Appendix 1 Methodology for studies on Pochonia chlamydosporia isolates (Rothamsted Research)

Cultures of P. chlamydosporia from infected nematode eggs in soil from different parts of the world have been freeze-dried and retained at Rothamsted Research, prior to use. Eight isolates (five collected from cyst nematode eggs and three from root-knot nematode eggs) were grown on 1.7% corn meal agar (Oxoid, Basingstoke, UK) at 25C prior to use. Chlamydospores used as inoculum were produced in 500 ml conical flasks containing (200 g) sterile rice. After 3 weeks incubation at 25C, the cultures were washed through 250 and 53 μm sieves to remove the grains, and the fungal propagules, mainly chlamydospores, were collected on a 10 μm aperture sieve and mixed with coarse sand as a carrier. The concentration of chlamydospores was estimated in diluted samples using a haemocytometer. To check the viability of the chlamydospores, 1 g of the inoculum was diluted in 9 ml of a 0.05% agar solution and 200 μl of dilutions at 10 and at 10 plated on 9-cm-diam Petri dishes containing sorbose (2 g l), agar (12 g l) and antibiotics (50 mg l each of streptomycin sulphate, chloramphenicol and chlorotetracycline). The proportion of germinated chlamydospores was estimated after incubation at 25C for 2 days; germination ranged between 85-95%.