Growth and Surface Sterilization of Plant-Parasitic Nematodes

Adults and juveniles of P. penetrans were multiplied for 2–4 months on carrot disks and extracted by Baermann funnel (EPPO, 2013). The root knot nematode M. incognita was multiplied on tomato cv. Moneymaker for 2 months in the greenhouse at 16 h photoperiod and 26°C. Second-stage juveniles (J2) were collected by picking egg masses from tomato roots and transferring them into sterile tap water at room temperature to facilitate hatch of J2. For surface disinfection, nematodes were placed first on 5 μm sieves (Cell-Trics1 filters, Sysmex, Norderstedt, Germany) and washed with 10 ml sterilized tap water. Nematodes were then treated with 0.02% HgCl₂ for 3 min and with 4000 ppm streptomycin sulfate for another 3 min. Next, nematodes were incubated for 4 h in 5 ml 1x CellCultureGuard (AppliChem, Darmstadt, Germany) on a rotary shaker at 150 rpm. Finally, the nematodes were washed on a 5 μm sieve and incubated overnight in sterilized tap water. Prior to use in the experiments, nematodes were checked for their sterility by plating them on R2A (Merck, Darmstadt, Germany) for bacterial growth and on potato extract glucose agar (Carl Roth, Karlsruhe, Germany) for fungal growth. Inoculation of plants with nematodes was done by digging eight ca. 5 cm deep half an inch wide holes in 5 cm distance around the shoot, and equally distributing the nematode suspension.