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First Report of Ilyonectria robusta Associated with Black Foot Disease of Grapevine in Southern Brazil

In August 2012, symptoms of black foot disease were observed on 21-year-old grapevines (Vitis labrusca cv. Bordô; own-rooted cultivar) at Nova Pádua city, Rio Grande do Sul state, Brazil. Symptomatic plants showed reduced vigor, vascular lesions, decline and death of vines, and necrotic lesions on roots. Isolation of fungi associated with necrotic root tissue was made on potato dextrose agar (PDA) medium containing 0.5 g L⁻¹ streptomycin sulfate. Cultures were incubated at 25°C for 7 days in darkness, and single-spore cultures were obtained from the colonies emerging from the diseased tissue. For morphological characterization, cultures were transferred to PDA and spezieller nährstoffarmer agar (SNA) medium with addition of two pieces of 1 cm² filter paper. One representative isolate (Cy9UFSM) was used for morphological and molecular characterization and pathogenicity confirmation. After 10 days growth on PDA at 20°C in the dark, colonies were umber to chestnut in color (3), appeared cottony to felty in texture, and sporulated profusely. After 5 weeks on SNA and under dark conditions at 20°C, cultures formed macroconidia predominantly on simple conidiophores, 1 to 3 septate, with both ends slightly rounded. Macroconidia varied in size depending on the number of cells as follows: one-septate (23-) 27.7 (-31) × (4.5-) 5.8 (-7) μm; two-septate (26-) 30.1 (-34) × (5-) 5.6 (-6) μm; and three-septate (24-) 31.2 (-35) × (5-) 5.8 (-6.5) μm. Microconidia were observed and did not have a visible hilum (6-) 11.2 (-17) × (3.5-) 4.2 (-5) μm (n = 30 observations per structure). Brown, thick-walled globose to subglobose chlamydospores were produced abundantly on PDA, (8.5-) 13.8 (-17) μm. To confirm the species, primer pairs H3-1a and H3-1b (2) were used to amplify a portion the histone H3 gene. Sequence of this region showed 98% similarity with a reference sequence for Ilyonectria robusta (A.A. Hildebr.) A. Cabral & Crous (GenBank Accession No. JF735530). Thus, both morphological and molecular criteria supported identification of the strain as I. robusta. This isolate was deposited in GenBank as accession KF633172. To confirm pathogenicity, 4-month-old rooted cuttings of Vitis labrusca cv. Bordô were inoculated by immersing roots in a conidial suspension (10⁶ ml⁻¹) for 60 min. After inoculation, the cuttings were planted in 1-L bags containing commercial substrate (MecPlant). Thirty days later, each plant was re-inoculated by applying 40 ml of a conidial suspension (10⁶ ml⁻¹) to the commercial substrate. Ten single-vine replicates were used for each isolate, and 10 water-inoculated vines were included as controls. After 4 months, the inoculated plants showed a 22.5% reduction of root mass, with root and crown necrosis, browning of vessels, and 20% mortality. Control plants treated with water remained symptomless. The fungus was re-isolated from blackened tissue of wood from the basal end of rooted cuttings, thereby satisfying Koch's postulates. I. robusta was first associated with black foot disease of grapevine in Portugal in 2012 (1). To our knowledge, this is the first report in southern Brazil of I. robusta associated with black foot disease of grapevine.

References: (1) A. Cabral et al. Mycol. Prog. 11:655, 2012. (2) N. L. Glass et al. Appl. Environ. Microbiol. 61:1323, 1995. (3) R. W. Rayner. A mycological colour chart. Commonwealth Mycological Institute and British Mycological Society, 1970.