Innovative Delivery of Cu(II) Ions by a Nanostructured Hydroxyapatite: Potential Application in Planta to Enhance the Sustainable Control of Plasmopara viticola.

Downy mildew caused by Plasmopara viticola is probably the most serious disease affecting grapevine (Vitis vinifera), and it is capable of causing consistent yield losses. In organic viticulture, the only acceptable and effective means to control the disease is by applications of copper-based fungicides. However, the use of copper in agriculture is expected to be further restricted by European countries because of its critical ecotoxicological and phytotoxicological profile. Research on ways to reduce the effective amounts of copper by developing innovative formulations as well as optimization of the distribution and persistence of copper-based pesticides for downy mildew control seems to be a promising approach. This research investigated the delivery properties of biomimetic synthetic hydroxyapatite (HA) to enhance the biological activity of Cu(II) ions. To this aim, four Cu(II) compounds were formulated with the innovative HA component and applied in an in vitro antifungal assay against Botrytis cinerea, a common grapevine pathogen suitable for in vitro activity tests, and finally, in in planta efficacy assays against P. viticola under greenhouse conditions. The in vitro results highlighted a different inhibition activity for each Cu(II) compound and indicated a different interaction between the cupric compounds and HA, potentially related to different delivery mechanisms of Cu(II) from HA. Under greenhouse conditions, additional findings on the biological activity of the applied formulations were gained, especially on the efficacy of various concentrations of HA in the formulations, the influence of dose variation of the formulation and the treatment efficiency, and the persistence under rain-washing effect. This study revealed promising findings on the formulation based on the HA particles and the soluble Cu(II) compound, which resulted in reduced disease severity and incidence in all of the experimental conditions, including the lower Cu(II) dosage and the rain-washing effect. This suggests that coformulation of the three insoluble Cu(II) compounds with HA might significantly enhance the adsorption and release of Cu(II) ions by HA particles.

Grapevine Trunk Diseases: A Review of Fifteen Years of Trials for Their Control with Chemicals and Biocontrol Agents.

Grapevine trunk diseases (GTDs) represent one of the most important problems for viticulture worldwide. Beyond the original causes of this outbreak in some countries like France, the lack of efficient control protocols and the prohibition of using active ingredients such as sodium arsenite and benzimidazoles, until recently used to reduce the impact of some GTDs but deleterious for humans and the environment, have probably worsened the impact of the diseases, leading to increasing economic losses. Since 1990, searches have been made to find efficient tools to control GTDs, testing a wide range of active ingredients and biocontrol agents. This review provides readers with an overview of the results reported in the scientific literature over the last 15 years. In particular, the review focuses on the trials carried out applying chemicals or microorganisms to control Esca complex diseases, Botryosphaeria dieback, and Eutypa dieback, the most widespread GTDs.

Xylem anatomy and hydraulic traits in Vitis grafted cuttings in view of their impact on the young grapevine decline.

Grapevine grafting is an essential practice in viticulture and over the years, various bench grafting techniques have been developed to mechanize the nursery process and to increase the yield in number of viable cuttings. Bench grafting is a fundamental nursery practice that can potentially affect the quality of propagation material also in young decline associated to grapevine trunk diseases and has been recently reported to influence leaf symptoms development associated with diseases of Esca complex. The study aimed to investigate how three bench grafting methods [i.e., (i) Omega graft as mechanical technique, (ii) Whip and Tongue graft as manual technique and (iii) Full Cleft graft as semi-mechanical technique] can influence these phenomena. Specifically, the different methods were compared for their effect on the anatomical development of the grafting point and the functionality of the xylem, also considering two factors: the grapevine cultivar (Cabernet Sauvignon, Glera and Teroldego) and the scion/rootstock diameter (thin and large). Observations by light microscopy on the anatomical evolution and measurements on the xylem morphology and hydraulic traits were correlated with the grafting methods and the investigated varieties. The anatomical observations revealed that the mechanical (Omega) and semi-mechanical (Full Cleft) grafting methods have a faster callusing response while the manual technique (Whip and Tongue) has a slower but greater vascularization of the differentiated callus. Significant differences between cultivars and/or grafting types were also detected in necrotic area on the grafted tissues. Statistical analysis of the grapevine vessels suggested differences in xylem parameters between cultivars, while grafting type had no significant effects. On the other hand, the grafting type significantly affected the intrinsic growth rate. The study confirms the potential incidence of lesions and dysfunctionalities correlated with the grafting method applied, which can potentially induce grafted vine declines in vineyards due to the necrotic area detected on the grafted tissues.

In planta Activity of Novel Copper(II)-Based Formulations to Inhibit the Esca-Associated Fungus Phaeoacremonium minimum in Grapevine Propagation Material.

Grapevine trunk diseases (GTDs) are a serious and growing threat to vineyards worldwide. The need for innovative control tools persists since pesticides used against some GTDs have been banned and only methods to prevent infections or to reduce foliar symptoms have been developed so far. In this context, the application of imaging methods, already applied to study plant-microbe interactions, represents an interesting approach to understand the effect of experimental treatments applied to reduce fungal colonization, on GTD-related pathogens activity. To this aim, trials were carried out to evaluate the efficacy of copper-based treatments, formulated with hydroxyapatite (HA) as co-adjuvant with innovative delivery properties, loaded with two different copper(II) compounds (tribasic sulfate and sulfate pentahydrate), and applied to grapevine propagation material to inhibit fungal wood colonization. The treated rootstock (Vitis berlandieri × Vitis riparia cv. K5BB) and scion cuttings (Vitis vinifera L., cv. Chardonnay) had been inoculated with a strain of Phaeoacremonium minimum (Pmi) compared to uninoculated rootstocks. Experimental treatments were applied during the water-soaking process, comparing the copper(II) compounds pure or formulated with HA, to hydrate the cuttings. After callusing, grafted vines were grown under greenhouse conditions in a nursery and inoculated with Pmi::gfp7 or with Pmi wild-type. Fifteen weeks post-inoculation, woody tissues close to the inoculation site were sampled to evaluate the efficiency of the treatments by studying the plant-microbe interaction by confocal laser scanning microscopy (CLSM). Copper and further elements were also quantified in the same tissues immediately after the treatments and on the CLSM samples. Finally, the grapevine defense responses were studied in the leaves of cuttings treated with the same formulations. The present investigation confirmed the relevant interaction of Pmi and the related transformed strain on the vascular tissues of grafted vines. Furthermore, in vitro assay revealed (i) the fungistatic effect of HA and the reduced effect of Cu fungicide when combined with HA. In planta assays showed (ii) the reduction of Pmi infection in propagation material treated with HA-Cu formulations, (iii) the movement of HA-Cu formulations inside the plant tissues and their persistence over time, and (iv) the plant defense reaction following the treatment with pure HA or Cu, or combined.

Activity of Trichoderma asperellum Strain ICC 012 and Trichoderma gamsii Strain ICC 080 Toward Diseases of Esca Complex and Associated Pathogens.

Grapevine trunk diseases are widespread in all grape-growing countries. The diseases included in the Esca complex of diseases are particularly common in European vineyards. Their distinctive foliar symptoms are well known to be associated not only with losses in quantity, as with all grapevine wood diseases, but also with losses in the quality of the crop. Protection of pruning wounds is known to reduce infections in artificial inoculations and, to some extent, reduce the external leaf symptoms. The application of biological control agents in the field is typically started at the first appearance of symptoms. In this article, the two strains belonging to two different species, Trichoderma asperellum ICC 012 and T. gamsii ICC 080, which are present in a commercial formulation, were tested in vitro, in vivo in artificial inoculation, and in the field in long-term experiments where the wounds on four young asymptomatic vineyards were protected since 1 or 2 years after planting. The in vitro trials highlighted the different temperature requirements of the two strains, the direct mycoparasitizing activity of T. asperellum, and the indirect activity shown by both Trichoderma strains. The in vivo trials confirmed the ability of the two strains to reduce the colonization following artificial inoculations with the high, unnatural concentration of spores used in artificial infections, even if with variable efficacy, and with long persistence as they could be reisolated 7 months post-application. The preventive applications carried out over 9 years showed a very high reduction in symptom development in the treated vines, on annual and cumulated incidence and on the death of vines, with disease reduction varying from 66 to almost 90%. Early and annual application of protection to the pruning wounds appears to be the best method for reducing damages caused by grapevine leaf stripe disease (a disease of the Esca complex of diseases). Trichoderma appears to offer an efficient, environmentally friendly, and long-lasting protection in the presence of a natural inoculum concentration.

Functionalization of a nanostructured hydroxyapatite with Cu(II) compounds as a pesticide: in situ transmission electron microscopy and environmental scanning electron microscopy observations of treated Vitis vinifera L. leaves.

BACKGROUND: The present study evaluated a biocompatible material for plant protection with the aim of reducing the amount of active substance applied. We used a synthetic hydroxyapatite (HA) that has been studied extensively as a consequence of its bioactivity and biocompatibility. An aggregation between HA nanoparticles and four Cu(II) compounds applied to Vitis vinifera L. leaves as a pesticide was studied. Formulations were characterized by X-ray diffraction (XRD), dynamic light scattering (DLS) and electron microscopy and applied in planta to verify particle aggregation and efficiency in controlling the pathogen Plasmopara viticola. RESULTS: The XRD patterns showed different crystalline phases dependig on the Cu(II) compound formulated with HA particles, DLS showed that nanostructured particles are stable as aggregates out of the nanometer range and, in all formulations, transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM) microscopy showed large aggregates which were partially nanostructured and were recognized as stable in their micrometric dimensions. Such particles did not show phytotoxic effects after their application in planta. CONCLUSION: A formulation based on HA and a soluble Cu(II) compound showed promising results in the control of the fungal pathogen, confirming the potential role of HA as an innovative delivery system of Cu(II) ions. The present work indicates the possibility of improving the biological activity of a bioactive substance by modifying its structure through an achievable formulation with a biocompatible material. © 2018 Society of Chemical Industry.