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Pyrenophora teres f. teres is well known for causing net form net blotch on barley, but its pathogenicity on wheat has also been reported in Hungary (Tóth et al. 2008) and east Russia (Mikhailova et al. 2010). The report from Hungary mentioned symptoms of dark brown necrotic lesions with or without chlorotic margins, while the report from Russia described symptoms similar to tan spot. A survey of fungal pathogens causing leaf spot diseases on wheat in the humid subtropical areas of Paraná State, southern Brazil, was conducted in 2016. Twenty four isolates, representing different wheat fields in the municipalities of Guarapuava and Pinhão, in the state of Paraná, were collected from wheat plants at the soft dough stage. The isolates analyzed in our study were obtained from leaf fragments of wheat cv. Toruk and were removed from the edge of lesions resembling tan spot, a major fungal disease of wheat in that region, caused by Pyrenophora tritici-repentis . The leaves presented oval, diamond, or elongate-shape lesions with tan necrotic spots. Still, they did not always show the brown center, which seemed not very characteristics of tan spot caused by P. tritici-repentis . Yellow halos surrounded the necrotic lesions. The fragments were superficially disinfected for 30 sec in 0.5% sodium hypochlorite, washed in sterile water, dried with a sterile paper towel, and transferred to petri plates containing potato dextrose agar (PDA) medium. After four days of incubation at 24±2ºC and a photoperiod of 12 h, hyphal tips were removed from the PDA medium and transferred to petri plates containing V-8 medium. Plates were incubated for 14 days in alternate cycles of 12 h at 23±2ºC under light and 12 h at 19±2ºC in darkness for sporulation. Pure cultures were obtained from single conidia produced on V-8 medium. Single spore isolates, representing different fields, were obtained from individual leaves. Conidia measuring 38-82 µm × 16-20 µm were smoothly cylindrical and straight, with rounded ends, subhyaline or yellowish brown, with four to seven pseudosepta. Genomic DNA extracted from cultures grown in yeast extract broth medium was used for PCR amplification of the ribosomal internal transcribed spacers and the 5.8S ribosomal DNA gene (ITS-5.8S rDNA) using the primers ITS4 and ITS5 described by White et al. (1990). A BLAST search of the GenBank database revealed that the ITS-5.8S rDNA sequence fragments (505 bp) from 12 out of 67 isolates were 99-100% similar to P. teres accession number EF452452 (Andrie et al. 2008). The DNA sequences of three P. teres isolates (20C3, 20A3, and 20A1) were deposited in GenBank (accession nos. MH594043.1, MH594042.1, and MH594041.1, respectively). These isolates were obtained from different fields in the municipality of Pinhão (25° 42 '26 "S; 51° 38' 0" W), in Paraná State. To test the pathogenicity of the isolates 20C3, 20A3, and 20A1, a conidial suspension for each isolate with 2-3 × 103 conidia per mL was sprayed at the boot stage (BBCH-scale 45) on leaves of wheat cv. Toruk and on susceptible barley cv. Ana 01 plants growing in pots. Each pot containing 5-7 plants was considered as replicate. Wheat plants were also inoculated with a P. tritici-repentis isolate with a conidial suspension at the same concentration described above to serve as reference of symptoms for tan spot. Wheat and barley plants were also inoculated with sterile water to serve as a control. Six replications were inoculated for each treatment. After inoculation, the plants were maintained in a dew chamber for 24 h and then moved to a growth room at 20±2ºC and 16 h photoperiod. Initially, the leaves of wheat plants inoculated with P. teres exhibited small, irregular, dark lesions surrounded by yellow halos, and later exhibited oval to elongate, pale brown, necrotic lesions, and yellow halos. Differently from tan spot caused by P. tritici-repentis , the wheat leaves inoculated with P. teres f. teres presented a more diffuse yellowing, and the center of necrotic lesions was pale brown. The symptoms observed in the barley leaves were the net form net blotch, which allowed us to infer that the disease was caused by P. teres f. teres isolates. All inoculated plants presented symptoms, with some variation among them. Pyrenophora teres f. teres was reisolated from the inoculated wheat and barley plants. This is the first report of P. teres f. teres causing leaf disease on wheat in Brazil. The finding contributes to a better understanding of the etiology of wheat leaf spots in humid subtropical areas of southern Brazil.

Greater plantain or common plantain (Plantago major L.) is an herbaceous plant native to most of Europe, northern and central Asia, and it has adapted well to tropical regions where it is used as a medicinal plant. Between November 2011 and April 2012, greater plantains cultivated in the Medicinal Plant Garden at the Umuarama Advanced Campus of the State University of Maringa (UEM-CAU) suddenly died off. A visual examination revealed the presence of white mycelium and sclerotia on the lower third of the plant. These sclerotia were collected and deinfested by immersion in 70% alcohol and 0.5% sodium hypochlorite for 45 s, and in sterilized water for 1 min. Next, the sclerotia were placed on 10 petri dishes containing potato dextrose agar (PDA) culture medium and incubated at 29°C. After 7 days, the culture medium was entirely coated with a cottony white mycelial growth and, 15 days after isolation, sclerotia began to form. Healthy seedlings were transplanted individually into pots containing autoclaved soil (120°C/2 h). After 10 days, eight seedlings were inoculated with 8-mm mycelia disks deposited on the base of the plant, and eight seedlings inoculated with fungus-free PDA disks (control). The plants were irrigated and the pots placed in with plastic bags and kept at an average temperature of 28°C. Three days after inoculation, we observed a cottony white mycelial growth and symptoms of rot on the plants. The plastic bags were then removed and the plants kept under the same temperature, relative humidity of 80% and 12 h of light. Seven days after inoculation, the plants treated with the fungus died, whereas the plants treated with PDA developed normally. The fungus was re-isolated from the symptomatic plants and slides evaluated under a light microscope, revealing that the mycelium was thick, septate, and hyaline. The sclerotia formed were spherical, initially white or light brown, becoming dark brown or black, with diameters ranging from 0.5 to 1.5 mm. The fungus was subjected to DNA analysis using ribosomal region oligonucleotides ITS4 (5'-TCCTCCGCTTATTGATAT-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') (1) to amplify the target region. The segment including the 5.8S gene and rDNa regions ITS1 and ITS2 was 630 bp long. DNA analysis revealed that it was 99% identical to Athelia rolfsii (Curzi) Tu and Kimbr (anamorph: S. rolfsii) (GenBank Accession No. HM222638.1). The isolate was deposited in the fungus collection at the UEM-CAU Phytopathology Laboratory under code F-Sr-01-UMU. Reference: (1) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications, M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

Syagrus oleracea (Mart.) Becc. (bitter coconut), a palm tree species that is native to central Brazil, has been increasingly cultivated in this country for heart-of-palm production. Epidemics of a necrotic leaf spot of unknown etiology have been recorded on bitter coconut plants in transplant nurseries and plantation since 2008. The first symptoms appear as small, yellow, hydrotic flecks on young or mature leaves that evolve to necrotic brown streaks that run parallel to the leaf veins. Usually, yellow halos occur around the lesions and hydrosis is common during lesion expansion. Necrotic lesions can reach up to 40 mm in length and 10 mm in width, and the lesions often coalesce, causing extensive tissue damage. During a survey in a 3-year-old bitter coconut plantation in Maringá County (coordinates: 23°23'51.25″ S, 51°57'02.09″ W; elevation: 507 m) in the state of Parana, a dozen symptomatic leaves were collected with the aim of elucidating the etiology of this disease. Conidia and conidiophores typical of Cladosporium were frequently observed on the diseased leaf tissue under natural field conditions as well on the surfaces of disinfected leaf tissues kept in a humid chamber for 48 h at 25 ± 2°C with a 12-h photoperiod. Five monoconidial cultures growing on potato dextrose agar (PDA) medium were obtained from different leaves showing leaf spot symptoms. The cultures were grown on PDA to induce sporulation. At 7 days after incubation at 25 ± 2°C and a 12-h photoperiod, gray to gray-olive colonies were observed. The conidiophores were macronematous, erect, oblong, branched, 1 to 5 septate, and 75.0 to 120.0 × 1.90 to 3.20 μm. The ramoconidia were cylindrical or oblong, 0 to 2 septate, and 28.0 to 40.0 × 2.8 to 3.6 μm, with a truncate base of 1.9 to 2.2 μm; secondary ramoconidia were cylindrical or oblong, 0 to 2 septate, 8.0 to 31.0 × 2.2 to 3.1 μm, with 3 to 5 distal conidial hila; intercalary 1-septate conidia were 5.5 to 17.0 × 2.1 to 3.4 μm, with 1 to 3 distal conidial hila; terminal 1-septate conidia were catenulate and 2.2 to 4.2 × 1.8 to 3.1 μm. Species identification was performed based on morphology and DNA sequence data (1). Portions of the elongation factor 1α (551 bp; TEF) and actin (213 bp; ACT) genes were amplified by PCR. A BLAST search of the GenBank database revealed that the TEF (KC484658 to KC484662) and ACT (KC484663 to KC484667) sequence fragments from isolates Gua1, Gua2, Gua3, Gua4, and Gua5 had 100% identity with the accessions HM148616 and HM148371 of Cladosporium perangustum (1). Isolates were tested for pathogenicity against bitter coconut. Ten potted plants with 4 to 6 fully expanded leaves were inoculated with each isolate by spraying a suspension of conidia (10 5 spores per ml) onto leaves until runoff using a handheld spray bottle. Non-inoculated controls (10 plants) were sprayed with distilled water. The plants were kept in a humid plastic chamber at 20 to 26°C. All examined isolates were pathogenic to bitter coconut, causing symptoms identical to those described above 12 days after inoculation. All inoculated tissues were plated onto PDA to confirm the presence of the pathogen. Live cultures are being maintained at the Micoteca/URM/UFPE ( www.ufpe.br/micoteca ), Brazil. To our knowledge, this is the first report of a disease caused by C. perangustum on S. oleracea worldwide, and the study provides valuable plant disease diagnostic information for the palm hearth industry in Latin America. Reference: (1) K. Bensch et al. Stud Mycol. 67:1, 2010.