Occurrence of Botryosphaeria obtusa, B. dothidea, and B. parva Associated with Grapevine Trunk Diseases in Castilla y León Region, Spain.

Between 2000 and 2004, 176 vineyards were surveyed for disease symptoms throughout the main grapevine-production areas of Bierzo, Cigales, Ribera del Duero, Rueda, and Toro in the Castilla y León region of Spain. Symptoms resembling Eutypa dieback, such as stunted chlorotic shoots, deformed leaves with necrotic areas, and typical wedge-shaped cankers in the wood, were observed in 80% of surveyed vineyards. The second most common disease observed was esca. The mild form of esca, interveinal chlorosis or reddened patterns on the leaves, was observed in 35% of surveyed vineyards. Severe esca symptoms that include sudden defoliation of some or all parts of the vine followed by shriveling of fruit clusters were observed in vineyards during very hot and dry summer periods. Wood from vines with esca was yellowish, soft, and often partially or completely surrounded by necrotic wood. Black vascular streaking in the wood was also observed in some vines with esca. Samples of wood from vines with symptoms of Eutypa dieback or esca were collected from different cultivars (Tempranillo, Cabernet Sauvignon, Mencía, Garnacha, Viura, and Verdejo). Small pieces of symptomatic wood were placed on 4% potato dextrose agar amended with tetracycline hydrochloride (0.01%) (PDA-tet) and incubated at room temperature. Pure cultures were obtained by excising hyphal tips and transferring to PDA-tet. Species of Botryosphaeria were most frequently isolated from wedge-shaped cankers as well as from wood with necrosis or black vascular streaking. Botryosphaeria spp. also were isolated from the soft yellowish wood, however, Fomitiporia punctata, Stereum hirsutum, and Phaeoacremonium spp. were the most common fungi associated with this symptom. On the basis of morphological characteristics in culture (1), three species were isolated (B. obtusa, B. dothidea, and B. parva). Colonies of B. obtusa were green to dark green with moderate aerial mycelium. Pycnidia developed after 6 days and conidia (n = 50) measured 19 to 27 × 9 to 17 μm and were hyaline and light brown, becoming dark brown when mature, mostly aseptate, and rounded in shape. Colonies of B. dothidea were white, becoming dark green with age and with copious aerial mycelium. Pycnidia started to develop after 10 days, and conidia measured 17 to 31 × 4 to 8 μm, were hyaline, aseptate, and fusiform in shape. Colonies of B. parva were similar in appearance to those of B. dothidea but pycnidia developed after 5 weeks. Conidia measured 11 to 21 × 4 to 9 μm, were hyaline when immature, becoming light brown with two septa with age, and ellipsoidal in shape. Identity of the three Botryosphaeria species was confirmed by comparing morphology with growth of the following identified California isolates: B. obtusa (UCD352Mo and UCD666Na), B. dothidea (UCD1066So), and B. parva (UCD642So) and by comparing sequences of the internal transcribed spacer region (ITSI-5.8S-ITS2) rDNA, and a partial sequence of the β-tubulin gene (BT2) of our isolates with those of previously identified and sequenced isolates deposited in GenBank. Sequences of B. obtusa (UCD343Spa, UCD461Spa, UCD468Spa, and UCD621Spa), B. dothidea (UCD303Spa), and B. parva (UCD577Spa and UCD578Spa) were deposited in GenBank. To our knowledge, this is the first report of B. obtusa, B. dothidea, and B. parva on grapevines in the Castilla y León region in Spain. Reference: (1) A. J. L. Phillips. Phytopathol. Mediterr. 41:3, 2002.