Las v 4.3 image analysis software

Cysts of H. schachtii were harvested from monoculture of mustard (Sinapsis alba ‘Albatros’) plants growing on modified Knop nutrient medium (2% w/v). The hatching of the nematodes was stimulated by adding 3 mM ZnCl2. Arabidopsis plants were grown in Petri dishes containing agar supplemented with Knop nutrient medium (2% w/v) under sterile conditions. Two plants were grown in one Petri dish. 60−70 J2s were inoculated to the surface of agar medium containing 12-days-old Arabidopsis plants. The numbers of nematodes per plant were counted using a stereomicroscope (Leica Microsystems) at 14 dpi. The female nematodes and female‐associated syncytia were outlined, and the area was calculated using an M165C stereomicroscope equipped with LAS v. 4.3 image analysis software (Leica Microsystems) at 14 dpi. Cyst sizes were outlined, and the area was calculated using an M165C stereomicroscope equipped with LAS v. 4.3 image analysis software (Leica Microsystems) at 42 dpi. Cysts were randomly selected and crushed and were then transferred into a counting dish and the number of eggs/cysts were counted using a stereomicroscope (Leica Microsystems). All infection assays were repeated a minimum of three times.

H. schachtii cysts were harvested from monoculture on mustard (Sinapis alba ‘Albatros’) roots growing on Knop medium (0.2% wt/vol). The hatching of the juveniles was stimulated by adding 3 mM ZnCl₂. On three consecutive washes with sterile water, 60–70 H. schachtii second‐stage juveniles (J2s) were inoculated onto the Knop medium plate containing 12‐day‐old Arabidopsis plants under sterile conditions. Two plants were used in one Petri dish and experiments were repeated at least three times independently, with 20–30 plants per genotype in each replicate. The numbers of female nematodes per plant were counted using a stereomicroscope (Leica Microsystems) at 14 dpi. Subsequently, the infection rate per centimetre of root length was determined after scanning roots with the WinRhizo root image analysis system. The female nematodes and female‐associated syncytia were outlined, and the area was calculated at 14 dpi using an M165C stereomicroscope equipped with LAS v. 4.3 image analysis software (Leica Microsystems). At 42 dpi, cysts were randomly selected and crushed in between slides. The contents were then transferred into a counting dish and the number of eggs/J2s were counted using an S4E stereomicroscope (Leica Microsystems). Cyst sizes were measured at 42 dpi.