*3.1. Efficacy of Pruning Wound Protection Products Applied Alone*

For 2019, the highest MPDC values observed for Cabernet Sauvignon were found for the treatment with Tessior, while the lowest values were recorded for the application of Esquive alone. Nevertheless, the same pattern was not observed in 2020 when, in general, both treatments were able to reduce MPR to a similar extent. Pitt et al. [12] reported similar results in Australia, where liquid and paste formulations showed better efficacy against *Diplodia seriata* and *Diplodia mutila* than *Trichoderma*-based products. More recently, Martínez-Diz et al. [41] also described a higher efficacy of Tessior compared to Esquive, on pruning wounds infected with *D. seriata* and *Phaeomoniella chlamydospora*. A slightly different trend could be observed for cultivar Touriga Nacional in 2019 with the highest MPDC values for grapevines treated with Esquive alone. In 2020, this cultivar showed similar efficacy of the Esquive treatment comparing with the Tessior treatment, the only exception being the MDPC values found for isolate CBS124060 which were significantly lower than those reported for Tessior. In fact, all the 2020 treatments induced a slightly higher efficacy than observed in 2019, except for the Esquive treatment applied alone. Similarly, chemical fungicides, namely benomyl, were less effective than *Trichoderma* spp. treatments on wounds inoculated with *Diaporthe ampelina*, *D. seriata*, *E. lata*, *Neofusicoccum australe*, *Neofusicoccum parvum*, *L. theobromae* and *P. chlamydospora* [1,59]. This specific *T. atroviride* strain I-1237 has shown efficacy in reducing GTD incidence and severity in preliminary assays carried out in both Portuguese [55] and French [54] vineyards. Nevertheless, Martínez-Diz et al. [41] did not recently find significant differences between pathogen re-isolation from pruning wounds treated with *T. atroviride* strain I-1237 and inoculated non-treated controls.

The variable effectiveness of Trichoderma-based treatments has been previously reported by authors [34,41,51,60,61] and could be attributed to several reasons. The main advantage of using Trichoderma-based products as pruning wound protectants is the long-term protection conferred by the fungus growing in the wood. Therefore, the success of the protection provided by these products is dependent on the establishment of *Trichoderma* spp. within the wound. The influence of grapevine cultivar to wound colonization by *Trichoderma* spp. has also been highlighted by Mutawila et al. [52], probably related to the plant defense responses that differ between cultivars. These different defense responses to *T. atroviride* have been recently reported by Leal et al. [62], where the authors observed that *T. atroviride* may act as a priming stimulus for Tempranillo plantlets, while no stimulus could be verified for Chardonnay. This is in agreement with our PCA analysis which showed a negative correlation among treatments with *T. atroviride* (Esquive), likely due to a difference of efficacy amongst cultivars, which reinforces the importance of the cultivar in wound colonization by *Trichoderma* spp. It is known that not only cultivar but also meteorological conditions, such as temperature and precipitation, as well as the time of application influence the establishment and persistence of *Trichoderma* spp. [63]. Both cultivar and meteorological conditions factors could explain the differences of *T. atroviride* strain I-1237 colonization in our study. According to Esquive manufacturer, this specific *T. atroviride* strain I-1237 can grow at temperatures above 5 ◦C. In our study, the average temperatures recorded during pruning and treatments performed were 12.2 ◦C and 14 ◦C in 2019 and 2020, respectively. This may explain the apparent higher success on the colonization of the pruning wounds, especially verified for all the treatments with Esquive on Cabernet Sauvignon. Martínez-Diz et al. [41], while using the same commercial formulation of *T. atroviride* strain I-1237 (Esquive), found significantly lower colonization in Spanish vineyard trials. Nevertheless, this study was conducted on a different cultivar (Godello) and under different meteorological conditions. In this latter study, temperatures recorded during pruning and treatment application were significantly lower than those recorded during our study (6.5 ◦C and 9.8 ◦C in Spain to 12.2 ◦C and 14.0 ◦C in Portugal). The PCA analysis performed during this study also showed that most of the treatments where *T. atroviride* strain I-1237 was used were more correlated with precipitation than with temperature, being the only exceptions of treatments 12 and 13 in 2019. Nevertheless, in

2020, there were higher precipitation levels during the months of April and May, and on the PCA analysis performed for this year, all treatments with *T. atroviride* showed a high correlation with precipitation. This seems to reinforce the hypothesis that cultivar and meteorological conditions, especially rainfall, may apparently impact both the establishment and persistence of *T. atroviride* strain I-1237 in pruning wounds. As expected, for both treatments with Esquive, the highest percentages of colonization by *T. atroviride* strain I-1237 led to the highest mean percentage of disease control values. Further research is still needed to prove this hypothesis in GTDs which is characterized by extremely high complexity.

During our study, the application of Tessior (pyraclostrobin + boscalid + liquid polymer) was able to provide some reduction in infection by *Lasiodiplodia* spp. The highest mean percentages of disease control were obtained for Cabernet Sauvignon during 2019, with values as high as 78.3%, while during the next growing season, MPDC values only reached values as high as 55.5%. For Touriga Nacional, as referred to earlier, Tessior application was apparently not as effective as that of *T. atroviride*. The maximum level of MPDC was 45.2% for 2020, and MPDC values as low as 13.3% could be found during the previous year. Application of pyraclostrobin alone was effective in reducing infection by Botryosphaeria dieback fungi under field conditions [35,42], and a mixture of pyraclostrobin with metiram in the nursery when diluted in the soaking water prior to grafting [64]. Prior to this work, only preliminary studies and one in-depth study have been conducted by applying Tessior to pruning wounds, but all of them for controlling *Diplodia* spp. and *P. chlamydospora*. Preliminary field studies were conducted in Greece [65,66], Germany [64,67], and Spain [64] where Tessior was effective in reducing infection by the referred fungi. Recently, a more extensive study was conducted in Spanish vineyards by Martínez-Diz et al. [41] where Tessior showed a high MPDC for both *D. seriata* and *P. chlamydospora* compared to several other commercially available fungicides and BCAs. No previous studies have been conducted using Tessior as a pruning wound protectant against *Lasiodiplodia* spp., and although on the referred study, a botryosphaeriaceous fungi was used, *D. seriata* is considered a less aggressive species in comparison to *Lasiodiplodia* spp. [7]. In our study, we used two isolates of *L. theobromae* and one isolate of *L. mediterranea*. The establishment of the pathogens within grapevines depends on several factors, including not only meteorological variables and cultural practices, but also pathogen intrinsic properties such as aggressiveness [42]. Van Niekerk et al. [68] attributed the higher pathogen infection level on inoculated wounds to higher percentages of rainfall in the Stellenbosch area. This is in agreement with the PCA analysis of most of the treatments in our field study, which were strongly correlated with precipitation. The only treatment that for both years had a higher correlation with temperature was T13 corresponding to the inoculated control of isolate Bt105 (*L. theobromae*). It has been suggested that the expression of virulence factors in *L. theobromae* can be modulated by temperature [8,69]. This isolate was collected in Portugal, and it was previously considered to be highly virulent against both cultivars under study [70], suggesting that it may be better adapted to the local conditions than the other isolates under study. Moreover, our PCA analysis showed a high correlation between all the treatments in which plants were inoculated with isolate LA-SOL3 and between plants inoculated with isolate CBS124060 in 2020. Consequently, the difference in MPDC values found herein, comparatively to previous studies, may be due to the meteorological variables but also related to the pathogen used, and the difference in aggressiveness between them. Another factor that may be noteworthy is the difference in susceptibility between the two cultivars used. In fact, on average higher MPDC values were obtained for Cabernet Sauvignon during both growing seasons of the field assay, compared to the values found for Touriga Nacional. This tendency is verified, not only for the treatments with Tessior, but for most of the treatments. Previous studies conducted on the same cultivars using the same isolates, proved their high aggressiveness towards grapevine in both field and greenhouse assays [70], suggesting that the difference in efficacy in reducing infection by *Lasiodiplodia* spp. may also depend on cultivar susceptibility. Moreover, Sofia et al. [71] showed that from a set of four different cultivars, Touriga Nacional was one of the most susceptible to *P. chlamydospora*, suggesting that this particular

cultivar may be highly susceptible to GTDs in general. Therefore, and given the differences found between both growing seasons and both cultivars, further research is recommended to evaluate each of the components of these products to understand how their efficacy may be affected by factors such as time of application, pathogen species aggressiveness and cultivar susceptibility, pruning wound size, and vineyard terroir.
