**1. Introduction**

Botryosphaeria dieback is currently among the most significant grapevine trunk diseases (GTDs) in all the grape-growing regions of the world [1]. It represents one of the major threats to sustainable and economically viable viticulture due to the reduction in yield, increased crop management costs, and shortened life span of vines and vineyards [1–3]. This disease is caused by fungi of the family Botryosphaeriaceae and more than 26 taxa of this family have been associated with Botryosphaeria dieback in grapevine [4–8]. *Lasiodiplodia* spp. and *Neofusicoccum* spp. were previously proven as being amongst the fastest wood colonizing genera, and therefore considered as some of the most virulent GTD fungi [9–12]. The genus *Lasiodiplodia* comprises 34 species [13,14], from which ten have been reported from the grapevine [13–19]. *Lasiodiplodia theobromae* is the most commonly isolated species in grapevine and, although it is most common in tropical and sub-tropical regions, it can be found in vineyards around the world, such as Australia [20], Algeria [21], Brazil [17], Bolivia [22], China [19], Italy [23], Mexico [9], Peru [24], Portugal [25], Spain [26], Turkey [27],

**Citation:** Reis, P.; Gaspar, A.; Alves, A.; Fontaine, F.; Rego, C. Combining an HA + Cu (II) Site-Targeted Copper-Based Product with a Pruning Wound Protection Program to Prevent Infection with *Lasiodiplodia* spp. in Grapevine. *Plants* **2021**, *10*, 2376. https://doi.org/10.3390/ plants10112376

Academic Editors: Carlos Agustí-Brisach and Eugenio Llorens

Received: 30 September 2021 Accepted: 1 November 2021 Published: 4 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and the USA [9]. Common external symptoms caused by infection with botryosphaeriaceous fungi on grapevine, include leaf spots and wilting, dry fruit rots, bud necrosis and perennial cankers, cordon dieback, and eventually the sudden death of the plant [7,28,29]. The internal wood symptoms usually consist of wedge-shaped necrotic sectors and brown streaking below the bark, sometimes beginning in the pruning wounds [30]. These fungi are air-borne and infect grapevine through any type of wound, but primarily the infection occurs through the pruning ones [1]. Pycnidia of different Botryosphaeriaceae species associated with Botryosphaeria dieback can be found within old pruning wounds, in dead or cankered wood, embedded on the bark of cordons and trunks of infected grapevines. They can also be found on pruning debris left in the vineyard, constituting a potential source of inoculum for new infections [7,31,32]. Up to now, there are no curative methods to mitigate infection by Botryosphaeriaceae species. Preventive control methods such as pruning wound protection are currently the practice proving to be more efficient [1,28], especially if carried out from the early stages of the vineyard lifespan [3,33]. The field efficacy of chemical wound protectants against botryosphaeriaceous fungi has been demonstrated in several grape-growing regions of the world, namely Australia [34], Chile [35], New Zealand [36,37], Portugal [38], South Africa [39,40], Spain [41], and the USA [42]. Another method considered to be the most effective strategy for controlling infection by GTD pathogens is the application of pastes and paints amended, or not, with fungicides. This latter can provide a physical barrier, preventing spore germination to occur in the wound, but if this barrier is altered by any external factor, the supplementation with a fungicide will act on the pathogen, inhibiting its growth [1]. Their efficacy has also been shown, specifically against Botryosphaeriaceae [34,35,41–43]. The currently existing active ingredients (AIs) are effective in protecting pruning wounds but have limited systemic activity. These AIs usually do not penetrate well enough into the grapevine tissues to effectively control pathogens inside the vascular system [28,44] and limit the colonization by pathogens. 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 [44–46]. 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. This last formulation has also shown an elicitation ability towards several genes related to plant defense [48]. However, increasing consumer demands for reduced chemical use and growing restrictions on the use of synthetic pesticides have increased interest in the use of natural active ingredients such as biocontrol agents (BCAs). Research on BCAs has greatly increased over the last years, being *Trichoderma* species the most studied as bio-pesticides [49]. Several *Trichoderma*-based products are currently used as pruning wound protectants against GTDs (for review see [1] and [28]) and several studies have been conducted using these products, showing encouraging results, against Botryosphaeriaceae species [28,40,50–56]. However, although several promising solutions currently exist, it seems impossible to manage Botryosphaeria dieback using only a single approach. An integrated pest management (IPM) strategy has been recommended including cultural practices, organic products, BCAs, responsible use of chemical fungicides, and control management that may combine both chemical and biological products [28,33].

To the best of our knowledge, no studies on strategies integrating pruning wound protection with chemical or biological products, and the application of site-targeted fungicides, with elicitor properties, have been conducted against Botryosphaeria dieback pathogens, on established vineyards. The main objectives of this work were thus to (i) test the efficacy of one *Trichoderma* based formulation, namely Esquive, single or in combination with LC2017, a new site-targeted copper-based treatment, formulated with hydroxyapatite loaded with copper (II) sulphate pentahydrate (CuSPHy + HA) which has a fungistatic and elicitor

effect and, (ii) to compare their efficacy with a strategy of combining the application of commercially available chemical pruning wound protectant, Tessior®, a liquid polymer containing boscalid and pyraclostrobin alone and with LC2017, to prevent both infection and colonization by *Lasiodiplodia* spp. in field trials.
