*4.4. In Vitro Preliminary Screening for Selecting the Most Effective Extract*

The antifungal activity of the "dry-powder", "EtOAc-extract", "MetOH-extract" and "Nitric-extract" were preliminarily checked in order to select, among them, the most promising one to be used in further tests.

For testing the effectiveness of the "dry-powder" in affecting mycelial growth, 16 g of shrimp waste powder were homogenized with 1 L of autoclaved PDA and poured in 90 mm Petri dishes. For each pathogen, a mycelial plug (diameter 3 mm) from a 7-day-old culture grown on PDA at 25 ◦C was transferred in the center of a "dry-powder"—amended PDA plate; control cultures of each pathogen, obtained by subcultures in "dry-powder" non amended PDA plates, were included in the test. The plates were incubated at room temperature (20 ± 2 ◦C) for three days (for fungal pathogens) or for 15 days (for oomycete pathogens). At the end of the incubation period, no negative effects were observed in mycelial growth compared with controls for any of the pathogens. The "dry-powder" was not further tested.

The effect of "EtOAc-extract", "MetOH-extract" and "Nitric-extract" on the mycelial growth of the pathogens was tested at different concentrations. To this purpose, "EtOAcextract" and "MetOH-extract" were separately diluted in 1% dimethyl sulfoxide to obtain, for each substance, four solutions at the following concentrations 10, 25, 50 and 100 mg/mL; "Nitric-extract" was diluted in water to obtain the following concentrations: 25, 50, 75 and 100%.

"EtOAc-extract", "MetOH-extract", "Nitric-extract" and each fungal pathogen were tested separately in a 90 mm PDA plate as it follows: 500 μL of a suspension of conidia of the fungal pathogen (concentration 10<sup>4</sup> conidia/mL) were homogeneously spread on the surface of a PDA plate; by using a cork borer, five wells (diameter 3 mm, each) were then realized on the PDA plate; then, 60 μL of each concentration of the substance were pipetted into the respective well; the plates were finally incubated at 25 ◦C for three days. For the oomycete pathogens (*Phytophthora* spp.), the influence of "EtOAc-extract", "MetOHextract" and "Nitric-extract" was tested separately as follows: for each *Phytophthora* strain, a mycelial plug (diameter 3 mm) from a 7-day-old culture grown on PDA at 25 ◦C was transferred in the center of a PDA plate and surrounded by 5 wells at a distance of 3 cm from the plug; then, 60 μL of each concentration of the substance tested were pipetted into the respective well. The plates were then incubated at 25 ◦C for 15 days.

In all the experiments, the possible mycelial growth inhibitory activity induced by each solvent used for the preparation of the respective extract was verified by *in vitro* tests performed as described above. For all pathogens and substances at each concentration, all the tests were performed in triplicate.
