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.
- 9 fully resistant LS = 0.
- 8 highly resistant
- 7 resistant
- 6 partially resistant
- 5 intermediate
- 4 partially susceptible
- 3 susceptible
- 2 very susceptible, LS = size of lesions on cultivar Bintje.
- 1 extremely susceptible, more susceptible than Bintje
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
- Caten, C. E., and Jinks, J. L. 1968. Spontaneous variability of single isolates of Phytophthora infestans I. Cultural variation. Canadian Journal of Botany 46:329-347.
- Murashige, T., and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15:473-497.
- Vleeshouwers, V. G. A. A., van Dooijeweert, W., Keizer, L. C. P., Sijpkes, L., Govers, F., and Colon, L. T. 1999. A laboratory assay for Phytophthora infestans resistance in various Solanum species reflects the field situation. European Journal of Plant Pathology 105:241-250.