Infection Assay of Cyst Nematodes on Arabidopsis Roots

Abstract

Plant parasitic nematodes are devastating pests on many crops. Juveniles (J2) of cyst nematodes invade the roots to induce a syncytium. This feeding site is their only source of nutrients. Male nematodes leave the roots after the fourth molt to mate with females. The females stay attached to their syncytia throughout their life and produce hundreds of eggs, which are contained in their bodies. When the females die their bodies form the cysts, which protect the eggs. Cysts can survive for many years in the soil until favorable conditions induce hatching of the juveniles. The beet cyst nematode Heterodera schachtii (H. schachtii)is a pathogen of sugar beet (Beta vulgaris) but can also complete its life cycle on Arabidopsis roots growing on agar plates under sterile conditions. We present here protocols for a stock culture of H. schachtii and an infection assay on agar plates.

Figure 1. Funnel for hatching.

Glass beakers, funnels, silicone tubes, and clips can be bought from local suppliers.

Figure 2. 15 μm and 100 μm sieves are needed for sterilization of J2s.

Only the 100 μm sieves are shown on this picture.

Figure 3. Template for Arabidopsis seeds

Figure 4. Male (A), female (B) nematode and the cyst (C)

Figure 5. Size measurement of female nematodes and syncytia. Numbers are the sizes calculated by the contour tool of the AxioVision software for nematodes (blue) and syncytia (red).

Shown are syncytia induced in transgenic lines overexpressing a WRKY gene and the wild type (Col) [from Ali et al. (2014)]

Figure 6. Growth classes of Arabidopsis plants.

This figure can be used to classify the Arabidopsis lines if there are differences in plant growth to adjust the number of nematodes [from Jürgensen (2001)].