Phytophthora cinnamomi (which causes 'root and crown rot' in a variety of hosts, including Castanea, conifers, Eucalyptus, Fagus, Juglans, Quercus, etc.), obtaining EC90 values of 322, 295, and 75 µg·mL-1, respectively, much lower than those attained for a commercial strobilurin fungicide (azoxystrobin). The extract was further tested ex situ against P. cinnamomi on artificially inoculated, excised stems of 'Garnem' almond rootstock, attaining complete protection at a dose of 782 µg·mL-1. The results suggest that holm oak bark extract may be a promising source of bioactive compounds against invasive forest pathogens, including the oomycete that is causing its decline, the so-called 'seca' in Spain.

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.

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.

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.

Burn effects on soil properties associated to heat transfer under contrasting moisture content.

The aim of this work is to investigate the topsoil thickness affected by burning under contrasting soil moisture content (field capacity versus air-dried conditions). A mollic horizon of an Aleppo pine forest was sampled and burned in the laboratory, recording the temperature continuously at the topsoil surface and at soil depths of 1, 2, and 3cm. Changes in soil properties were measured at 0-1, 1-2, 2-3, and 3-4cm. Both the maximum temperature and the charring intensities were significantly lower in wet soils than in air-dried soils up to 3cm in depth. Moreover, soil heating was slower and cooling faster in wet soils as compared to dry soils. Therefore, the heat capacity increase of the soil moistened at field capacity plays a more important role than the thermal conductivity increase on heat transfer on burned soils. Burning did not significantly modify the pH, the carbonate content and the chroma, for either wet or dry soil. Fire caused an immediate and significant decrease in water repellency in the air-dried soil, even at 3cm depth, whereas the wet soil remained hydrophilic throughout its thickness, without being affected by burning. Burning depleted 50% of the soil organic C (OC) content in the air-dried soil and 25% in the wet soil at the upper centimeter, which was blackened. Burning significantly decreased the total N (TN) content only in the dry soil (to one-third of the original value) through the first centimeter of soil depth. Soluble ions, measured by electrical conductivity (EC), increased after burning, although only significantly in the first centimeter of air-dried soils. Below 2cm, burning had no significant effects on the brightness, OC, TN, or EC, for either wet or dry soil.