A genetic linkage map was constructed using a pseudo-testcross strategy based on a cross between the seedless Vitis vinifera 'Crimson Seedless' and the complex hybrid 'Villard Blanc', resistant to downy mildew. A total of 315 DNA markers, including 262 AFLP, 48 simple sequence repeats (SSR), 2 SCARs (sequence characterized amplified region) and 3 minisatellite markers were used to generate a map for each parent. For both parents, 19 linkage groups were obtained, covering 1,111.0 cM and 926.0 cM for 'Villard Blanc' and 'Crimson Seedless', respectively. The position of SSR loci in the obtained maps is consistent with the genomic sequence. Quantitative Trait Loci (QTLs) for seedlessness and resistance to downy mildew were investigated. Two major effect QTLs for downy mildew resistance and seedlessness were mapped on the same region of linkage group 18. These QTLs explain 25.0-55.7% and 54.0-62.,4% of the total variance, respectively. The MIKC C-Type MADS box gene VvAG3, whose orthologue is involved in Arabidopsis carpel and ovule development, and located in the confidence interval of the seedlessness QTL detected on the LG 18, could be considered as a candidate gene to control seed development in grape. Colocalizations were found in the same region, between the position of the Rpv3 locus, which is very rich in TIR-NBS-LRR genes, and the main QTL identified for downy mildew resistance. Our results demonstrate that the same region of LG 18 contains important genetic determinants for seedlessness and downy mildew resistance in grapevine. Moreover, assessing the allelic variation at these agronomically important loci provides a basis for the development of marker-assisted selection for seedlessness and downy mildew simultaneously.

Cultivated grapevine (Vitis labrusca and V. vinifera) is of considerable economic importance to the Brazilian fruit industry for both fresh market consumption and for the production of wines, sparkling beverages, and juices. Black foot disease is caused by fungi of the genera Ilyonectria P. Chaverri & C. Salgado (anamorph: Cylindrocarpon Wollew.), Campylocarpon Halleen, Schroers & Crous, and Cylindrocladiella Boesew. In 2012, 4- to 40-year-old grapevines (Vitis spp.) showing reduced vigor, vascular lesions, necrotic root lesions, delayed budding, vine decline, and death were collected from seven locations at Rio Grande do Sul state, Brazil. Fungal isolations were made from root fragments and crown lesions (at least 2 cm above the bottom) on potato dextrose agar (PDA) medium added with 0.5 g L–1 streptomycin sulfate. Eight isolates were obtained and identified on the basis of morphological features and multi-gene analysis (rDNA-ITS, β-tubulin, and histone H3) as Ilyonectria macrodidyma (Halleen, Schroers & Crous) P. Chaverri & C. Salgado. One representative isolate (Cy5UFSM) was used for more detailed morphological and molecular characterization, and pathogenicity confirmation. When incubated in the dark at 20°C for 7 to 10 days, colonies of felty straw-colored mycelium (3) 4.79 cm diameter on average were observed. No sporodochia or other fruiting bodies were produced on carnation leaf agar (CLA) medium after 30 days. Microconidia that were produced after 5 weeks on spezieller nährstoffarmer agar (SNA) medium with addition of two pieces of 1 cm2 filter paper showed ovoid and ellipsoid shape (6.4 × 3.6 μm) and one-septate macroconidia (17.3 × 4.1 μm). To confirm the species, primer pairs ITS1 and ITS4 (4); Bt2a and Bt2b; and H3-1a and H3-1b (2) were used to amplify the ITS1-5.8S rRNA-ITS2, part of the β-tubulin and histone H3 genes, respectively. Sequences of these three regions showed 99, 100, and 100% of homology with I. macrodidyma, respectively. To confirm pathogenicity, 4-month-old rooted cuttings of V. labrusca cv. Bordô were inoculated by immersing them in a conidial suspension of the isolate (106 conidia ml–1) for 60 min (1). Thirty days later, inoculation was performed again by drenching the crown with 40 ml of 106 conidia ml–1 suspension to ensure infection of the roots. In the control treatment, plants were inoculated with sterile distilled water. Plants inoculated with I. macrodidyma showed necrosis of the leaf ribs, reduction in root mass, root and crown necrosis, browning of vessels, drying of shoots, and death. I. macrodidyma was re-isolated from the crown necrosis and vascular lesions, confirming Koch's postulates. To our knowledge, this is the first report of I. macrodidyma associated with black foot disease of grapevine in Brazil, which poses considerable threat to the industry unless management options are realized. References: (1) A. Cabral et al. Phytopathol. Mediterr. 51:340, 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. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Since 1999, the decline of American grapevines (Vitis labrusca L.) has been common in Rio Grande do Sul, Brazil (1). In August 2012, V. labrusca with black foot symptoms were collected in vineyards in the Serra Gaúcha Region. Symptomatic plants had low vigor, vascular lesions, delayed budding, and decline and death of vines. Symptomatic roots had necrotic lesions and reduced biomass. Fungal isolations were made from necrotic root and crown fragments (own-rooted cultivar) on potato dextrose agar (PDA) medium amended with 0.5 g L–1 streptomycin sulfate. Putative colonies of “Cylindrocarpon” pauciseptatum Schroers & Crous were obtained from single macroconidia isolations. Two isolates were used to confirm the identity of isolated colonies: Cy12UFSM and Cy13UFSM. After incubation in the dark for 10 days at 20°C, the isolated mycelial colonies, which were cottony white to felty in texture, became dark orange to brown. Both isolates produced chlamydospores in chains at 40 days. Chlamydospores of Cy12UFSM and Cy13UFSM were 9 to 12 μm and 5 to 11.5 μm in diameter. Sporodochia formation on carnation leaf agar (CLA) medium was observed after 30 days. To encourage development of conidia, the isolates were grown on spezieller nährstoffarmer agar (SNA) medium for five weeks at 20°C with addition of two pieces of 1 cm2 filter paper. Microconidia of Cy12UFSM were 4 to 8.5 × 3.5 to 5 μm and those of Cy13UFSM were 3.5 to 7.5 × 3 to 5 μm. Macroconida were predominantly 3-septate (Cy12UFSM was 36 to 45 × 7.5 to 9 μm and Cy13UFSM was 30 to 38 × 7.5 to 8 μm), but 1-, 2- septate macroconidia were observed. The sizes of the three spore types and colony morphology for our isolates were similar to those described by Schroers et al. (3) for “C.” pauciseptatum. To further confirm the identity of Cy12UFSM and Cy13UFSM, multi-gene DNA sequence analysis (rDNA-ITS, β-tubulin, and histone H3) was conducted using primer pairs ITS1 and ITS4 (4), Bt2a and Bt2b, and H3-1a and H3-1b (2), which amplify the ITS1-5.8S rRNA-ITS2 genes, part of the β-tubulin gene, and the histone H3 gene, respectively. Sequences of these three regions had 99, 99, and 97% similarity with references sequences of “C.” pauciseptatum (isolate Cy238; accessions ITS [JF735307]; β-tubulin [JF735435], and histone H3 [JF735582], respectively). To evaluate pathogenicity, 4-month-old rooted cuttings of V. labrusca cv. Bordô were inoculated with two isolates by immersing them in a conidial suspension (106 conidia ml–1) for 60 min. Ten single-vine replicates were used for each isolate, and 10 water-inoculated vines were included as controls. Thirty days after inoculation, vines were re-inoculated with 40 ml of a 106 conidia ml–1 suspension to ensure root infection. After 4 months, the inoculated plants had reduced root mass relative to controls (39.18% for Cy12UFSM and 18.27% for Cy13UFSM). Inoculated plants also had root and crown necrosis, vascular lesions, shoot decline, and vine mortality (60 and 80% mortality for Cy12UFSM and Cy13UFSM, respectively). All water-inoculated control plants remained symptomless. The fungi Cy12UFSM and Cy13UFSM were re-isolated from infected woody tissues, confirming Koch's postulates. To our knowledge, this is the first report of “C.” pauciseptatum associated with black foot disease of grapevine in Brazil, which may potentially impact the sustainability of grapevine nurseries and vineyard productivity. References: (1) L. R. Garrido et al. Fitopatol. Brasil. 29:548, 2004. (2) N. L. Glass et al. Appl. Environ. Microbiol. 61:1323, 1995. (3) H. J. Schoers et al. Mycol. Res. 112:82, 2008. (4) T. J. White et al. Amplification Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

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The long-term use of vineyards and their renewal causes numerous chemical changes in the bioavailable copper (Cu), zinc (Zn) levels, organic carbon and microbial properties of the soil. However, there are few works with an approach to the use of vineyards in subtropical soils. Thus, the objective of this work was to evaluate the relationship between physical-chemical properties, microbial basal respiration, activity, richness, and diversity from vineyards and nearby forest reference soils in three different areas in southern Brazil. Each area underwent three treatments: old cultivation, i.e., a well-established vineyard; renewed vineyard; and reference, a native forest located adjacent to the vineyards. The physical-chemical properties clay content, moisture, pH, organic matter (OM), carbon (C), nitrogen (N), phosphorus (P), potassium (K), copper (Cu), and zinc (Zn) level; the basal respiration (BR), microbial biomass Carbon (MBC), microbial biomass Nitrogen (MBN), hydrolysis of fluorescein diacetate (FDA), microbial quotient (qMic), and metabolic quotient (qCO2); and microbial richness and Shannon diversity index were determined. The organic C level differently influenced the microbial activity and richness in each of the three experimental areas. The change in microbial diversity indexes impacted soil biological activity in two locations. Vineyard renewal promotes the reduction of Cu (- 48.8%) and zinc (- 20%) bioavailability and increases microbial diversity in subtropical soils in southern Brazil.

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