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

Abstract

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.

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

“…Upon the Botryosphaeriaceae infections, the LC2017 treatments significantly increased the plant defenses in comparison with the NT conditions. Similar results were presented in the recent paper Battiston et al (2021) with in planta assay against Phaeoacremonium minimum, a causal agent in Esca disease, another relevant GTDs.…”

“…LC2017 exerted a marked fungistatic effect at 0.5% (175 µg/mL of Cu 2+ ) against four Botryosphaeriaceae strains (Figure 6) as observed against P. minimum (Battiston et al, 2021). These results confirm the concentration range sensibility of Botryosphaeriaceae to copper, as observed in vitro, by Bester et al (2007) with copper ammonium acetate (ineffective below 20 µg/mL) and by Amponsah et al (2012) with copper hydroxide (EC 50 84 µg/mL).…”

“…Despite being carried out under controlled condition, our study nonetheless highlights several interesting aspects of LC2017 treatments in view of their potential use in vineyards. Its low copper content and innovative delivery system (Battiston, 2018;Battiston et al, 2018Battiston et al, , 2019Battiston et al, , 2021 can improve the AI's efficiency, thereby respecting limitation in copper delivery while simultaneously enabling the control of different fungal and bacterial diseases. Beside the well-known copper fungicide effects, LC2017 acted much like BTH against downy and powdery mildew through its elicitor effect.…”

“…Against Phaeoacremonium minimum, an Esca complex pathogen on grapevine, HA + Cu(II) limits this pathogen both in vitro and in the nursery when applied during the misting of cuttings (Battiston et al, 2018). Work by Battiston et al (2018Battiston et al ( , 2019Battiston et al ( , 2021, beside the ability of HA + Cu(II) to control both Botrytis cinerea in vitro and P. viticola on grapevine cuttings under greenhouse conditions, has also assessed the elicitation properties of this formulation toward genes linked to the host-plant defense system.…”

Combining an HA + Cu (II) Site-Targeted Copper-Based Product with a Pruning Wound Protection Program to Prevent Infection with Lasiodiplodia spp. in Grapevine

“…Therefore, new methods to efficiently deliver fungicides to specific targeted areas of the plant are considered to have great potential to improve GTD control. The application of site-targeted fungicides to protect vascular tissues against GTD pathogen colonization has been recently investigated by several authors [44][45][46]. Lignin nanocarriers loaded with pyraclostrobin, as a targeted drug delivery system [45], phloem mobile derivative of fenpiclonil in combination with beneficial endophyte [44], and copper-based treatments, formulated with hydroxyapatite (HA) as co-adjuvant with innovative delivery properties [47,48] have shown promising results in controlling both esca and Botryosphaeria dieback pathogens.…”