Pantelis I. Natskoulis

Effect of temperature and water activity on growth and ochratoxin A production boundaries of two Aspergillus carbonarius isolates on a simulated grape juice medium

Aims:  To develop and validate a logistic regression model to predict the growth and ochratoxin A (OTA) production boundaries of two Aspergillus carbonarius isolates on a synthetic grape juice medium as a function of temperature and water activity (aw).

Modelling the effect of temperature and water activity on the growth of two ochratoxigenic strains of Aspergillus carbonarius from Greek wine grapes.

Aims:  To develop descriptive models for the combined effect of temperature (10–40°C) and water activity (0·850–0·980) on the growth of two ochratoxin A producing strains of Aspergillus carbonarius from Greek wine grapes on a synthetic grape juice medium.

Biodiversity and ITS-RFLP Characterisation of Aspergillus Section Nigri Isolates in Grapes from Four Traditional Grape-Producing Areas in Greece

A study on the occurrence of Aspergillus section Nigri species on grapes from four traditional grape-producing areas in Greece during the 2011/2012 vintage, and their capability to produce OTA was conducted. One hundred and twenty-eight black aspergilli isolates were characterised at the species level initially by the use of morphological criteria in accordance with appropriate keys, followed by molecular characterisation performed with Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) of the 5.8 ribosomal RNA gene Internal Transcribed Spacer region (5.8 rRNA ITS). Restriction enzyme digestion of the ITS amplicons using the HhaI, HinfI and RsaI, endonucleases distinguished eleven different patterns of restriction fragment length polymorphism (RFLP), four for each of the HhaI and RsaI digests and three for HinfI. From a total number of 128 individual isolates, 124 were classified into four Aspergillus species corresponding to A. carbonarius, A. tubingensis, A. japonicus and A. ibericus, and the remaining 4 were classified as members of the A. niger aggregate. A. carbonarius and A. tubingensis being the main representative species were equally counted, with higher geographical representation of the former in southern and the latter in northern regions, respectively. All isolates were tested for their ochratoxigenic potential by use of High Performance Liquid Chromatography (HPLC) and Enzyme Linked Immuno Sorbent Assay (ELISA), resulting in significant interspecies differences in OTA production.

A survey of ochratoxin A occurence in Greek wines.

A total of 150 wines, including 123 dry wines (64 red, 49 white and 10 rosé) and 27 dessert wines (14 red and 13 white), were obtained from various viticulture and oenological practices across Greece during the period 1999–2006 and analyzed for ochratoxin a (OTA) using immunoaffinity clean-up and HPLC with fluorescence detection. There was a high frequency of OTA in commercially available wines (69% positive samples). However, the level of contamination was relatively low, with only one sample marginally reaching the EU permitted maximum level (2.0 µg l−1). A total of 91% of the samples had OTA concentrations <1.0 µg l−1. The higher concentrations were found in wines from the southern regions, especially in dessert-type wines. There were no significant differences based on wine color or production years. Furthermore, there was no difference between conventional or organic cropping systems in terms of OTA presence.

Impact of water activity and temperature on growth and ochratoxin A production of two Aspergillus carbonarius isolates from wine grapes in Greece

The aim of this study was to determine the effects of water activity (a(w); 0.85 to 0.98) and temperature (10 to 40 degrees C) on the radial growth rate and ochratoxin A (OA) production of two Aspergillus carbonarius isolates in vitro. The isolates were obtained from wine grapes cultivated in Greece, and the trial was conducted with a synthetic grape juice medium similar in composition to grapes between veraison (beginning of color change) and ripeness. Fungal growth and OA production data were collected for 55 days. Response surface curves and cardinal values for a(w) and temperature were obtained using multiple regression analysis. The lag phase lasted from less than 1 to 10 days. Both isolates grew optimally at 30 to 35 degrees C and 0.96 a(w), but maximum OA production occurred under suboptimal growth conditions (15 to 20 degrees C and 0.93 to 0.96 a(w)). Growth also was observed at 0.85 a(w) and 25 degrees C, however at this same a(w) the fungus failed to produce mycelium at any other temperatures tested. The isolates produced OA at 15 to 30 degrees C and 0.90 to 0.98 a(w). Maximum OA production was detected after 25 days of incubation at 20 degrees C and 0.96 a(w) and was 3.14 and 2.67 microg g(-1), respectively, for the two strains. The isolated strains used in this study were more xerotolerant than others from the Mediterranean basin. These data will allow producers to identify and thus monitor critical environmental conditions effectively in wine grapes. These data also increase the knowledge base concerning the ability of A. carbonarius to grow and produce toxin under different ecological conditions and can contribute to the development of secondary models for the prediction and risk assessment of OA in wine production.

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 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.

Comparative study of growth responses and screening of inter-specific OTA production kinetics by A. carbonarius isolated from grapes

The aim of this work was to assess OchratoxinA (OTA) production of different Aspergillus carbonarius isolates, evaluate their growth profile through different growth measurements, and reveal any underlying correlation between them. Ten different isolates of A. carbonarius isolated from Greek vineyards located in different geographical regions were examined in vitro for their OTA production potential after an incubation period of up to 11 days. All fungal isolates grew on a synthetic grape juice medium (SGM) similar to grape composition at optimum conditions of temperature and water activity (25°C and 0.98 aw). Samples for OTA determination were removed at 3, 5, 7, 9, and 11 days of growth and analyzed by HPLC. Based on OTA measurements the isolates were characterized by diverse OTA production ranging from 50 to 2000 ppb at day 11. The different fungal growth responses (colony diameter, colony area, biomass, biomass dry weight, and colony density) have been measured and correlated with toxin production by means of principal components analysis (PCA), confirming satisfactory correlation and explained over 99% of data variability. Leudeking-Piret model was also used to study OTA production with time, revealing a mixed-growth associated trend and pointing a fail-safe model with slightly better prediction through colony area. This approach contributes to the assessment of correlation between mycotoxin production and different methods of fungal growth determination in relation to time.

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.

Evaluating the efficacy of turbimetric measurements as a rapid screening technique to assess fungal susceptibility to antimicrobial compounds as exemplified by the use of sodium metabisulfite

The in vitro susceptibility to sodium metabisulphite (NaMBS) was investigated in 10 different food spoilage filamentous fungi, namely Aspergillus flavus, A. carbonarius, A. niger, A. ochraceus, A. tubingensis, A. westerdijkiae, Cladosporium cladosporioides, Fusarium oxysporum, Penicillium commune and P. expansum. The fungi were inoculated in sterile 96-well microtiter plates containing Yeast-extract Sucrose (YES) semi-solid agar supplemented with NaMBS in concentrations ranging from 2000 to 3.9 mg l-1 and incubated at 25 °C. Growth was monitored by absorbance measurements at 600 nm using a multi-spectrophotometer. The surface areas under the optical density (OD) vs. time growth curves obtained were used to calculate the fractional area f(a), from which the non-inhibitory (NIC) and minimum inhibitory concentrations (MIC) of the antifungal agent were calculated for each fungus using the Gompertz model for decay. Most Aspergillus species showed remarkable resistance to NaMBS, presenting NIC and MIC values higher than 250 and 2500 mg l-1, respectively. The most susceptible fungi were the two Penicillium species and A. carbonarius, which presented very low NIC (<100 mg l-1) and MIC (<1300 mg l-1) values, whereas C. cladosporioides and F. oxysporum presented intermediate values. The method has the advantage of good repeatability and accuracy, rapid results within 48-72 h, growth detection and susceptibility to the antifungal agent for several fungi at the same time, and optimal use of microbiological media by using small volumes of consumables.