SolRgene

Deatached leaf assay

The detached leaf assay is a generally well-accepted, and a routinely used technique to assess resistance to P. infestans isolates under laboratory conditions. Details on the method have been described in Vleeshouwers et al. (1999).

Experimental set up

A series of detached leaf experiments were performed. In each experiment, the susceptible potato cultivar Bintje was included as a susceptible control.

Plant material

All plant genotypes were propagated in vitro in sterile jars containing MS  medium (Murashige and Skoog 1962) supplemented with 3% (w/v) sucrose at 20 °C. After 1-2 weeks of rooting, the in vitro plantlets were transferred to pots with sterilized soil in the greenhouse for 8 – 10 weeks.

Inoculation

Phytophthora infestans isolates were grown on rye agar medium supplemented with 2% (w/v) sucrose (Caten and Jinks 1968) and incubated at 15°C in the dark for 1-2 weeks. For inoculation, a plate covered with mycelium was flooded with water and the sporangiospore suspension was pipetted into a test tube. After 1-2 hours incubation at 4°C, zoospores were released. Leaves from mature greenhouse-grown plants were detached and placed in water-saturated florists foam (Oasis®) in a tray. The leaves were spot-inoculated by pipetting 10 µl droplets of the spore suspension (5 x 104 spores/ml) on the abaxial side.Incubation intact plants climate chamber The trays were covered with transparent lides, and incubated in a climate chamber at a 16h/8h day/night photoperiod at 15°C.

Disease assessment

Lesion sizes (LS) were measured at 6 days after inoculation using an electronic caliper connected to a desktop computer. LS were transformed to a linear scale from 1 – 9, relative to potato cultivar Bintje, to relate the independent experiments to each other. The rates 1-9 correspond to the following criteria.

Symptoms on detached leaves. Left: Resistant. hypersensitive (HR) spots is delimiting the lesion on the leaves. Right: Susceptible. expanding lesions and sporulation.
A population is segregating “black & white” for resistance to P. infestans. Clear distinction between resistant and susceptible progeny plants can be made.

References