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.
Keywords: Causal Agent; Crop Type; Etiology; Field crops; Fungi; Subject Areas; cereals and grains.