Culturing and long-term storage of virulent races of the rose blackspot pathogen, diplocarpon rosae wolf

Blackspot disease of roses is the most destructive of rose diseases worldwide. Breeding for resistance to blackspot is made difficult by multiple races of the causal fungus, Diplocarpon rosae. In orDer to efficiently and accurately screen roses for resistance, a protocol must be developed or isolating, culturing, and maintaining races in a virulent state in long-term storage. Diplocarpon rosae was isolated from leaf pieces and cultured on potato dextrose agar (PDA). Colonies growing from single conidia were selected and increased. Plants of the universally susceptible cultivar,Rosa hybrida 'Chorale', were then inoculated with spores of three different isolates. Infected leaves were harvested twelve days post inoculation and immediately stored at -20°C,-80°C, and without any cryoprotectant in the gas phase of liquid nitrogen. Colonies isolated from the infected leaves and cultured on PDA were also stored in sterile distilled water at 4°C. At ten week intervals, the fungus was isolated from hyphae growing within the stored leaves, sub-cultured on PDA, and re-inoculated to 'Chorale' plants. Differences in response to storage treatments were observed between isolates. Vigorous growth in culture and consistent virulence was observed for the cryogenic storage treatment, even though vials were partially thawed and re-frozen at each inoculation. Successful application of the cryogenic storage protocol to a race test inoculation using fourteen isolates collected across eastern North America confirmed the effectiveness of this method. These isolates are maintained in a virulent state in cryogenic storage and are available to the scientific community.