**1. Introduction**

Wheat is susceptible to *Fusarium* head blight (FHB) world-wide which causes both yield and quality losses, in addition to contamination of harvested grain with mycotoxins, particularly zearalenone (ZEN) and type B trichothecenes such as deoxynivalenol (DON). There are strict regulations in place for maximum contamination levels of these two toxins to reduce accumulation in the human and animal food chains. Both mycotoxins can be produced pre- and post-harvest. Although *Fusarium graminearum* requires higher moisture content levels compared to other more xerotolerant or xerophilic spoilage fungi (e.g., *Penicillium* and *Aspergillus* species, respectively), delayed or ine fficient drying and unsafe storage practices can allow fungal growth and potential mycotoxin accumulation.

Recent studies have shown that under conducive moisture contents and over a wide range of temperatures, *F. graminearum* colonisation of stored wheat grain can cause significant dry matter losses (DMLs) and result in DON and ZEN contamination exceeding the EU maximum limits for unprocessed cereals for human food consumption (DON: 1.750 and ZEN: 100 μgKg−1) [1] and animal feed (DON: 8.000 and ZEN: 2.000 μgKg−1) [2]. Overall, <1% DML can result in significant impacts on quality losses and toxin contamination exceeding the EU limits [3–5].

Stored cereals in silos represent an ecosystem in which there are significant interactions between the abiotic and biotic factors [6]. Indeed, Limay-Rios et al. [7] showed that the main sources for contamination of stored wheat in grain silos are at the top of the silo, where the inlet for grain filling is placed, and the outlet, where the grain is removed from the silo. These areas and the silo walls represent areas where higher moisture content can occur due to moisture migration, leading to increased contamination with mycotoxins.

However, there is very little information on how fungal growth is initiated and the way in which mycelial colonisation occurs, especially in three dimensions. Most previous studies have been conducted by measuring colonisation in two directions or dimensions only [8,9]. However, in reality, filamentous fungi such as *F. graminearum* grow in stored grain in three dimensions extending their mycelium in all directions to obtain nutrients from the rich wheat substrate. Du et al. [10] and Vidal-Diez de Ulzurrun et al. [11] recently reported novel lattice-based models for a three dimensional study of fungal growth, but there are no publications that have examined *Fusarium* colonisation in stored cereal grain.

Measurement of ergosterol, fungal biomass and colony diameter have been positively correlated on agar media previously suggesting that the three parameters may be useful for primary modelling [12–14]. Colony diameter is extensively used to assess fungal growth. However, in aged colonies (>30 days) ergosterol did not increase exponentially, suggesting that despite the colony expansion in terms of area, there was a decrease in the rate of ergosterol accumulation [12]. Similarly, fungal diameter measurements may not be a perfect indicator of fungal biomass, especially in aging colonies where biomass may increase despite limitation in actual colony extension [13]. Other studies have used ergosterol content as a measure of the overall mould contamination levels of commodities and related this to safe or unsafe storage conditions [15,16]. In addition, the use of ergosterol was found to be an effective biochemical marker for early indications of fungal contamination as well as of predicted DON contamination levels in stored cereals [17,18].

However, few studies, if any, have examined the colonisation of cereals by a mycotoxigenic species in three dimensions to try to understand the growth kinetics and the relationship between growth and toxin production. There is also a lack of knowledge of how temporal fungal colonisation occurs in a 3D matrix such as stored grains via the intergranular spaces, and how this may be a ffected by the initial inoculum position or the prevailing environmental factors.

The objectives of this study were to examine the kinetics of 3D spatial colonisation of stored wheat grain by *F. graminearum* and the accumulation of DON and ZEN when inoculated in di fferent positions and under di fferent water availabilities in laboratory microcosm storage systems. This allowed information to be obtained on (a) spatial patterns of colonisation depending on the di fferent initial inoculum positions, (b) the changes in respiration and associated DMLs, (c) quantification of fungal biomass, using ergosterol, (d) the temporal DON and ZEN mycotoxin accumulation during colonisation and (e) the correlation between these di fferent biological and physical parameters.
