Assessment of a New Copper-Based Formulation to Control Esca Disease in Field and Study of Its Impact on the Vine Microbiome, Vine Physiology and Enological Parameters of the Juice.

Copper-based preparations have been used for more than 100 years in viticulture to control downy mildew caused by Plasmopara viticola. LC2017, and a new low-copper-based formulation, has been developed to control grapevine trunk diseases (GTDs). Previous greenhouse studies showed the potential of LC2017 to control GTDs by both fungistatic and plant defense elicitor effects. Here, we further characterize the effects of LC2017 in the field determining its impact on: (i) incidence of Esca, (ii) the vine microbiome, (iii) the vine physiology and (iv) enological parameters of juices. We observed a progressive decrease of cumulate Esca incidence in treated vines over the years with annual fluctuation related to the known erratic emergence of GTD symptoms. Neither harmful effects of LC2017 on the vine microbiota, nor on vine physiology were observed (at both transcriptomic and metabolomic levels). Similarly, no impact of LC2017 was observed on the enological properties of berries except for sugar content in juice from esca-diseased vines. The most important result concerns the transcriptomic profiles: that of diseased and LC2017 treated vines differs from that of disease untreated ones, showing a treatment effect. Moreover, the transcriptomic profile of diseased and LC2017-treated vines is similar to that of untreated asymptomatic vines, suggesting control of the disease.

In planta Activity of the Novel Copper Product HA + Cu(II) Based on a Biocompatible Drug Delivery System on Vine Physiology and Trials for the Control of Botryosphaeria Dieback.

The growing concerns on human and environment health are forcing the plant protection industry toward the formulation of more eco-sustainable plant protection products (PPP), both efficient and innovative in their approach to disease control. A large number of these innovative formulations now rely on a combination of pathogens antagonistic properties and stimulation of natural plant defense to pathogens. The formulation HA + Cu(II), in which copper is delivered to the plants by the drug-delivery molecule hydroxyapatite (HA), was found efficient against the grapevine pathogens Plasmopara viticola and Phaeoacremonium minimum and able to induce the host-plant defense system. We investigated the HA + Cu(II) impacts on grapevine physiology, both in uninfected and when infected by the Botryosphaeria dieback agents Diplodia seriata and Neofusicoccum parvum. This study of plant physiology and disease impact were addressed to evaluate both the HA + Cu(II) potential as a plant defense elicitor and its possible and future use as PPP in vineyard. Our results showed that HA + Cu(II) induced several key-defense genes without negatively affecting plant growth and photosynthetic activity. In addition, fungistatic effect on the two Botryosphaeriaceae at the in planta tested concentrations is reported. Altogether, our results obtained under controlled conditions fully support the potential of HA + Cu(II) as a promising PPP toward grapevine trunk diseases in vineyard.

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.

Epidemiological Investigations and Molecular Characterization of 'Candidatus Phytoplasma solani' in Grapevines, Weeds, Vectors and Putative Vectors in Western Sicily, (Southern Italy).

Bois noir is caused by 'Candidatus Phytoplasma solani', and it is one of the most important and widespread diseases in the Euro-Mediterranean region. There are complex interactions between phytoplasma and grapevines, weeds, and vectors. These ecological relationships can be tracked according to molecular epidemiology. The aims of the 2-year study (2014-2015) were to describe incidence and spatial distribution of Bois noir in a vineyard with three grapevine varieties in Sicily, and to identify the molecular types of the tuf and vmp1 genes in these naturally infected grapevines, according to the potential reservoir plants and vectors. Disease incidence in 2015 was significantly higher in 'Chardonnay' (up to 35%) than for 'Nero d'Avola' and 'Pinot noir' (<5%). All grapevine, weed, and insect samples were infected by 'Ca. P. solani' tuf-type b. Most of the collected insects were strictly related to Vitis spp. and belonged to Neoaliturus fenestratus, Empoasca spp., and Zygina rhamni. The characterization of the vmp1 gene revealed six different vmp types in grapevines (V1, V4, V9, V11, V12, V24), three in weeds (V4, V9, V11), and four in insects (V4, V9, V11, V24). Notably, V4, V9, appear both in hosts and vectors, with V9 predominant. Virtual restriction fragment length polymorphism (RFLP) analysis based on the nucleotide sequences supported the data of the conventional RFLP. Connections between the molecular data recorded in the vineyard ecosystems and the application of innovative tools based on the geostatistical analysis will contribute to further clarification of the specific ecological and epidemiological aspects of 'Ca. P. solani' in Sicily.

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.

Defense Responses in Grapevine (cv. Mourvèdre) after Inoculation with the Botryosphaeria Dieback Pathogens Neofusicoccum parvum and Diplodia seriata and Their Relationship with Flowering.

As a result of the increasing economic impact of grapevine trunk diseases on viticulture worldwide, efficient and viable control strategies are urgently needed. However, understanding both plant-pathogen interactions and plant physiological changes related to these diseases is fundamental to such an achievement. In this study, we analyzed the effect of inoculation with the Botryosphaeria dieback fungal agents, Neofusicoccum parvum and Diplodia seriata, with and without inflorescence removal at the onset of G stage (separated clusters), I stage (flowering) and M stage (veraison). A measure of lesion size and real-time reverse-transcription polymerase chain reaction-based analysis were carried out. The results clearly show the importance of inflorescences in the development of lesions associated with Botryosphaeria dieback pathogens inoculated on green stems of adult vines, especially at the onset of flowering. At flowering, the biggest necroses were observed with the inflorescences present, as well as an activation of the studied defense responses. Thus, an ineffective response to the pathogen could be consistent with a possible metabolic reprogramming linked to the host phenophase.

Pathogenicity bioassays of isolates of Beauveria bassiana on Rhynchophorus ferrugineus.

BACKGROUND: The control of Rhynchophorus ferrugineus (Olivier), the main palm pest in the Mediterranean Basin, is problematic because of its biology and the current restrictions in many European countries on the use of chemical insecticides in urban areas. Entomopathogenic fungi have been studied as potential biological control agents, but information on their natural incidence is limited. Strains of Beauveria bassiana (Balsamo) Vuillemin were isolated from symptomatic insects collected on dead palms, and their pathogenicity against different instars of R. ferrugineus was evaluated in the laboratory. RESULTS: The overall percentage of infected insects found in Canary palms was 7%. In laboratory bioassays, hatching of eggs treated with three different isolates of B. bassiana was 41.2, 26.8 and 29.9%, significantly lower than the control (62.4%). Larvae and adults were treated with a single isolate in two ways: spraying each insect with a conidial suspension or feeding them with fruit portions previously immersed in the same conidial suspension. At the end of the two trials, the mortality of treated larvae was 88 and 92%, and the mean survival time was 10.4 and 11.8 days, significantly different from the control, where no insect died during the trials. Mortality and survival time recorded in either trial on adults did not significantly differ between treatment and control. CONCLUSION: This study shows that the pathogenicity of wild isolates of B. bassiana differs among the tested R. ferrugineus instars. The low mortality of treated adults supports their use as vectors of B. bassiana as a potential tool for reducing R. ferrugineus populations.