*2.3. Colonisation Pattern Assessment*

Fungal colonisation of the wheat grain was followed by recording the superficial mycelial extension visible through the six sides of the square clear jars (supplementary Figure S2). Clear labels with a mesh of 0.5 × 0.5 cm were aligned and pasted on the bottom and sides of the jars. To avoid external contamination of the grains, jar lids were opened under sterile conditions and a plastic sheet with the same mesh was aligned on the top. Visual fungal colonisation was followed every 24 h until the grain was fully colonised.

The colonisation data were used to compute volumetric colonisation (cm3) by *F. graminearum* from the three inoculation treatment positions. In two dimensional studies, fungal growth is known to expand in circular colonies following a radial pattern [21]. In the present study, we expanded the approach assuming that mycelial colonisation followed an ellipsoidal shape before being constrained by the cubic shape of the jar. The detailed procedure followed to compute the volumetric colonisation is presented in Appendix A.

### *2.4. Respiration Determination and Dry Matter Loss Estimation*

Every 24 h, samples were sealed for 1 h at 25 ◦C with injection lids that contained polytetrafluoroethylene (PTFE)/silicone septa (20 mm diameter × 3.175 mm thickness) to allow for CO2 accumulation in the headspace. Headspace volumes of jars containing grain at 0.95 or 0.97 aw were, respectively, 90.67 and 88.33 mL. In both growth conditions, 5 mL of headspace air were withdrawn with a syringe and directly inserted into the Gas Chromatograph (GC) for CO2 analysis. An Agilent 6890N Network GC (Agilent Technologies, Cheshire, UK) with a Thermal Conductivity Detector and helium as a carrier gas was used. A Chromosorb 103 packed column was usedand the data were analysed using Agilent Chemstation Software (Agilent Technologies, Cheshire, UK).

GC was calibrated with a standard gas bottle of 10.18% CO2 and 2% O2 in nitrogen (British Oxygen Company, Guilford, Surrey, UK). *F. graminearum* CO2 production was obtained by subtracting the sample respiration of a blank culture that had not been inoculated with the fungal species. The percentages of CO2 production were used to calculate the Respiration (R) in mg CO2 (kg·h−1), total cumulative production CO2 and total DML as described in Mylona and Magan [3].
