4.2.4. Product Application and Pathogen Inoculation

For both seasons, immediately after harvest, one application of LC2017 was carried out as recommended by the manufacturer (Table 4; Figure 5). Harvest occurred during the month of September for both years of the assay. For both cultivars, one-year-old canes with a similar appearance, namely length, were selected for treatment followed by inoculation and were pruned at 2 cm above the third bud. After pruning, both products were prepared according to the label's rate. Esquive was weighted and mixed with water in a 200 mL spray bottle with a concentration of 4 kg/ha (Figure 5). Tessior (ready to apply solution) and was applied using the equipment specially designed for this product application (Figure 5). Untreated controls were mock-treated with SDW, and the wound protectants were allowed to dry for a few hours. This was followed by application with LC2017, as indicated by the manufacturer (Table 4, Figure 5), using a backpack sprayer. One day after the treatment, inoculation with the selected was performed, by applying 20 μL of the spore suspension (≈2000 spores) on each wound using a micropipette (Figure 5). After inoculation, the pruning wounds were protected for one week using Parafilm M® (Bemis, Sheboygan Falls, WI, USA) to prevent dehydration and promote spore germination. Pruning and artificial inoculation were performed on the 14 and 15 February 2019, and on the 20 and 21 February 2020, during the winter dormancy, taking into consideration that all the procedures were made during favorable meteorological conditions, namely cloudy and humid, but avoiding rain periods. The same precautions were taken for the remaining applications with LC2017, with the consideration of also avoiding days with strong winds to minimize spray drift.

#### 4.2.5. Pathogen Recovery and Identification

For both years, canes were recovered after harvest, during the month of October, and stored in a cold chamber (4 ◦C) until further processing. For pathogen re-isolation, the bark of each cane was removed, and a sample was collected from about 1 cm below the pruning wound (Figure 5). Four pieces of wood were collected from the border of necrotic internal tissue, surface disinfected with a 7% sodium hypochlorite solution, rinsed in SDW, and plated onto 9 mm Petri dishes containing PDA amended with chloramphenicol (PanReac, AppliChem, Darmstadt, Germany) at 250 mg/L. Plates were incubated at 25 ◦C, in the dark, and assessed for counting Botryosphaeriaceae and *Trichoderma* spp. colonies (Figure 5). A representative set of *Lasiodiplodia* spp. and *Trichoderma* spp. isolates was selected for identity confirmation. A DNeasy Plant Mini Kit from Qiagen® (Venlo, The Netherlands) was used to extract genomic DNA from 8-day-old cultures grown in PDA and incubated at 25 ◦C, in

the dark, following the manufacturer's instructions. The identity of *Lasiodiplodia theobromae* and *L. mediterranea* was confirmed by sequencing part of the translation elongation factor 1α gene (tef1-α) by using the primers EF1-688F and EF1–1251R [79], while *T. atroviride* strain I-1237 was confirmed by sequencing the internal transcribed spacer region (ITS) using the universal primers ITS5 and ITS4 [80]. Amplified DNA was visualized on agarose gels stained with GreenSafe Premium (Nzytech, Lisbon, Portugal), and was visualized using a UV transilluminator to assess PCR amplification. PCR products were purified using an Illustra ExoProStar Enzymatic PCR and Sequencing Clean-up Kit (GE Life Sciences, Buckinghamshire, UK). PCR products were sequenced both ways at STABVIDA (Lisbon, Portugal) and compared with sequences from GenBank in BLAST searches.

**Figure 5.** Diagram showing the several steps of the field assay. Information on the products used can be found in Table 4, and a description of the different treatments can be found in Table 5. (**A**) Marked sample collected from the field prior to analysis; (**B**) Sample with the bark removed showing sign of necrosis; (**C**) Sample collected about 1 cm below the pruning wound, to be divided into four pieces and plated onto PDA; (**D**) Isolate Bt105 (*Lasiodiplodia theobromae*) recovered from infected pruning wounds; (**E**) Isolate LA-SOL3 (*Lasiodiplodia theobromae*) recovered from infected pruning wounds; (**F**) Isolate CBS124060 (*Lasiodiplodia mediterranea*) recovered from infected pruning wounds; (**G**) Petri dish containing wood obtained from non-inoculated control plants showing no signs of pathogen growth.
