Root-dip inoculation protocol
Starting material: Verticillium cultures maintained on solid Czapek-dox medium in petri plates.
Two to three weeks before inoculation: start plant cuttings, start Verticillium liquid cultures. Each plant will ultimately be inoculated with 20 ml of Verticillium spore suspension.
1. Prepare an appropriate volume of liquid Czapek-Dox medium for the number of plants to be inoculated. Autoclave to sterilize.
2. Working in a laminar flow hood, use sterilized forceps to cuf ~0.5cm squares of Verticillium from petri plates (solid medium covered with a combination of spores and hyphae).
3. Add 2 Verticillium squares to each 100 ml of liquid medium.
4. Incubate on shaker at 180 RPM, at room temperature, until day of inocuation.
On the day of inoculation, prepare the inoculum:
1. Strain liquid cultures through a single layer of Miracloth in order to isolate conidia.
2. Centrifuge filtrate at 10,000xg 5 minutes to pellet conidia. Pour off liquid medium. This step is done to remove any substances that were secreted by the fungus during the culture period.
3. Resuspend conidia in the same volume of sterile, distilled water.
4. Determine the concentration of conidia. We use a hemacytometer for this purpose.
5. Dilute the conidia to a concentration of 10^7 per ml. Inoculate the plants:
Other materials needed:
clean 50-ml beakers
sterile distilled water
sterile (autoclaved) soil.
1. Each 50ml beaker can be used to simultaneously dip 2 plants. Therefore, the number of beakers needed is one half the number of plants inoculated.
2. For dipping, pour ~20ml sterile water into a beaker for control plants, or ~20ml spore suspension for inoculated plants. Remove plants from soil and shake as much soil as possible off roots. Swirl roots around in the beaker, then leave plants in beaker 5 minutes. Make sure that leaves don't get dipped in inoculum.
3. Re-plant the treated plants in sterile soil.
4. Post-inoculation, plants are not watered for at least 24h to allow maximum exposure to conidia.