Sporulation of Aspergillus flavus scletoria in different ecological regimes.

Aspergillus flavus is an important pathogen of maize, one of the most hazardous fungi able to produce aflatoxins (AFs), mycotoxins with high toxicity for animals and humans and with immunosuppressive and carcinogenic properties. Different in vitro and in situ studies have been conducted to describe and improve the knowledge on the infection cycle of this fungus. Surprisingly, very little information is available on sporulation, which is the crucial stage to trigger fungal infection. The most important inoculum source is represented by sclerotia since they can survive for a long time in soil or on debris, even in extreme conditions of temperature (T) and water activity (aw). Both these ecological factors are critical in all stages of the infection cycle, including sporulation. The aim of this research was to study the dynamics of sporulation, both on artificial media and on maize stalk residues, under different T and aw regimes. Sclerotia produced by a strain of A. flavus (MPVP 2062, Giorni et al., 2007) on Czapek agar (CZ) were collected and used as inoculum source. CZ agar and maize stalk pieces (approximately 3 x 2 cm) were selected as substrates and inoculated with a single sclerotium. CZ was added with required amounts of glycerol to obtain a wide range of aw conditions (0.50, 0.60, 0.70, 0.80, 0.85, 0.90, 0.93, 0.95, 0.97, 0.99); similarly, stalk portions were incubated in chambers with salt solutions (target aw levels: 0.90, 0.93, 0.95, 0.97, 0.99) to maintain the required equilibrium relative humidity. The experiment with modified aw was incubated at 25°C. CZ and stalk portions at 0.99 aw were incubated at different temperature conditions (5-45°C, with 5°C step). Sporulation was checked after 0.25, 1, 2, 4, 6, 8, 12, 16 and 20 days incubation. Results were reported as number of spores produced per sclerotium in each studied condition. The analysis of variance was applied to logarithm transformed data. The daily sporulation rate was also computed as function of T and aw. Sporulation on CZ and on stalk debris was significantly influenced by temperature, aw and time of incubation. Spore production was observed in all aw conditions studied, with wide variability. The maximum sporulation (number of spores/sclerotium) was obtained at 30-35°C and 0.99 aw after 8 days incubation in both CZ and on stalks. The number of conidia produced was higher in the artificial media when compared with that on stalk pieces. Sporulation rate in different T and aw regimes will be included as fundamental information in the development of a mechanistic model able to predict the infection cycle of A. flavus in maize in relation to meteorological conditions. This will provide valuable support to farmers in the prediction of AFs to optimise maize management pre- and post-harvest.