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.
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