During a survey for grapevine decline, five young grapevines (cvs. Tempranillo and Viura) with low vigor and reduced foliage were collected (June and August 2009). Fungal isolations were performed from vascular and brown wood. Small pieces of brown wood were placed onto malt extract agar supplemented with 0.25 g/liter of chloramphenicol and incubated at 25°C in darkness. Five resulting colonies were transferred to potato dextrose agar (PDA). Isolates were characterized by abundant, gray, aerial mycelium that reached a radius of 45 mm after 4 days. Pycnidia induced on water agar with pine needles and UV light contained conidia that were hyaline, smooth, thin walled, fusiform, (20-) 22 to 26 (-28) × (5.5-) 6 (-6.5) μm, with granular cytoplasm. On the basis of morphological characteristics Neofusicoccum mediterraneum was suspected (1). Single-conidial cultures were generated from each isolate. DNA analyses were described in Martin and Cobos (2). Sequences of the internal transcribed spacer (ITS) region confirmed the identification and revealed 99% genetic identity with N. mediterraneum (GenBank Accession No EU040221). A sequence of the ITS fragment was deposited with Accession No. JF437919. Partial sequences of β-tubulin and 1-α elongation factor genes were amplified and deposited in the GenBank with Accession Nos. JF437921 and JF437923, showing 100 and 99% similarity to Accession Nos. GU292786 and GU251350, respectively. Pathogenicity tests were conducted with two isolates. The inoculations were carried out on a fresh wound on which an agar plug was applied; on 110R-rootstock woods of 12 young vines with N. mediterraneum and 12 other control plants were treated with agar only. Grapevines were maintained in a greenhouse at 20 to 25°C. After 4 months, N. mediterraneum was reisolated from vascular and brown tissues in 92% of inoculated plants, fulfilling Koch's postulates. Control plants were asymptomatic and N. mediterraneum was not recovered. With the same methodology, isolate Y264-21-1 reached a radius of 43 mm after 4 days at 25°C on PDA, presented colonies becoming olivaceous with a moderately dense mycelia, mat in center, and aerial around. Conidia were hyaline, fusiform, base subtruncate (19-) 23 to 26 (-31) × 5 to 6 (7.5) μm, unicellular, and smooth with granular contents. Based on these descriptions, N. australe was suspected (3). ITS sequence comparison revealed 99% genetic identity with N. australe (Accession No. FJ150697), a sequence of the fragment was deposited with Accession No. JF437920. Partial sequences of β-tubulin and 1α-elongation factor were deposited in the GenBank (Accession Nos. JF437922 and JF437924) showing 100 and 99% similarity to Accession Nos. AY615149 and GU251352, respectively. Koch's postulates were completed as described above. After 4 months, N. australe was reisolated from internal brown lesions in 92% of inoculated plants. Control plants were asymptomatic and N. australe was not recovered. The streaking length average from inoculation point for N. mediterraneum was 42 ± 22 mm and 53 ± 7 mm for N. australe. To our knowledge this is the first report of N. mediterraneum and N. australe in Castilla y León (Spain).
References: (1) P. W. Crous et al. Fungal Planet 19:2, 2007. (2) M. T. Martin and R. Cobos. Phytopathol. Mediterr. 46:18, 2007. (3) B. Slippers et al. Mycologia 96:1030, 2004.