First Report of Ilyonectria macrodidyma Associated with Black Foot Disease of Grapevine in Brazil.

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, ß-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 ß-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.

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.

First Report of Fusarium equiseti Associated on Pecan (Carya illinoinensis) Seeds in Brazil.

Pecan [Carya illinoinensis (Wangenh.) K. Koch] is an important producing nut tree that has been intensively cultivated in the state of Rio Grande do Sul (Brazil) in recent decades. This species is commonly grown in association with other crops and more often with cattle or sheep. An elevated incidence of the fungal genus Fusarium was observed during a quality control seed assay of pecan seeds obtained from orchards in the city of Anta Gorda (28°53'54.7″ S, 52°01'59.9″ W). Concomitantly, seedlings of this species, cultivated in a nursery, showed foliar necrosis, wilt, and root rot. The fungus was thereafter isolated from the seeds (from original seeds lots) and subcultured from single spores. Cultures were purified in order to perform pathogenicity tests. The isolated Fusarium sp. was increased on autoclaved wet corn kernels that were incubated for 14 days (1), and then were mixed with commercial substrate (sphagnum turf, expanded vermiculite, dolomitic limestone, gypsum, and NPK fertilizer) in plastic trays (capacity 7 L), with drainage holes. Twenty seeds were sowed and 90 days later, evaluations were undertaken. Forty percent of the seedlings presented symptoms, i.e., foliar necrosis and wilt owing to root rot. Fusarium sp. was re-isolated from the affected roots by transferring hyphal tips to potato dextrose agar (PDA) and carnation leaf agar (CLA) medium in petri dishes in order to identify the species morphologically. On PDA, the colony pigmentation was yellowish brown and the aerial mycelium was whitish to peach; macroconidia were relatively long and narrow (31.75 × 4.02 µm), with 5 septa on average, and whip-like bent apical cells (2). Chlamydospores were not observed on PDA or CLA. Primer pairs ITS1 and ITS4 (3) and EF1-T and EF1-1567R (4) were employed to amplify the internal transcribed spacer (ITS) and elongation factor-1α (TEF 1-α) regions, respectively. The resulting DNA sequences showed 99% for ITS and 98% for TEF 1-α similarity with Fusarium equiseti (Corda) Sacc. and phylogenetic analysis grouped it with sequences of this species. The consensus sequence was submitted to GenBank and received the accession numbers KC810063 (ITS) and KF601580 (TEF 1-α). The pathogen was re-isolated on PDA and CLA substrate in order to complete Koch's postulates. The pathogenicity test was repeated with the same conditions described before and the results were confirmed. No symptoms were observed on the control seedlings. This species is considered a weak parasite (2); however, it has been reported causing wilt in Coffea arabica in Brazil (5). This pathogen could cause serious damage and high losses to seedling in commercial nurseries. Besides that, it could also carry the disease to the field causing further damage on established plants. To our knowledge, this is the first to report of F. equiseti causing foliar necrosis and wilt on C. illinoinensis in Brazil. References: (1) L. H. Klingelfuss et al. Fitopatol. Brasil. 32:1, 2007. (2) W. Gerlach and H. Nirenberg. The Genus Fusarium - a Pictorial Atlas. Biologische Bundesanstalt für Land- und Forstwirtschaft, Braunschweig, Germany, 1982. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA, 1990. (4) S. A. Rehner and E. A. Buckley. Mycologia 97:84, 2005. (5) L. H. Pfenning and M. F. Martins. Page 283 in: Simpósio de Pesquisa dos Cafés do Brasil, 2000.

First Report of "Cylindrocarpon" pauciseptatum Associated with Black Foot Disease of Grapevine in Brazil.

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, ß-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 ß-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]; ß-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.