First Report of Sclerotinia sclerotiorum on Periwinkle ( Catharanthus roseus ) in Ontario, Canada

Periwinkle (Catharanthus roseus) is cultivated ornamentally and also as an herbal medicine in many parts of the world. Many vinca alkaloids were isolated from this plant and have been used against numerous human diseases (Noble 1990). Leaf and stem rot was observed on periwinkle in early September 2015 in Guelph, Ontario, Canada (43[degrees]11' N, 79[degrees]24' W). Approximately 20 to 80% of the plants (Pacifica Mix Xp) in five locations were infected. Occasionally, white mycelia and black sclerotia (3 to 5 mm long) were present on infected tissues. Several symptomatic leaves and stems were collected, washed in sterilized water, and cut into small pieces (3 x 2 mm). The tissue pieces were surface-sterilized in 70% ethanol for 50 s plus 1% sodium hypochlorite for 2 min, and then washed with sterilized water. Three types of fungi morphology were repeatedly isolated after surface-sterilized sections were placed on potato dextrose agar (PDA) plates and incubated at 25[degrees]C in dark for 2 days. To confirm Koch's postulates, 1-month-old periwinkle plants were grown in pots (20 cm diameter) containing sandy soil in greenhouse. Ten plants were used as controls. Leaves and stems were inoculated with a hyphal (5 mm diameter) plug separately for each isolate (10 plants per isolate). Control plants were inoculated with clean agar plugs. The inoculated plants were placed at 25[degrees]C (12/12 h light/dark with over 90% humidity). The experiment was repeated three times. After 2 days, only one of the isolates inoculated was found to induce disease similar to that observed in the field, showing water-soaked brown symptoms and after 5 days, leaf and stem necrosis, small, black, irregularly shaped sclerotia on the infected surface along with white mycelia were observed, while the control treatment remained symptomless. The fungus was consistently reisolated from inoculated plants but not from the control, and was culturally identical to the fungus originally observed on symptomatic plants. The internal transcribed spacer (ITS) region and a portion of the translation elongation factor 1-alpha (TEF-1 alpha ) regions were sequenced after PCR amplification using rDNA universal primers ITS1/ITS4 (White et al. 1990) and EF-728F/EF-986R (Carbone et al. 1999), respectively. ITS (KU170707) and TEF-1 alpha (KU170709) sequences both showed 100% identity to Sclerotinia sclerotiorum(KT935403.1 AF398888.1), respectively. S. sclerotiorum is virulent on a wide range of hosts, was re