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.