No abstract

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.

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 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.

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.

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.

Lime-induced chlorosis (LIC) is an important abiotic constraint affecting the growth and yield of grapevines growing in calcareous soils in the Mediterranean region, and the sensory properties of the produced wine. In the work presented herein, the impact of LIC on the nutritional status and chlorophyll content was assessed for eleven varieties and a clone (Merlot, Pinot Noir, Cabernet Sauvignon, Tempranillo, Parraleta, Moristel, Aglianico, Macabeo, Sauvignon, Chardonnay, and Riesling), grafted to the same rootstock (1103 Paulsen). Macro- and micronutrient contents were determined in the fruit set and veraison stages by petiole analyses, while chlorophyll content in young leaves was monitored by SPAD. Significant differences were detected amongst varieties for all nutrients (including Fe), and inverse relationships between Fe and P contents in the petiole and chlorophyll concentration in the young leaves were found. Regarding LIC resistance, the Fe and chlorophyll contents suggest that Cabernet Sauvignon, Tempranillo and Aglianico varieties would show the best performance, while Sauvignon would be the least tolerant.

Grapevine is highly susceptible to fungal diseases, whose incidence and severity increase due to climate change. The present work focuses on the assessment of eight combinations of natural products with chitosan oligomers with fungicidal capacity that may be effective in the integrated control of powdery mildew, in compliance with Article 14 of the European Directive 2009/128/EC. Their efficacy was evaluated in field conditions against natural infections, in a plot with high disease pressure during a growing season (assaying both foliar or root application), and against overwintering inoculums (chasmothecia) through in vitro tests. In addition, their possible biostimulant capacities were evaluated based on harvest yields. Treatments based on chitosan oligomers in combination with secondary metabolites of Streptomyces spp. and chitosan oligomers combined with hydrolyzed gluten showed the best results in terms of disease control. Given the high efficacy of these formulations, comparable to that of conventional antifungals, they constitute an interesting alternative for the control of this disease whose treatment can, in some cases, represent almost half of the production costs.