Pinus radiata surface-disinfested seeds were externally inoculated at doses of 104 or 106 spores/mL, stored at room temperature for one day until sowing in forest sowing 35-trays containing substrate composed of 80% peat plus 20% coconut fiber, and incubated at constant temperature 20 °C, and 12 h day/night light. Each treatment was applied to three trays, and trays were arranged in a randomized block design. The same conditions were repeated in a second experiment at the dose of 104 spores/mL.
There was a periodical monitoring and recording of seedling emergence and appearance of symptoms produced by F. circinatum seedling infection, such as girdling of root collar, wilting and die-back. Dead plants were sampled and some sections were plated on SFA medium to corroborate the presence of the pathogen. Identification of F. circinatum was confirmed morphologically on SNA [26] and PDA media based on morphological features in pure culture [27]. The last day of recording pre-emergence damping-off was at the date of the last emergence event. Post-emergence damping-off was recorded until 281 dpi, considering the first two months after emergence as damping-off and later on, damage of established seedlings. Analysis of variance was used to estimate the effect of inoculum dose on the proportion of pre- and post- emergence damping-off as well as proportion of dead plants at the end of the experiment. Data were transformed prior to the analysis using arc sine of the square root of the proportion. Mean values were compared by Student-Newman-Keuls multiple range tests.
Disease progress for the low inoculum dose (104 spores/mL) was regressed using different models (linear, monomolecular, logistic and Gompertz) [28]. The best model was chosen according to the lowest mean square error value (MSE) and the highest coefficient of determination value (R2); then, disease infection rate was obtained from the slope parameter of the selected model [28]. Best fit of data was achieved using the linear model, that is, disease incidence regressed with time (dpi). Two lines were adjusted, one for post-emergence damping-off, and other for damage of established seedlings. The disease infection rate is the average of both replicates.