First Report of Diaporthe eres Associated with Cane Blight of Grapevine (Vitis vinifera) in Italy

In March 2014, in a ‘Trebbiano’ vineyard in the province of Florence (Tuscany, Italy), 15% of the grapevines (Vitis vinifera L.) showed 1-year old canes with bleached areas, sometimes surrounded by dark margins, irregular dark blotches, and several dead buds. Canes were covered by black pycnidia, and occasionally cracks on the cortex were evident. Woody tissues under the bark were necrotic and canes broke easily. Fifteen symptomatic canes were collected from different vines and cane segments of 20 cm length were placed in a moist chamber and incubated at 25°C. After 72 h, a cream-white cirri mass of conidia was observed from pycnidia and it was plated onto potato dextrose agar (PDA) plates. After one week incubation at 22 ± 1°C in the dark, grown fungal colonies showed a white mycelium with overlapping regions of denser mycelium with rosulate margins. From collected pycnidia, length and width of 100 conidia of each type (alpha and beta) were measured, and the 95% confidence interval, mean, and standard deviation were calculated. Alpha conidia were unicellular, hyaline, fusiform, with one or both ends tapered, not guttulated or unibiguttulate (sometimes with more than two guttules), measuring (5.85) 7.71 to 8.21 (13.56) μm × (1.48) 2.12 to 2.20 (2.70) μm (mean 7.96 ± 1.29 μm × 2.16 ± 0.22 μm). Beta conidia were hyaline, aseptate, filiform, from curved to hamate, measuring (26.61) 25.70 to 26.71 (32.86) μm × (1.03) 1.34 to 1.39 (1.64) μm, mean 26.15 ± 2.33 μm × 1.37 ± 0.14 μm. Based on the current revisions of the genus Diaporthe Nitschke (Gomes et al. 2013; Udayanga et al. 2014), a multigene phylogenetic analysis was carried out. Internal transcribed spacer (ITS1-5.8S-ITS2) region and parts of the translation elongation factor 1-α (TEF1) and β-tubulin (TUB) genes of two isolates were sequenced (GenBank Accession No. KT369109 to KT369114). ITS, TEF1, and TUB partial gene alignments were generated by including the corresponding alleles of 22 strains of D. eres along with 27 most closely related Diaporthe species (Gomes et al. 2013; Udayanga et al. 2014) retrieved from the NCBI databases. Maximum likelihood analyses were performed on individual and concatenated data sets using MEGA6 (Tamura et al. 2013). According to the morphological and phylogenetic characters, the two isolates were identified as belonging to the species D. eres Nitschke. A pathogenicity test was carried out on excised, lignified, one-year old canes collected from grapevines cv. Sangiovese, cut into 20-cm-long segments. Canes were surface sterilized with 2% sodium hypochlorite, rinsed, dried, and wounded with a sterile scalpel creating a 1-cm-long incision on an internode. A 4-mm agar disc with mycelium grown on PDA at 25°C for 7 days was placed into each wound and sealed with Parafilm. Sterile PDA plugs were used as a negative control. Ten canes were inoculated with each strain and placed in sterile, glass petri dishes containing wet, sterile paper towels. After one month of incubation at 25°C, necrotic lesions of the wood had developed on the inoculated canes with an average length of 5.42 ± 1.66 cm. Negative control canes remained symptomless. Diaporthe eres strains were reisolated from inoculated canes while no fungal colonies were recovered from the control canes. To the best of our knowledge, this is the first report of D. eres causing cane blight of grapevine in Italy