**2. Materials and Methods**

### *2.1. Fungal Isolate and Spores Preparation*

*F. graminearum* strain Fg 08/111 isolated from wheat in the UK was cultivated on V8 vegetable juice plates (V8 ®, 175 mL; CaCO3, 3 g; ZnSO4·7H2O, 0.01g; CuSO4·5H2O, 0.005g; agar, 20 <sup>g</sup>·L−1) and incubated in the dark at 25 ◦C for ten days to obtain heavily sporulating cultures [19]. Following incubation, 3 mL of sterile Tween 80 ® (0.05%) was poured on the Petri plates previously incubated and

then gently scratched with a sterile Drigalsky's spatula. The liquid was transferred to a second Petri plate containing fungi and the procedure repeated with the remaining 4 Petri plates. The resulting liquid was recovered and collected in a sterile falcon tube. After homogenizing, the number of spores per mL was determined using a Thoma counting chamber (Celeromics, Grenoble, France) and the final concentration adjusted to 1 × 10<sup>4</sup> spores·mL−<sup>1</sup> in sterile water + Tween 80 (0.005%).

### *2.2. Grain Preparation, Inoculation and Incubation*

Dry wheat grain (0.71 water activity (aw), 13.5 % moisture content (m.c.)) was irradiated with 12–15 kGy (Synergy Health, Swindon, UK) to reduce microbial contaminants while maintaining germinative capacity of the grain, and stored at 4 ◦C until use. The grain aw levels were modified to 0.95 and 0.97 by adding known amounts of sterile water and reference to an adsorption curve for this batch of wheat grain, which was detailed in Garcia-Cela et al. [20], and verified using the Aqualab 4TE aw meter.

Square jars of 136 mL capacity (Pattersons Glass Ltd., Grimsby, UK) (see supplementary Figure S1) with a 9 mm diameter hole in the jar lid, which was closed with a cotton wool plug to allow gaseous exchange while avoiding contamination, were autoclaved at 121 ◦C for 20 min. Single grains inoculated with a drop of 5 μL of the 10<sup>4</sup> spores mL−<sup>1</sup> suspension (approx. 50 spores per grain) were placed in distinct positions to provide initial inoculum of *F. graminearum* for initiating growth in the different positions within the jars: (a) top-centre, (b) bottom-side and (c) bottom-centre. For the bottom inoculations, an 8 μL drop of organic clear nail polish was used to fix the grain on the jar bottom. Fixed grains were inoculated with the stated spore solution drop and 1 min later, the remaining grains were added to the jar. For the top-centre position, the spore solution drop was placed over a single grain in the centre of the top layer of grains. In all cases, 15 jars per treatment combination were used, including 3 samples without *F. graminearum* inoculated per aw tested.

Inoculated jars were incubated at 25 ◦C for 10 days. To maintain atmospheric equilibrium relative humidity, jars were placed in food storage containers (10 L volume) containing beakers of glycerol–water solution at the treatment aw level (6 × 100 mL per container). Glycerol–water solutions were renewed once (day 5) during the experimental period.
