*2.2. Field Assays*

The efficacy of the treatments was assessed based on the mean percentage of pathogen recovery (MPR) and, the mean percentage of disease control (MPDC) was calculated according to Sosnowski et al. [57,58] and Martínez-Diz, et al. [41]. Statistically significant differences were found amongst the treatments for both cultivars and years of the study (Table 2) when compared to the inoculated controls. For cultivar Cabernet Sauvignon, during the 2019 assay, treatments with Esquive + LC2017 reduced the MPR of isolate CBS124060 showing an MPDC of 70.8%, while Tessior + LC2017 was able to significantly reduce the MPR for both isolates Bt105 and CBS124060, with MPDCs of 81.5% and 75.0%, respectively. Tessior alone significantly reduced the MPR of isolate LA-SOL3 with an MPDC of 78.3%, and although an MPDC of 34.8% for isolate LA-SOL3 was recorded for the treatment with Esquive alone, no significant reduction was observed for any isolate. During 2020, a significant reduction in MPR of isolate Bt105 was verified for all the treatments, being the highest MPDC of 67.7% for the treatment with Tessior + LC2017. The only remaining significant reduction in MPR was on the pruning wound treatment with Tessior challenged with the isolate CBS124060 (MPDC 55%). Regarding cultivar Touriga Nacional, in 2019, no significant reduction in MPR was found for any treatment, when compared with the inoculated control, with the sole exception of Esquive inoculated with isolate CBS124060 (MPDC 53.3%). The 2020 assay showed a significant reduction in the MPR on treatment Esquive + LC2017 for isolates LA-SOL3 and CBS124060, with MPDCs of 48.4% and 58.6%, respectively. Isolate Bt105 provided a significant reduction in MPR with an MPDC of 52.5% on treatment with Tessior + LC2017, while treatment with Tessior was able to provide an MPDC of 45.2%. No significant reduction in MPR was verified for none of the remaining treatments when compared to the inoculated controls. Trichoderma spp. was only isolated from samples treated with Esquive. In 2019, recovery rates on Cabernet Sauvignon ranged from 24% to 16% for Esquive + LC2017 and Esquive treatments, respectively. For Touriga Nacional, the recovery rate for Esquive + LC2017 treatment was 20%, while a 30% recovery rate was recorded for Esquive applied alone. During 2020, Trichoderma spp. recovery rates for treatments with Esquive + LC2017 and Esquive alone were 38% and 28% for Cabernet Sauvignon and 25% and 22% for Touriga Nacional.


**Table 2.** Efficacy of treatments used solely as pruning wound protectants and in a combination with the copper-based product, LC2017, to reduce *Lasiodiplodia* sp. as part of an integrated disease management.



<sup>y</sup> Efficacy of all the treatments based on the mean percentage of recovery (MPR) of all the isolates used in this study, from the inoculated pruning wounds. Values in the same column with the same letter (a, b, c, d, e) do not significantly differ according to Tukey's test (*<sup>p</sup>* = 0.05). <sup>z</sup> Mean percentage of disease control (MPDC) of all the treatments calculated according to the formula MPDC = 100 × [1 − (MPR treatment/MPR inoculated control)].

> Meteorological data recorded during the time of pruning treatments and pathogen inoculation for both years are presented in Figure 3. In 2019, the average daily temperature in the week of treatment and inoculation (11–15 February) was 10.05 ◦C with no rain events recorded. In 2020, treatments and pathogens inoculations were performed during the week from the 16–20 February, with an average temperature of 13.3 ◦C and only one rain event recorded on the 17th with 1.1 mm of precipitation. Regarding the total rainfall and average temperature for the month of February of both years, 2019 recorded an average temperature of 12.2 ◦C and a total precipitation of 12.7 mm spread throughout five rain events. In 2020, the average temperature was 14.0 ◦C, and a total precipitation of 8.4 mm with eight rain events.

**Figure 3.** Monthly rainfall and average temperature for the two years of the trial.

The principal component analysis (PCA) was performed to better visualize the relationship between all the variables (Figure 4A,B). For 2019 (Figure 4A), the two first principal components, accounted for 59.9% of the total variability of the data (PC1—41.78%; PC2— 18.12%), while for 2020 (Figure 4B) the two first principal components represented 53.98% of the total variability of the data (PC1—35.34%; PC2—18.64%). For both years, we could also observe a clear distribution of the RP of the treatments according to the cultivar, which probably results from the influence of the different susceptibility that both cultivars may have towards the different pathogens studied. The relationship between the treatments and the meteorological variables could be observed in the vector plots of Figure 4A,B. For both years, meteorological variables showed similar behavior with precipitation being more related to component 1, while temperature showed a higher relation with component 2. In the vector plots, it was observed that the two meteorological variables were very weakly correlated, especially in 2020, where almost no correlation occurred between the variables.

Since the vineyard was in an area with a Köppen climatic classification of Csa (hot-summer Mediterranean climate), it makes sense that these variables are negatively correlated since higher temperatures are equivalent to less precipitation. This is again evident for the lower correlation verified in 2020, as less rainfall was recorded during the summer months compared to 2019 (Figure 3). For 2019 (Figure 4A), most of the treatments showed a much higher correlation with precipitation than with the average temperature recorded throughout the duration of the assay. The only exception was for treatments 2, 12, and 13 which showed a higher correlation to the average temperature recorded. A negative correlation was verified between some treatments, such as T4 and T12 and, T2 and T10. T4 was a treatment with Tessior + LC2017 inoculated with isolate Bt105 while T12 was a treatment with Esquive inoculated with isolate CBS124060. This negative correlation may be due to not only these treatments having very different modes of action (chemical combination vs BCA), but also because the behavior of the two cultivars was completely opposite. T4 appeared to have higher efficiency than T12 on Cabernet Sauvignon, with MDPC values of 81.5% and 20% respectively, while the exact opposite was verified for Touriga Nacional, with 39.3% and 53.3% (Table 2). Treatments T2 and T10 also showed a negative correlation with each other, and although they are both treatments with Esquive (T2 is Esquive + LC2017 inoculated with isolate LA-SOL3 and T10 is Esquive applied alone inoculated with isolate Bt105) again the different efficacy of the treatments in both cultivars seems to have significance, since T2 seems to be more effective in Cabernet Sauvignon with higher MPDC values than T10, but the opposite was recorded for Touriga Nacional (Table 2). This trend is also verified for treatments T2 and T1, which also show a negative correlation. Both treatments are Esquive + LC2017 but inoculated with different isolates (T1-Bt105 and T2-LA-SOL3). MPDC values of T2 (26.1%) appear to show once again a higher efficacy in Cabernet Sauvignon than in Touriga Nacional (18.5%), while T1 appears to have a better impact in protecting Touriga Nacional (25%) than Cabernet Sauvignon (18%). This behavior of treatments with Esquive (alone or in combination) where T1 and T10 showed higher MPDC levels for Touriga Nacional comparatively to Cabernet Sauvignon was also verified for T11 and was confirmed by the high correlation showed by these three variables (Figure 4A). It is also noteworthy that all the treatments with chemical formulations, namely Tessior + LC2017 (T4, T5, T6) and Tessior applied alone (T7, T8, T9) showed high correlation amongst themselves, all being positively correlated with PC1. The only treatment with a BCA (Esquive +LC2017) showing a high correlation with the chemical-based ones was T3. The other *Trichoderma* treatments (Esquive + LC2017 and Esquive applied alone) showed mixed behavior, while the treatments T1, T10, and T11 were highly correlated with each other, but negatively correlated with T2 and almost uncorrelated with T12. For 2020 (Figure 4B), a similar behavior was observed for most of the treatments. Almost all the treatments showed a high correlation with precipitation, except for treatment 4, which revealed again a high correlation with precipitation during the previous year and for treatment 13 again. In this case, treatments 3 and 9 exhibited a similar correlation with both precipitation and temperature. Therefore, for both years of the assay, almost all isolates showed a higher correlation with precipitation rather than temperature, except for treatment T13 apparently more strongly related to temperature. This treatment also showed a negative correlation with T8, and very low to almost no correlation to the remaining treatments, which could be related to the fact that T13 corresponds to the inoculated control of isolate Bt105. During this year, treatments T2 and T4 showed a negative correlation. These are again treatments with different modes of action since T2 is a treatment of Esquive + LC 2017 inoculated with isolate LA-SOL3 and T4 is a treatment of Tessior + LC2017 inoculated with isolate Bt105. As was observed for the 2019 analysis, this negative correlation may be attributed to T2 being apparently slightly more effective in Touriga Nacional (MPDC = 58.6%) than in Cabernet Sauvignon (MPDC = 53.8%) while the opposite result is found for T4, with an MPDC of 67.7% in Cabernet Sauvignon opposed to 52.5% in Touriga Nacional. On this 2020 analysis, it is also noteworthy that all the treatments inoculated with isolate LA-SOL3 (T2, T5, T8, T11, T14) appear to have a high correlation amongst themselves, showing a

low correlation with the remaining treatments. The exception was T5 which also showed a high correlation with the referred treatments, but nonetheless, this correlation shows that not only the variety and the type of product applied but also the isolate inoculated during this assay, may have some influence on the efficacy of the treatments tested.

**Figure 4.** Principal component analysis (PCA) for the interactions of all treatments, 15 variables including all the product/isolate combinations (T represents the treatment numbers which are stated in Table 2), as well as inoculated controls on both variables and the meteorological variables (Figure 2). Red marks represent the observations for Cabernet Sauvignon (CS) and green marks represent the Touriga Nacional (TN) observations. (**A**)-PCA analysis for 2019; (**B**)-PCA analysis for 2020.
