Differentiation and identification of grape-associated black aspergilli using Fourier transform infrared (FT-IR) spectroscopic analysis of mycelia.

The purpose of this study was to evaluate the potential of FT-IR spectroscopy as a high-throughput method for rapid differentiation among the ochratoxigenic species of Aspergillus carbonarius and the non-ochratoxigenic or low toxigenic species of Aspergillus niger aggregate, namely A. tubingensis and A. niger isolated previously from grapes of Greek vineyards. A total of 182 isolates of A. carbonarius, A. tubingensis, and A. niger were analyzed using FT-IR spectroscopy. The first derivative of specific spectral regions (3002-2801cm-1, 1773-1550cm-1, and 1286-952cm-1) were chosen and evaluated with respect to absorbance values. The average spectra of 130 fungal isolates were used for model calibration based on Discriminant analysis and the remaining 52 spectra were used for external model validation. This methodology was able to differentiate correctly 98.8% in total accuracy in both model calibration and validation. The per class accuracy for A. carbonarius was 95.3% and 100% for model calibration and validation, respectively, whereas for A. niger aggregate the per class accuracy amounted to 100% in both cases. The obtained results indicated that FT-IR could become a promising, fast, reliable and low-cost tool for the discrimination and differentiation of closely related fungal species.

Modelling the influence of temperature, water activity and sodium metabisulphite on the growth and OTA production of Aspergillus carbonarius isolated from Greek wine grapes.

The purpose of the present study was to develop a modelling approach to quantify the effect of temperature (15-38 °C), a(w) (0.88-0.98) and sodium metabisulphite (NaMBS) concentration (0-200 mg L(-1)) on the growth and OTA production of Aspergillus carbonarius on a Grape Juice based Medium (GJM). Growth responses of the fungus were recorded over time in terms of colony diameter changes, and fitted to the primary model of Baranyi and the estimated maximum growth rates (µ(max)) and lag phases (λ) were subsequently modelled as a function of temperature, a(w) and NaMBS concentration using the cardinal values model with inflection (CMI). Moreover, OTA production was measured during fungal growth and modelled as a function of the same parameters through a quadratic polynomial model. Results showed that NaMBS increased the lag phase of A. carbonarius, particularly at 38 °C/0.98 a(w) and 38 °C/0.96 a(w), as well as at lower a(w) levels regardless of temperature. In the lowest NaMBS concentration (50 mg L(-1)) there was no inhibitory effect, while at higher concentrations (100 and 150 mg L(-1)) fungal growth was delayed. No growth was observed at 200 mg L(-1) of NaMBS irrespective of temperature and a(w) levels. The optimum values for growth were found in the range 30-35 °C and 0.96 a(w), while for OTA production at 20 °C and 0.98 a(w). The developed models were subjected to internal and external validation and presented satisfactory performance as inferred by graphical plots and statistical indices (bias and accuracy factors). The present study will complement the findings on the ecophysiology of A. carbonarius using NaMBS as an inhibitory agent.

Effect of water activity, temperature, and mixed fungal spore interactions on ochratoxin A production by Aspergillus carbonarius.

The purpose of this work was to investigate the potential of two nontoxigenic Aspergillus section Nigri species (Aspergillus tubingensis and Aspergillus japonicus) to influence the in vitro ochratoxin A (OTA) production of three toxigenic Aspergillus carbonarius isolates (Ac-28, Ac-29, and Ac-33) from Greek vineyards of different geographical areas. OTA accumulation was evaluated by inoculation of 0:100, 25:75, 50:50, 75:25, and 100:0 ratios of mixed spore suspensions on a synthetic grape juice medium for up to 28 days at different temperatures (15, 20, and 25°C), water activity (aw) levels (0.95 and 0.98 aw) and incubation time (7, 14, 21, and 28 days). Results confirmed that environmental factors and fungal species had a significant effect on OTA production. Specifically, maximum OTA concentration for Ac-28 (3.21 µg g(-1)) and Ac-29 (7.69 µg g(-1)) was observed at 20°C/0.98 aw and for Ac-33 (9.13 µg g(-1)) at 15°C/0.95 aw, regardless of incubation time. Moreover, A. tubingensis had no significant influence on OTA concentration of all toxigenic isolates assayed, regardless of temperature, aw, and incubation time. On the other hand, the presence of A. japonicus slightly inhibited OTA production of Ac-29 and Ac-33, while for Ac-28, stimulation of OTA was observed in some cases. Overall, lower aw levels reduced OTA accumulation for Ac-28 and Ac-29, regardless of temperature, inoculum ratio, and time. On the contrary, for Ac-33, low aw increased OTA production, regardless of the investigated parameters. The importance of this study concerns the understanding of interspecific interactions on OTA diffusion by A. carbonarius in an attempt to find ways to prevent the presence of toxins in grapes and their derivatives.

Effect of interaction between Aspergillus carbonarius and non-ochratoxigenic grape-associated fungal isolates on growth and ochratoxin A production at different water activities and temperatures.

The effect of water activity (0.90, 0.94, and 0.98 aw) and temperature (15, 20, and 25 °C) on the in vitro interactions between three ochratoxigenic strains of Aspergillus carbonarius (Ac-28, Ac-29, and Ac-33) and eleven non ochratoxigenic grape-associated fungal strains was assessed in this study. Fungal strains were allowed to grow in dual cultures on Synthetic Grape-juice Medium (SGM) for 15 days and fungal interactions were given a numerical score to obtain an Index of Dominance (ID) for each fungus. Results showed that in most cases A. carbonarius toxigenic strains were dominant against other fungal species. However, A. carbonarius presented mutual antagonism with A. section Nigri strains regardless of water activity (aw) and temperature. Moreover, interactions with Penicillium spinulosum and Cladosporium spp. at 15 °C, as well as Botrytis cinerea at 20 °C, showed that the antagonists were more competitive against A. carbonarius. In some cases, growth rates of A. carbonarius strains were either slightly stimulated or inhibited after interaction in dual cultures, depending on temperature, aw and competing species. Regarding OTA production, the presence of other species sometimes decreased the production or slightly enhanced it, depending on fungal competitor and environmental conditions. Overall, OTA production was higher at 15 °C/0.98 aw and 20 °C/0.98 aw for all target strains and at 20 °C/0.94 aw for Ac-33 strain only, but decreased at higher temperatures regardless of aw and interacting species.