SolRgene

In vitro inoculation assay

The in vitro resistance test is a quick, space-effective, and accurate assay that can be used for high throughput disease assessment to P. infestans in Solanum species. This assay is particularly suitable for screening collections that are being maintained in vitro, and has provided comparable data as obtained with routine detached leaf assays.

Experimental setup

Presented data in this database were obtained from a high throughput screening of wild Solanum genotypes. A series of inoculation experiments were performed. To be able to reliably related the independent experiment, 4 control genotypes were included, i.e. resistant S. demissum 344-14 (CGN20571-14) and S. huancabambense 354-1 (CGN17719-1), and susceptible S. tuberosum cv. Bintje and S. tuberosum RH89-039-16, in each inoculation experiment.

Preparation of plant material

For each Solanum genotype, 5 fresh shoots or nodes were cut and transplanted to a plastic jar (d=15 cm) with on Murashige and Skoog (MS) medium supplemented with 30 g per liter sucrose (MS30). A 2 - 3 cm space was left between the cuttings and the inner wall of the jars. In vitro plantlets were grown at 24°C and 16 h light/8 h dark, and plantlets with 3-4 fully developed leaves are used.

Inoculation

Inoculum preparation and inoculation were performed under sterile conditions. From each in vitro plantlet, three largest leaves were inoculated by pipetting 10 µl droplets of a zoospore suspension of 5 x 10⁴ P. infestans spores/ml on the adaxial side, one spot per leaf (Figure 1). The jars were incubated in a climate chamber at a 16h/8h day/night photoperiod at 15°C.

Figure 1: The inoculation of vitro plants from S. andreanum. Inoculum droplets are placed on the leaves.

Disease assessment

Plantlets were macroscopically examined for disease symptoms at 7 and 14 days post inoculation, and genotypes were rated for resistance on a 5-interval scale, as below:

9 highly resistant No symptoms or localized HR, comparable to control genotype S. demissum 344-14 and S. huancabambense 354-1.
7 resistant HR resistance responses, but minor symptoms can be noted on some parts. The infection is stopped at 14 dpi, and plantlets stay green and continued to grow.
5 intermediate Plantlets show some resistance, but also sporulating leaves or stems. Variation between plantlets may occur.
3 susceptible Sporulation on leaves and stems, plantlets collapsed at 14 dpi.
1 highly susceptible Heavy sporulation at 7 dpi, collapsed plantlets, and completely blighted at 14 dpi. More susceptible than control genotypes Bintje and RH89-039-16.