First Report of Ilyonectria robusta Causing Black Foot Disease of Grapevine in Spain.

In June 2017, symptoms of black foot disease were observed on 2-year-old grapevines (Vitis vinifera L.) cultivar Malvasia grafted on 110 Richter rootstock and on 2-year-old grapevines cultivar Garnacha Tintorera grafted on 110 Richter rootstock in Mendavia (Navarra Province, northern Spain) and Requena (Valencia Province, eastern Spain), respectively. Affected plants showed delayed budding with low vigor and leaf chlorosis. Roots showed black discoloration and necrosis of wood tissues. Root fragments were cut, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small pieces of discolored or necrotic tissues were plated onto malt extract agar supplemented with 0.4 g/liter of streptomycin sulfate. Plates were incubated at 25°C in the dark for 10 days, and all colonies were transferred to potato dextrose agar (PDA). A Cylindrocarpon-like asexual morph fungus was consistently isolated from necrotic root tissues. Single conidial isolates were obtained and grown on PDA and incubated at 25°C for 10 days in darkness. The isolates developed buff to cinnamon and felty abundant mycelium. All isolates produced slightly curved one-septate (16.0 to) 21.6 (to 26.5) × (4.5 to) 6.1 (to 7.8) μm, two-septate (19.5 to) 25.9 (to 35.0) × (5.0 to) 6.6 (to 7.9) μm, and three-septate (22.5 to) 31.3 (to 47.5) × (5.5 to) 7.1 (to 8.9) μm macroconidia, and aseptate (4.0 to) 7.7 (to 12.5) × (2.5 to) 3.3 (to 5.4) μm and one-septate (8.5 to) 12.5 (to 17.5) × (3.5 to) 5.1 (to 6.2) μm microconidia. DNA sequence analysis of the partial histone H3 gene was obtained for isolates BV-816 and GIHF-156 and deposited in GenBank (accession nos. MH229866 and MH229867). These sequences showed high similarity (99%) to the sequence of Ilyonectria robusta (A.A. Hildebr.) A. Cabral & Crous (GenBank accession no. JF735517), in agreement with morphological features (Cabral et al. 2012a). Pathogenicity tests were conducted on 2-month-old grapevine seedlings (cv. Tempranillo) using both isolates. Fifteen seedlings per isolate were inoculated when two to three leaves had emerged by immersing the roots in a 10⁶/ml conidial suspension for 30 s (Cabral et al. 2012b). Fifteen control plants were inoculated with sterile distilled water. Seedlings were maintained in a growth chamber at 25°C. Symptoms developed on all plants 15 days after inoculation and consisted in reduced vigor, interveinal chlorosis and necrosis of the leaves, necrotic root lesions with a reduction in root biomass, and plant death. The fungus was reisolated from the roots of affected seedlings and identified as I. robusta, completing Koch’s postulates. No symptoms were observed on the control plants. To our knowledge, this is the first report of I. robusta causing black foot disease of grapevine in Spain. I. robusta is one of the Cylindrocarpon-like asexual morphs included in the complex of fungal species belonging to the genera Campylocarpon, Cylindrocladiella, Dactylonectria, Ilyonectria, Neonectria, and Thelonectria, which are associated with black-foot disease, causing untenable economic losses to the grapevine industry in Spain and worldwide (Gramaje et al. 2018).