Seedling Flood Assay for Screening Bacterial Resistance in Tomatoes

To screen for bacterial resistance in different tomato strains, begin with 10-day-old tomato seedlings grown under sterile conditions.

These seedlings display emerging true leaves and established roots.

Flood both strains with pathogenic bacteria mixed with a surfactant to facilitate uniform spread of the inoculum on the plant surface. Tilt and swish the plate to maximize seedling contact with the inoculum.

Remove the excess inoculum, reseal the plates, and incubate them in a growth chamber.

The bacteria enter through stomata and colonize the apoplast, the extracellular space between leaf cells.

The bacteria inject effector proteins into plant cells to suppress plant immune responses.

The susceptible strains fail to detect these effectors, allowing bacterial proliferation and bacterial toxin-induced plant tissue damage.

The resistant strains recognize the effectors and produce reactive oxygen species to kill the bacteria.

Post-incubation, resistant strains show sustained growth, while susceptible strains display arrested development.

Transfer the plates with the ten-day-old seedlings from the growth chamber to the biosafety cabinet and remove the surgical tape. Then transfer six milliliters of inoculum to each plate.

Gently push the seedlings into the inoculum with a pipette tip and start a timer for three minutes. Hold one plate in each hand and tilt the front of the plate down to submerge the cotyledons and leaves of the seedlings. Swish the inoculum side to side five to seven times, then tip the plates back to cover the roots with the inoculum.

Tilt the plates down again and repeat the cycle for a total of three minutes. Pour the inoculum off the plates, set them down on a flat surface, and pour off any residual inoculum for a second time. Re-wrap the plates and place them back in the growth chamber.