Powdery mildew is an important plant disease. Accurate determination of the disease phenotype requires even inoculation of the pathogen. Since existing methods are inefficient, we have developed this protocol.
Our spore inoculation method can easily achieve even inoculation, and it is simple and scalable. Hence, any lab working with powdery mildew can easily use this method. The researchers can also mount mesh over a cardboard box and use it for spore inoculation.
How to dislodge spores and brush them through mesh is a critical step. So demonstrating this on video could be very helpful. For this protocol, get a roll of 50 micrometer nylon membrane mesh or 48 micrometer stainless steel mesh.
Cut large sized, medium-density plywood into pieces of 24.5X10 inches and 12X10 inches for making an inoculation box. To make a removable top lid with a mesh, cut the long wooden square dowel into two sets of 26 inches and 12.75 inches. Glue and nail the four pieces to fit around the box.
Take acrylic sheets and assemble all the acrylic sheets into a box, using eight corner clamps. Cut a 7X2 inch opening window on the front side of the acrylic sheet of the box. To prepare fresh powdery mildew spores as inocula, grow clean Arabidopsis immune-compromised pad4-1 mutant plants in a growth chamber at 22 degrees Celsius and 65%relative humidity for six to eight weeks.
Grow the plants to determine infection phenotypes in standard flats. Inoculate one or half of pad4-1 plants to build sufficient fresh powdery mildew spores. When fresh spores from infected plants are available, move a flat of plants into the inoculation box inside the inoculation chamber for an infection test.
Cut 15 to 20 heavily infected Arabidopsis leaves, and let them dry if there is condensed water. Hold one to two leaves upside down in one hand with a pair of long forceps. Then put both hands through the opening of the chamber and gently hit the arm of the forceps with a pair of scissors, while slowly moving the leaves above the mesh mounted on the inoculation box.
Brush the spores with fine fan blender brushes through the entire mesh surface in different directions to maximize the even distribution of spores onto the plants. Gently pat the mesh a few times with the brushes to shake off the spores stuck in the mesh. Once the spores settle, move out the flat containing the inoculated plants from the inoculation box.
Cover the flat with a plastic dome and place it in a growth chamber at 22 degrees Celsius and 65%relative humidity. Disease phenotyping can be performed at 5 or 8 to 12 days post inoculation. Convert cardboard boxes to an inoculation box by removing the top and bottom sides of the box, taping the corner to firm it, and mounting a 50 micrometer mesh on the top with glue.
Move the flat containing the plants inside the inoculation chamber, and place the smaller inoculation box on top of the flat to cover the plants. Then dislodge and brush the spores. Make a mini inoculation box slightly bigger than a Petri dish.
Store the plates at four degrees Celsius in a fridge until use. Cut one or two fully expanded leaves from the base of the petiole for each plant to be tested. Insert the petiole of the detached leaves into the agar medium at an angle of 30 degrees.
Inoculate the leaves inside the inoculation chamber, and move the plate with the lid to a growth chamber. For assessing infection phenotypes, transfer the leaves to a fresh aseptic MS agar plate at four or five days post inoculation. Cut the base of the petiole before inserting the leaves into the fresh agar medium.
Assess the disease phenotypes at eight or nine days post inoculation. The microscopic analysis displayed even distribution of spores on the microscopic slides on the bottom of the flat. The calculated density of spores, distributed in six different locations after light inoculations, showed no significant differences.
Similar results were achieved following heavy inoculations with spores. The inoculation evenness was examined among different plant genotypes at 12 days after heavy inoculation. The pad4-1 plants showed enhanced disease susceptibility compared to Columbia 0, the wild type plants.
Using adequate fresh spores, produced around 10 days after inoculation, on susceptible plants, are important, because fresh spores are easier to dislodge and more likely to establish colonization. Powdery mildew only colonizes plant epidermal cells. Isolation of powdery mildew infected cells by peeling the epidermis, or laser micro dissection, would enable a more targeted transcriptomic and proteomic study of host responses.
This method will help researchers more efficiently determine powder mildew infection phenotypes, and therefore should facilitate more effective functional studies on plant powdery mildew interactions.
