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First Report of Dactylonectria torresensis Causing Black-Foot Disease on Grapevines in the Czech Republic

Pečenka, J., Eichmeier, A., Peňázová, E., Baránek, M., León, M., Armengol, J.
Plant disease 2018 v.102 no.10 pp. 2038
Vitis, agar, air drying, autoclaving, bark, biomass, conidia, conidiophores, cultivars, culture media, financial economics, fungi, genes, greenhouses, growth retardation, histones, inoculum, leaves, mycelium, necrosis, nucleotide sequences, pathogenicity, peat, photoperiod, planting, plastic bags, root growth, roots, rootstocks, seeds, sodium hypochlorite, streptomycin, sulfates, tap water, vineyards, wheat, wood, Czech Republic
In 2015, diseased grapevines, cultivar Sauvignon blanc on SO4 rootstock, were surveyed in a young vineyard in Lednice, South Morava, Czech Republic. One year after planting, 30% of plants showed characteristic symptoms of black-foot disease: necrotic root lesions, reduced root growth, and wood necrosis at the base of the rootstock. Bark was removed from affected parts of the rootstock and the rootstock washed under running tap water, surface disinfected for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with distilled water. Small pieces of discolored tissues, including root fragments, were placed on malt extract agar amended with 500 mg/liter of streptomycin sulfate. Plates were incubated for 7 to 10 days at 25°C in the dark. Growing fungi were transferred to potato dextrose agar (PDA) and cultivated under the same conditions for 10 days. Single spores were transferred from orange-brown colonies to PDA for morphological and molecular identification. For morphological identification, cultures were incubated under a 12-h day/night photoperiod and 25°C for 10 days on synthetic nutrient-poor agar, with the addition of filter paper pieces. Thirty conidia per morphological type (90 per isolate) of three isolates were measured. Macroconidia were predominantly three-septate, straight or curved, more or less cylindrical, with rounded ends, 38.5 to 51.3 (44.9) × 6.6 to 9.4 (8.1) µm. Two-septate macroconidia measured 34.1 to 50.4 (41.6) × 6.1 to 10.2. (8.3) µm, and microconidia were ellipsoidal, zero to one septate, 28.2 to 51.8 (41.0) × 6.5 to 9.4 (8.1) µm, formed in sparsely branched conidiophores. The isolates were tentatively identified as Dactylonectria torresensis (A. Cabral, Rego & Crous) L. Lombard & Crous (Cabral et al. 2012). The identification was confirmed by amplifying and sequencing the histone H3 gene using primers CYLH3F and CYLH3R (Cabral et al. 2012). The nucleotide sequence was 100% identical with histone H3 coding regions of D. torresensis (CBS 188.49), and the sequence was deposited in GenBank/NCBI under accession number MH000286. Pathogenicity tests were carried out as described by Agustí-Brisach et al. (2016). Twenty plants of 16-month-old grapevines (cv. Sauvignon blanc on SO4 rootstock) were used. Fungal inoculum of isolate CZ-2 was produced in bags with wheat seeds. Seeds were soaked for 12 h in distilled water, air dried, transferred to 3-liter plastic bags, and autoclaved three times. Four disks of mycelium from a 2-week-old culture were aseptically placed in separate bags containing 1 liter of seeds and cultivated for 20 days at 25°C and mixed up every 48 h. Plants were individually planted in plastic pots with a mixture of 4 liters of sterilized peat and 180 g of inoculum per pot. Controls were inoculated with sterile wheat seeds. For each of two trials, eight plants were inoculated and two plants were noninoculated controls. Plants were maintained in a greenhouse at 25 to 30°C. Black-foot disease symptoms, including necrotic lesions on roots, foliage stunting, and root biomass reduction occurred 2 months after inoculation. Fungal colonies recovered from roots of symptomatic, inoculated plants were identified as D. torresensis as indicated above. Black-foot disease caused by D. torresensis causes substantial economic losses in young vineyards owing to replanting costs because diseased plants must be removed (Agustí-Brisach and Armengol 2013). To our knowledge, this is the first report of D. torresensis associated with decline of young grapevines in the Czech Republic.