*4.1. Isolation and Identification of Actinomycetes Strains*

During a survey carried out from 2015 to 2019 on the root diseases of grapevine plants affected by decline and apoplexy, caused by fungal agents of grapevine root (Black foot) such as *Dactylonectria* spp., *Ilyonectria* spp. and trunk diseases (GTD) such as *Phaeomoniella chlamydospora* and *Phaeoacremonium* spp. [28], other unknown microorganisms were isolated including not-sporulated fungi, bacteria and *Actinomycetes*. Among the latter microbial agents, a consistent number (37 isolates, corresponding to 1.6% of microorganisms isolated) of presumptive *Streptomyces* strains were observed and recorded. All *Actinomycetes* cultures were subjected to purification techniques by spreading over Petri dishes of agar and water (AW). After an overnight incubation at 28 ± 3 ◦C, single germinating spores or small pieces of hyphae were transferred to Petri dishes with fresh potato-dextrose-agar (PDA; 39 g/L, Oxoid) for molecular identification. For this purpose, a representative strain of *Actinomycetes* showing a putative antimicrobial activity, called CARA17, was used for molecular characterization. Genomic DNA of the CARA17 strain was extracted from a 15-day-old culture growing on PDA at 28 ± 3 ◦C in the dark, according to Carlucci et al. [29].

For preliminary molecular identifications, the primer pairs used were 16SAct1F (5 CGC GGC CTA TCA GCT TGT TG 3 ) and 16SAct1R (5 CCG TAC TCC CCA GGC GGG G 3 ) of 16S rDNA for the amplification of the 16S ribosomal RNA (R RNA) region [30]. The amplification was made according to the following PCR protocol: 1× PCR buffer, 2.5 mM MgCl2, 200 μM of each nucleotide, 2.5 pmol of each primer, 0.25 U Taq polymerase, 0.5 μL DMSO, and a 30–50 ng DNA template taken to a total volume of 25 μL. The Taq polymerase, nucleotides and buffers were supplied by Eurofins Genomics (Milan, Italy). The amplification conditions were: initial denaturation for 5 min at 94 ◦C, followed by 35 cycles of denaturation for 1 min at 95 ◦C, annealing for 30 s at 58 ◦C, elongation for 1 min at 72 ◦C and the final extension step for 10 min at 72 ◦C. Five microliters of amplicon were analyzed by electrophoresis at 100 V for 30 min in 1.5 % (*w/v*) agarose gels in 1× TAE buffer (40 mM Tris, 40 mM acetate, 2 mM EDTA, pH 8.0). The gels were stained

with ethidium bromide and were visualized in a Gel Doc EZ System under UV light (Biorad, Hercules, CA, USA). The PCR products were purified before DNA sequencing, using Nucleo Spin Extract II purification kits (Macherey-Nagel, Germany), according to the manufacturer's instructions. Both strands of the PCR products were sequenced by Eurofins Genomics (Ebersberg, Germany). The nucleotide sequences obtained were manually edited using BioEdit v.7.0.9 (http://www.mbio.ncsu.edu/ BioEdit accessed on 8 Febrary 2021). The consensus sequence was compared with those available in the GenBank database, using the Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/ accessed on 24 March 2021) to confirm the preliminary morphological identification and to ascertain the sequence similarity searches.
