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.

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.