2.1 Plant material
A 20-year-old V. vinifera cv. Italia vineyard (1,600 vines) located in the countryside of Bari (Apulia, southern Italy), was used for symptoms survey and sample collection. The vines, grafted onto 140-Ru, were trained by the overhead system (‘tendone’) and grown under irrigation on alkaline-clayey soil. Since 2006, the vineyard was surveyed for incidence and development of “Esca complex” symptoms.
Wood cores were taken with a 95% ethanol pre-killed Pressler increment borer from the trunk at 30 and 110 cm above the ground, to assess internal infections. Holes were disinfected with copper oxychloride solution (20% in water) and filled with a mixture of 2.5 % copper oxychloride in linseed oil. For each core, slices of wood were cut, surface sterilised for 1 min in 70% ethanol, soaked 1-2 min in a sodium hypochlorite solution (3% active chlorine) and rinsed thrice in sterile distilled water. The pieces were then axenically cut to chips and seeded (five per plate) on 90-mm diameter Petri dish. Malt extract (2%) agar (MEA), MEA amended with 0.25% chloramphenicol (MEAC) and MEA adjusted with 0.25% benomyl (MEAB) were used. Isolation plates were incubated at 25±1°C, in the dark, for 4-6 weeks. The isolation frequency (IF) of each fungal taxon was calculated as IF = Ni/Nt × 100, where Ni is the number of wood-fragments from which the fungus was isolated and Nt the total number of seeded chips.
Fifteen vines were selected: five with brown wood-streaking (BWSV), five with brown wood-streaking and white rot (BWSWRV) and five healthy (HV) without internal and external symptoms.