3.2.1. Fungal Respiration Dynamics

Cumulative fungal respiration for the 10 day experimental period is shown in Figure 4a,b. In all inoculation positions and aw treatments, respiration increased over time as the *F. graminearum* mycelia colonised the wheat grain. The accumulated respiration at the end of the colonisation process (days 6 for bottom-side, and day 5 for top-centre and bottom centre inoculations) was significantly affected by the initial inoculation position (*p*-value = 0.022). Fungal colonisation from the bottom-side proceeded more slowly but produced more CO2 (4.96 ± 0.84 <sup>g</sup>·CO2·kg−1) than the top-centre (3.83 ± 0.79 <sup>g</sup>·CO2·kg−1) and bottom-centre (3.23 ± 0.52 <sup>g</sup>·CO2·kg−1). Bottom-side respiration was significantly higher (α = 0.05) than the bottom-centre respiration. However, these two treatments were indistinguishable from the top-centre cumulative respiration.

**Figure 4.** Temporal evolution of the indirect indicators of fungal growth from three different inoculum positions. Cumulative respiration of *F. graminearum* at (**a**) 0.95 aw and (**b**) 0.97 aw. The dry matter loss (DML; %) of wheat grain inoculated with *F. graminearum* at (**c**) 0.95 aw and (**d**) 0.97 aw. Ergosterol content of *F. graminearum* inoculated grain at (**e**) 0.95 aw, and (**f**) 0.97 aw. Bars show standard deviation of the three replicates. Upper case and lower case letters show statistically significant differences among treatments at the end of the colonisation and at the end of the experiment, respectively.

The ANOVA analysis at the end of the experiment (day 10), suggested a significant effect of the inoculation treatment position (*p*-value < 0.001) and an interaction amongs<sup>t</sup> the inoculation positions and aw factors (*p*-value = 0.003). The 0.95 aw top-centre, the 0.97 aw top-centre, and 0.97 aw bottom-centre treatments were similar. The other treatments: 0.95 aw bottom-side, 0.97 aw bottom-side, and 0.95 aw bottom-centre, were similar but significantly lower than the previous group (Figure 4a,b).
