*3.1. Fungal Colonisation*

*F. graminearum* showed a rapid colonisation rate at both tested aw levels with all of the wheat grain visually completely colonised after 5–6 days. A comparative example of the recorded superficial growth showed distinct colonisation patterns of *F. graminearum* depending on the initial inoculum position (Figure 1). Superficial colonisation data were well described using a two-phase linear model (Figure 2) providing estimates of the lag time prior to colonisation and the superficial colonisation rate (cm<sup>2</sup>·day−1) shown in Table 2. The lag phase length prior to superficial colonisation was shown to be significantly affected by the inoculation position but not by aw, with mean lag phase times higher for the bottom-side position (2.97 ± 0.22 h) than the bottom-centre (2.92 ± 0.31 h) and top-centre (2.94 ± 0.32 h) positions. No significant differences (α = 0.05) in the colonisation rate were detected among treatments. As a consequence of this increased lag time, the bottom-side inoculations were able to completely colonise the grain surface only by day 6 of the experiment. For the other inoculum positions (top-centre and bottom-centre) this occurred after 5 days (Figure 3a,b).

**Figure 1.** Example of the visual observation of the fungal colonisation dynamics at 0.95 aw starting from different inoculation positions: (**a**) top-centre, (**b**) bottom-side and (**c**) bottom-centre. In the figure, colours depict colonisation after day 2 ( ), day 3 ( ), day 4 ( ), day 5 ( ) and day 6 ( ).

**Figure 2.** Example of the two-phase linear model fit (blue line) for the colonisation data (dots) recorded for the sample grown on 0.95 aw, bottom-centre inoculation, second replication. (**a**) Model fit to surface colonisation data, and (**b**) model fit to volume colonisation data.

**Table 2.** Superficial and volumetric colonisation rates. Lag times and colonisation rates were obtained by adjusting the data to a two-phase linear model.


**Figure 3.** Temporal colonisation of the wheat grain by *F. graminearum* in both aw treatments from the three different initial inoculum positions. (**a**) Superficial colonisation for 0.95 aw treatment, (**b**) superficial colonisation for 0.97 aw treatment, (**c**) volumetric colonisation for 0.95 aw treatment, and (**d**) volumetric colonisation for 0.97 aw treatment. Bars show standard deviation of the three replicates.

Volumetric fungal colonisation (cm3) was obtained from the visible colonisation assuming that the fungal growth of the grain was consistent with a partial ellipsoidal expansion of the mycelia constrained by the cubic volume of the jar (see Appendix A). Volumetric colonisation (Figure 3b,c) was also well adjusted to a simple two-phase linear model, obtaining colonisation lag times and volumetric colonisation rate (cm<sup>3</sup>·day−1) estimates shown in Table 2. The length of the lag phase was shown to be significantly affected by the inoculation position (*p*-value = 0.003), with the bottom-side inoculation having an extended lag period (3.28 ± 0.16 h) distinguishable from that of the top-centre (2.89 ± 0.21 h) and bottom-centre (2.76 ± 0.14 h) inoculum positions. The volumetric colonisation rate were significantly affected by aw, and was more rapid at 0.97 aw (69.52 ± 15.24 cm<sup>3</sup>/day) than at 0.95 aw (51.10 ± 7.42 cm3·day−1).

### *3.2. Indirect Indicators of Fungal Growth*
