To assess the effects of inoculum location and doses on seed germination and germling growth, 50 surface-disinfested seeds of P. radiata were inoculated by immersion as described in Section 2.2, at doses of 102 or 104 spores/mL: (1) protrusion-stage pregerminated seeds on water agar that mimic embryo, gametophyte and external infestation; (2) carborundum rubbed seeds to crack their coat and favor fungal penetration into seeds (imitating gametophyte and external infestation); and (3) no previous seed treatment to imitate external infestation of seed coats. Inoculation with water was done for each respective inoculation method. Each group of 50 seeds were germinated on a tray, covered with plastic bags to maintain high humidity and incubated at 22 °C with 12 h of light. Periodically until 48 days post inoculation (dpi), germlings were evaluated in a scale (0–2) for growth stage, where 0 = no germination, 1 = radicle present, and 2 = radicle and hypocotyl present; and for presence/absence of F. circinatum mycelia in stages 1 and 2.
Logit analysis was performed to test effects of inoculation method and inoculum dose on germination percentage at 20 dpi. Likewise, proportion of damaged germlings in growth stages 1 and 2 at the end of the experiment was analyzed by logistic regression on inoculation method and inoculum dose and their interaction.