**4. Materials and Methods**

#### *4.1. In Vitro Assays*

#### 4.1.1. Mycelial Growth Inhibition Assay

Prior to field application of the LC2017 (CuSPHy + HA) product on the field, mycelial growth assays were conducted using three *Lasiodiplodia* spp. isolates, Bt105, LA-SOL3, and CBS124060 (Table 3). An assay was also performed using the *T. atroviride* strain I-1237 (Esquive®, product developed by Agrauxine S.A. and commercialized by Idai Nature S. L.), to test the compatibility between both products. To obtain the *T. atroviride* strain I-1237, a solution was made by directly suspending the Esquive® product, on 100 mL of sterile

distilled water. A 1.5 mL aliquot was transferred to a 90 mm Petri dish containing 20 mL of Potato-Dextro-Agar (PDA, Difco, Sparks, MD, USA), and spread onto the surface using a sterile plastic loop. Petri dishes were then incubated at 25 ◦C for 7 days in absolute darkness. For the mycelial growth assays, a stock solution of product LC2017 was made by suspending the product at the recommended field concentration (250 L/ha) to be applied immediately after pruning, in 1000 mL of sterile distilled water (SDW). Six different concentrations were made in SDW and added to 50 ◦C molten PDA, and 20 mL was poured into each 90 mm Petri dish, with six replicate plates allowed for each combination of LC2017 concentration and isolate (both *Lasiodiplodia* spp. and *T. atroviride* strain I-1237). The test range of LC2017 product concentration ranged from 0.025 to 12.5 mL L−<sup>1</sup> and the six concentrations tested were evenly distributed across that range. Four hours after preparing the plates, 3 mm diameter discs were cut from the actively growing margin of one-week-old colonies all the isolates and placed on the center of each plate. Control plates contained only PDA. Plates were incubated at 25 ◦C for 48 h, in complete darkness, after which the two perpendicular diameters of the colonies were measured using a digital caliper. Mycelial growth inhibition (GI) was calculated according to Battiston et al. [46]: GI = [(DC − DO)/DC] × 100, where DC is the diameter of mycelial growth in the control plates and DO is the diameter of mycelial growth in treated plates. To establish if the effect of LC2017 on the tested fungi was only fungistatic, inhibited fungal disks were reinoculated onto fresh PDA plates and their growth revival was observed after 48 h.

**Table 3.** *Lasiodiplodia* spp. isolates used for pruning wound inoculation.

