In a context in which the incidence and severity of grapevine fungal diseases is increasing as a result of both climate change and modern management culture practices, reducing the excessive use of phytosanitary products in viticulture represents a major challenge. Specifically, grapevine trunk diseases (GTDs), caused by several complexes of wood decay or xylem-inhabiting fungi, pose a major challenge to vineyard sustainability. In this study, the efficacy of chitosan oligomers (COS)–amino acid conjugate complexes against three fungal species belonging to the Botryosphaeriaceae family (Neofusicoccum parvum, Diplodia seriata, and Botryosphaeria dothidea) was investigated both in vitro and in planta. In vitro tests led to EC50 and EC90 effective concentrations in the 254.6−448.5 and 672.1−1498.5 µg·mL−1 range, respectively, depending on the amino acid involved in the conjugate complex (viz. cysteine, glycine, proline or tyrosine) and on the pathogen assayed. A synergistic effect between COS and the amino acids was observed against D. seriata and B. dothidea (synergy factors of up to 2.5 and 2.8, respectively, according to Wadley’s method). The formulations based on COS and on the conjugate complex that showed the best inhibition rates, COS−tyrosine, were further investigated in a greenhouse trial on grafted vines of two varieties (”Tempranillo” on 775P and “Garnacha” on 110R rootstock), artificially inoculated with the mentioned three Botryosphaeriaceae species. The in planta bioassay revealed that the chosen formulations induced a significant decrease in disease severity against N. parvum and B. dothidea. In summary, the reported conjugate complexes may be promising enough to be worthy of additional examination in larger field trials.
The use of nanocarriers (NCs), i.e., nanomaterials capable of encapsulating drugs and releasing them selectively, is an emerging field in agriculture. In this study, the synthesis, characterization, and in vitro and in vivo testing of biodegradable NCs loaded with natural bioactive products was investigated for the control of certain phytopathogens responsible for wood degradation. In particular, NCs based on methacrylated lignin and chitosan oligomers, loaded with extracts from Rubia tinctorum, Silybum marianum, Equisetum arvense, and Urtica dioica, were first assayed in vitro against Neofusicoccum parvum, an aggressive fungus that causes cankers and diebacks in numerous woody hosts around the world. The in vitro antimicrobial activity of the most effective treatment was further explored against another fungal pathogen and two bacteria related to trunk diseases: Diplodia seriata, Xylophilus ampelinus, and Pseudomonas syringae pv. syringae, respectively. Subsequently, it was evaluated in field conditions, in which it was applied by endotherapy for the control of grapevine trunk diseases. In the in vitro mycelial growth inhibition tests, the NCs loaded with R. tinctorum resulted in EC90 concentrations of 65.8 and 91.0 μg·mL−1 against N. parvum and D. seriata, respectively. Concerning their antibacterial activity, a minimum inhibitory concentration of 37.5 μg·mL−1 was obtained for this treatment against both phytopathogens. Upon application via endotherapy on 20-year-old grapevines with clear esca and Botryosphaeria decay symptoms, no phytotoxicity effects were observed (according to SPAD and chlorophyll fluorescence measurements) and the sugar content of the grape juice was not affected either. Nonetheless, the treatment led to a noticeable decrease in foliar symptoms as well as a higher yield in the treated arms as compared to the control arms (3177 vs. 1932 g/arm), suggestive of high efficacy. Given the advantages in terms of controlled release and antimicrobial product savings, these biodegradable NCs loaded with natural extracts may deserve further research in large-scale field tests.
In the work presented herein, we analyze the efficacy of three basic substances that comply with European Regulation (EC) No 1107/2009, namely chitosan, horsetail (Equisetum arvense L.) and nettle (Urtica dioica L.), for the control of grapevine trunk diseases (GTDs) in organic farming. The E. arvense and U. dioica aqueous extracts, prepared according to SANCO/12386/2013 and SANTE/11809/2016, have been studied by gas chromatography–mass spectrometry (GC-MS), identifying their main active constituents. The three basic substances, either alone or in combination (forming conjugate complexes), have been tested in vitro against eight Botryosphaeriaceae species, and in vivo, in grafted plants artificially inoculated with Neofusicoccum parvum and Diplodia seriata. A clear synergistic behavior between chitosan and the two plant extracts has been observed in the mycelial growth inhibition tests (resulting in EC90 values as low as 208 μg·mL−1 for some of the isolates), and statistically significant differences have been found in terms of vascular necroses lengths between treated and non-treated plants, providing further evidence of aforementioned synergism in the case of D. seriata. The reported data supports the possibility of extending the applications of these three basic substances in Viticulture beyond the treatment of mildew.
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.
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.
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.
Neofusicoccum parvum and Rhizoctonia solani are fungal pathogens with an increasing incidence in young grapevine plants. In this study, the antagonistic potential of some strains of the genus Trichoderma isolated from grapevine against these pathogens was investigated at the laboratory and greenhouse levels. In-plate confrontation assays showed that the selected Trichoderma strains could inhibit the mycelial growth of both taxa, being more effective against N. parvum. In the in vivo assays, the biocontrol activity of the mentioned strains against the pathogens, when applied either simultaneously or successively, was tested on both grafted plants and seedlings germinated from seed. The effectiveness of the treatments was evaluated by comparing biomass weight and vascular rot lengths data. In seedling trials, successive treatments resulted in higher root development and a lower colonization rate of the pathogens, especially against R. solani. In grafted plants, some disparity was observed against N. parvum: simultaneous treatments resulted in higher aerial biomass, but successive treatments resulted in higher root biomass and lower necrosis. Against R. solani, simultaneous treatments were clearly more effective, with higher root and aerial length values and lower necrosis. The obtained data suggest that the use of Trichoderma spp. isolates can constitute an alternative to conventional fungicides to control certain grapevine wood diseases.
Flavonoids and phenolic acids play roles in grapevine defence against pathogens causing grapevine trunk diseases (GTDs). Rutin is a major flavonoid in vegetative organs of the grapevines, and this compound, unlike other flavonoids, is non-toxic and non-oxidizable. Rutin was assayed in vitro and in vivo against two Botryosphaeriaceae taxa. The limited bioavailability of this compound was circumvented by conjugation with stevioside, a glycoside obtained from Stevia rebaudiana. Clear synergistic effects were observed for the stevioside-rutin adduct, resulting in EC50 and EC90 values of 306.0 and 714.9 μg·mL-1 against Neofusicoccum parvum and 241.6 and 457.8 μg·mL-1 against Dothiorella viticola. In greenhouse experiments, moderate inhibition of N. parvum growth and complete inhibition of D. viticola were observed. These inhibitory effects were greater than those of ferulic acid, which has been considered the most effective phenolic acid against GTDs. Conjugation with stevioside provided solubility enhancement of rutin, paving the way to the design of glycopesticides based on rutin-rich plant extracts as promising antifungals against GTDs.
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