Pathogen inoculation procedure

Protocol for Botrytis cinerea infection

Prepare the B. cinerea spores:

The mycelium of B. cinerea was grown on PDA medium in the Petri Dish for about 10 days (5 to 10 plates each time).

Add the sterile water or 0.8% NaCl solution (10 ml for each plate) in the Petri Dish and wash the mycelium with a glass pipet to harvest the B. cinerea spores.

Collect all the mycelium solution in a falcon 50 ml or a sterile bottle 200ml and mix vigorously.

Filter the mycelium solution with three layer sterile gauze in a new clean falcon or bottle.

Centrifuge 10 min, 2000 rpm, 4 °C and remove the supernatant.

Resuspend the spores pellet with sterile water and dilute to the final concentration 2*10⁷spores/ml and stock at -80°C.

For the B. cinerea infection:

Grow Arabidopsis plants in a clean microbe-free climate chamber under the short day condition for about 4 weeks.

Prepare a solution of 2.5*10⁵ spores/ml in the Vogel buffer (1 L: 15 g of Sucrose, 3 g of Na-citrate, 5 g of K₂HPO₄, 0.2 g of MgSO₄·7H₂O, 0.1 g of CaCl₂·2H₂O, and 2 g of NH₄NO₃) from the solution stock.

Infect the intact plants with two droplets of 2 ml each of this solution (on one leaf surface).  For mock treatment, Vogel buffer alone was used.

Put all the plants in sealed hoods to keep high humidity and photo plants until 3 days.

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