4.2.2. Fungal Isolates Used and Inoculum Preparation

Two *L. theobromae* and one *L. mediterranea* isolates were used for this assay (Table 3*). Lasiodiplodia theobromae* isolates Bt105 and LA-SOL3, were collected in Portugal and Peru, respectively, and were stored at the culture collection of Instituto Superior de Agronomia. Both were isolated from grapevine wood showing symptoms of cankers and wood necrosis. The *L. mediterranea* isolate used is from the CBS culture collection from the Westerdijk Fungal Biodiversity Institute, in Utrecht, Netherlands, with the accession CBS 124060. Although *L. mediterranea* is currently not reported in Portugal, it has been previously reported in other European countries and so, one isolate of this species was also included in this study, not only for comparison but also to investigate the efficacy of the studied products towards this species. Isolates were maintained in PDA and transferred to Petri dishes with PDA to promote colony growth. Cultures were incubated at 25 ◦C in complete darkness for 8 days. After incubation, cultures were plated onto 6 mm Petri dishes containing 2% water agar with autoclaved pine needles (*Pinus pinea*) and incubated at 25 ◦C under fluorescent light for a 12 h photoperiod, to pycnidia sporulation [76–78]. On the day of the inoculation, conidia were harvested by collecting pycnidia formed on the pine needles to a 1.5 mL Eppendorf tube containing sterile distilled water (SDW), crushing them with the help of a pestle, followed by shaking the tube in a vortex for one minute. Spore suspensions obtained were filtered through cheesecloth and the concentration was adjusted to <sup>1</sup> × 105 spores/mL with the use of a hemocytometer (Brand, Wertheim, Germany).
