**5. Conclusions**

In this study, the behaviour of *F. graminearum* in stored wheat in terms of grain colonisation and mycotoxin production (DON and ZEN) was evaluated in a 3D volume for a period of ten days. Primary inoculum position affected the initial growth significantly and therefore the colonised grain. Modern silos are currently monitored at different spatial levels, consequently, spatial modelling could be used to predict the level of risk. Respiration and DML indicators seem to be as reliable as ergosterol measurements (to indicate fungal colonisation) but with the advantage that they can be monitored in real-time. Thus, to perform efficient silo management, different approaches must be tailored to each of the spatial areas covered by the sensors in which the alert was detected.

The results of this study revealed that understanding the fungal growth pattern and the diffusion of multiple mycotoxins is essential for the development of accurate predictive models that can support effective post-harvest managemen<sup>t</sup> of grain. This is critical as grain is traded on a wet weight basis and very slight changes in the moisture can lead to an increase in the activity of mycotoxigenic spoilage moulds and mycotoxin contamination.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-2607/8/8/1170/s1, Figure S1: Clear square jars used in this experiment, Figure S2: Labelling system used to follow 3D colonisation of wheat grains by *F. graminearum*.

**Author Contributions:** Conceptualization, X.P. and E.G.-C.; Formal analysis, X.P., C.V.-V. and E.G.-C.; Funding acquisition, E.G.-C.; A.M. and N.M.; Investigation, R.T.-R.; Methodology, X.P., C.V.-V., A.M., W.O. and E.G.-C.; Supervision, X.P., A.M., W.O. and E.G.-C.; Writing–original draft, X.P., C.V.-V., R.T.-R. and E.G.-C.; Writing–review & editing, X.P., C.V.-V., R.T.-R, A.M., W.O., N.M. and E.G.-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by European Union's Horizon 2020 research and innovation programme under gran<sup>t</sup> agreemen<sup>t</sup> No. 678012 (MyToolBox), and BBSRC-SFI research gran<sup>t</sup> (BB/P001432/1) between the Applied Mycology Group at Cranfield University and the School of Biology and Environmental Science, University College Dublin, Ireland.

**Acknowledgments:** We thank Simon Edwards, Harper Adams University, for providing the strain used in this study. The authors would like to thank Dominique Vaessen for helping in the preparation of the jars for the experiment.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

### **Appendix A. Computation of the Grain Colonised Volume**

Three-dimensional fungal colonisation was assumed to follow an ellipsoidal shape of radii *a*, *b* and *c* (Figure A1). After the initial stages of colonisation, the ellipsoidal expansion was constrained by the finite volume of the jar and a number of assumptions were required. The procedure followed for the centre and side positions is described below.

**Figure A1.** Radii and spatial configuration assumed in the study. (**a**) Ellipsoid showing radii of the fungal colonisation, and (**b**) coordinate system and faces of the medium.

### *Appendix A.1. Top-Centre and Bottom-Centre Positions*

Volume computation could be generalized in two main cases:

1. Mycelial expansion just observable on the top (bottom) side. Volumetric colonisation at the time *t* (*Vt*) was computed assuming a half ellipsoidal shape (Equation (A1))

$$V\_t = \frac{2}{3}\pi \cdot a \cdot b \cdot c,\tag{A1}$$

where *a*, *b* and *c* are elliptic radii. Radii *b* and *c* where computed as the maximum minus the minimum coordinates showing colonisation along X and Y axis divided by two, and *a* was assumed to be the 0.75· *b*+*c* 2 . This assumes an upwards (downwards) radial reduction due to the fact that the mycelial expansion proceeds through a more tortuous path that the surface as observed experimentally [39].

2. Mycelial expansion was observable on the vertical sides of the grain volume. Colonised volume was approximated as cubic shape and a half ellipsoid (Equation (A2)) as follows:

$$V\_t = L\_{\hat{\mathcal{K}}} \cdot L\_{\hat{\mathcal{Y}}} \cdot L\_{\hat{\mathcal{Z}}} + \frac{2}{3} \pi \cdot \frac{L\_{\hat{\mathcal{K}}}}{2} \cdot \frac{L\_{\hat{\mathcal{Y}}}}{2} \cdot a\_\prime \tag{A2}$$

where *LX*ˆ , *LY*ˆ and *LZ*ˆ are mean distances obtained along axes X, Y and Z, respectively, showing colonisation. Radii *a* (in cm) was computed as 3.5 − *LZ*ˆ if the face opposite to the inoculation point (bottom and top, respectively, for top-centre and bottom-centre inoculations, respectively) showed any sign of colonisation, or as the minimum value between 0.75· *LX*ˆ −*LY*<sup>ˆ</sup> 2 and 3.5 − *LZ*ˆ if not.
