Maintenance and Sub-culturing of Melon Powdery Mildew Strains on Squash Leaves

  1. Squash seeds of variety Early summer Crookneck are germinated in the lab on moist paper towels. The seeds with germinated radicals (emerging after 2-days) are planted in gallon (c. 4 L) pots in the greenhouse.
  2. Squash plants are maintained with care preventing infection from extraneous powdery mildew and pests. Infected plants are routinely removed. For pest control, predatory mites (Hypoaspis miles, Amblyseius cucumeris) and parasitic wasp (Encarsia formosa) are used.
  3. Mature leaves are collected and brought to the lab in a box lined with moist paper towels.
  4. Assay plates (Square Bioassay Dishes, 245mm x 245mm, Corning Inc. 431272) with water agar supplemented with PIK solution (pimaricin, 1.5g; IAA, 150mg; and kinetin,300mg, in 100ml of DMSO) at the rate of 10µl/l of media are used to maintain the squash leaves. The PIK is added to cooled media before pouring the plates. Autoclaved blotting paper (VWR Cat. No. 28298-022) is placed over the media on the assay plate to keep the leaves dry.
  5. Petioles of the leaves are cut and inserted through the blotting paper into the media. Four medium sized leaves can fit into one plate each.
  6. Inoculation is done in a fume hood or secluded space to avoid cross contamination. Spores from the strain to be sub-cultured are transferred to new leaves by dabbing the new leaves on the colonies from previously infected leaves. Care is taken to avoid transferring mycoparasites from 7. The inoculated leaves in assay plates are maintained under constant fluorescent light at room temperature around 25C. Mildew colonies generally start appearing 3-5 days after inoculation.
  7. Transpiration from the leaves can cause excess moisture and lead to abundant growth of mycoparasites. To avoid this, the lids of plates are wiped carefully with paper towels in a fume hood to avoid cross contamination.
  8. The strains are subcultured every 14-days, or earlier, as required. A constant supply of squash plants should be maintained in the greenhouse.