*3.2. Efficacy of a Strategy Combining the Application of Pruning Wound Protectants with LC2017*

The great diversity of species currently associated with Botryosphaeria dieback, but also with all GTDs, combined with the intrinsic differences in hundreds of cultivars planted worldwide under different terroir conditions, makes the research for an effective management strategy to reduce the impact of GTDs an extremely difficult challenge. To this extent, in this work, we tried to integrate currently used products for pruning wound protection with the application of a site-targeted product based on a copper (II) compound (copper sulfate) and synthetic nanostructured particles of hydroxyapatite (HA) which have already shown interesting drug delivery properties in planta [46–48,72]. In our work, the application of this new site-targeted copper (II) formulation (LC2017), does not seem to greatly increase the efficacy of the pruning wound protection strategy. In some cases, the combination of the two strategies actually increased the mean percentage of disease control, but in others, the opposite was also verified. For example, in 2019, and for the inoculation of isolate CBS124060 on Cabernet Sauvignon, an MPDC of 70.8% was obtained for the treatment with Esquive + LC2017 while only 16.7% was recorded for the treatment with Esquive alone. During the next year (2020) the same value of 60% was found for both treatments. In 2019, on Cabernet Sauvignon, the application of Tessior + LC2017 caused also an MPDC of 81.5% for isolate Bt105, while only 37% was found for the treatment with Tessior alone. For the two other isolates under study, LA-SOL3 showed an increase in MPDC when Tessior was applied alone, while the isolate CBS124060 showed an opposite trend. Although the highest concentration of LC2017 used in the in vitro assay for the inhibition of mycelial growth showed fungistatic effect for all the isolates and *T. atroviride* strain I-1237, there is no apparent pattern on the influence of the LC2017 application as part of an IPM for any of the cultivars, or specific isolate. Moreover, the fungistatic effect verified for *T. atroviride* strain I-1237 did not seem to impact the colonization of pruning wounds by this BCA, since the highest values of re-isolation percentage could be found for the treatments combining Esquive and LC2017 except for Touriga Nacional during 2019. Moreover, no relevant correlation between the treatments using LC2017 was observed for both years by the PCA analysis, indicating a low influence of LC2017 application on the MPDC values found in this study.

The copper formulation used (LC2017) has already shown both fungistatic and fungicidal effects in vitro and *in planta* against *Phaeoacremonium minimum* [46]. Mondello et al. [48] have also reported that LC2017 showed the same in vitro fungistatic effect on *D. seriata* and *N. parvum* as it was verified for our *L. theobromae* isolates. The same authors described as well, the ability of this formulation to activate GTD-related plant defense reactions [48]. Di Marco et al. [73] also tested a copper formulation against *P. chlamydospora* and *P. minimum*, which revealed the ability to reduce conidial germination in in vitro assays but was not able to reduce *P. chlamydospora* colonization on young potted grapevines. Amposah et al. [36] tested a copper hydroxide formulation against Botryosphaeria dieback fungi, and were not able to obtain any significant control both in in vitro and in plant tests. More recently, Mondello et al. [48] performed greenhouse in planta assays that did not show significant differences in stem necrosis length of plants treated with LC2017 and inoculated with *D. seriata* and *N. parvum*. This agrees with our results since the same copper formulation used does not appear to provide a significant impact when combined with pruning wound protectants in controlling these specific Botryosphaeria dieback fungi. Therefore, further research is needed on testing all the components of this formulation, since HA applied alone has shown a non-fungitoxic and stimulant activity on *P. minimum* [48] and *Botrytis cinerea* [71]. However, the efficacy of copper (II) products has been previously related not only as

a fungicide but also as an elicitor of some plant defense responses. Aziz et al. [74] observed this eliciting effect for CuSO4 sprayed on leaves, and Battiston et al. [48] have also shown that this formulation of CUSPHy was able to strongly induce several plant defense genes. Mondello et al. [48] also showed that LC2017 (HA + Cu (II)) had the same elicitation potential as BTH (*S*-methyl benzo (1,2,3) thiadizole-7-carbohthioate), a commonly marketed elicitor (BION®, Syngenta, France) by inducing genes related with chitinase and glucanase synthesis and also genes related to the biosynthesis pathways of several phenolic compounds.

Therefore, new methods to efficiently deliver fungicides to specific targeted areas of the plant are considered to have great potential to improve GTD control, and further research is needed to investigate not only the impact that these formulations may have on several GTD pathogens but also on the plant microbiome. Further long-term field trials should also be undertaken to investigate the effectiveness that a prolonged management control combining pruning wound protection and site-targeted fungicides might provide in controlling GTDs.

During this work, to ensure a proper establishment of infection with the fungi under study, approximately 2000 spores of each of the *L. theobromae* and *L. mediterranea* isolates were used to challenge each pruning wound. This represents a high inoculum pressure compared to the levels that pruning wounds are usually exposed to under natural field conditions. This suggests that due to the higher inoculum applied to the pruning wounds, the real efficacy of all the treatments tested against *Lasiodiplodia* spp. may have been underestimated [28].

In conclusion, this study demonstrated the field potential of pruning wound protection formulations to control *Lasiodiplodia* spp., applied alone or in combination with a novel site-targeted copper (II) formulation. The efficacy of the studied products differed between the two cultivars used and between the two growing seasons of the duration of the assay. Nevertheless, some measure of control was achieved for all treatments studied, despite the application of the copper-based product LC2017 not showing clear evidence of improving the control obtained by the pruning wound protection products applied alone. Still, this new site target copper-based product may prove to be a viable way of reducing the amount of copper applied for diseases management on vineyards, especially with further European Union restrictions on copper use in agriculture. Furthermore, the combination of effective pruning wound protection management by using a combination of a BCA with a more sustainable copper-based product has already proven to have interesting results not only as an elicitor by strongly inducing plant defense genes but also in the control of *P. minimum*, as well as *Plasmopora viticola*, may lead, in the long term, to healthier grapevines, which may reduce the expression of GTD symptoms in vineyards. However, this hypothesis still needs to be clarified by conducting studies using the same management strategy over a longer period of time, targeting not only other grapevine cultivars but also other Botryosphaeriaceae species. Thus, good pruning practices and wound protection combined with sustainable management strategies, such as biostimulants and host resistance inducers, can reduce the impact of Botryosphaeria dieback, not only by controlling pathogens already common in a certain wine region but also by making it difficult for the potential establishment of new species.
